James S. Hardwick

gamma-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Abstract Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a histone deacetylase inhibitor active clinically in cutaneous T-cell lymphoma and preclinically in leukemia. A phase 1 study was conducted to evaluate the safety and activity of oral vorinostat 100 to 300 mg twice or thrice daily for 14 days followed by 1-week rest. Patients with relapsed or refractory leukemias or myelodysplastic syndromes (MDS) and untreated patients who were not candidates for chemotherapy were eligible. Of 41 patients, 31 had acute myeloid leukemia (AML), 4 chronic lymphocytic leukemia, 3 MDS, 2 acute lymphoblastic leukemia, and 1 chronic myelocytic leukemia. The maximum tolerated dose (MTD) was 200 mg twice daily or 250 mg thrice daily. Dose-limiting toxicities were fatigue, nausea, vomiting, and diarrhea. Common drug-related adverse experiences were diarrhea, nausea, fatigue, and anorexia and were mild/moderate in severity. Grade 3/4 drug–related adverse experiences included fatigue (27%), thrombocytopenia (12%), and diarrhea (10%). There were no drug-related deaths; 7 patients had hematologic improvement response, including 2 complete responses and 2 complete responses with incomplete blood count recovery (all with AML treated at/below MTD). Increased histone acetylation was observed at all doses. Antioxidant gene expression may confer vorinostat resistance. Further evaluation of vorinostat in AML/MDS is warranted.

Purpose Vorinostat, a histone deacetylase inhibitor, represents a rational therapeutic target in glioblastoma multiforme (GBM). Patients and Methods Patients with recurrent GBM who had received one or fewer chemotherapy regimens for progressive disease were eligible. Vorinostat was administered at a dose of 200 mg orally twice a day for 14 days, followed by a 7-day rest period. Results A total of 66 patients were treated. Grade 3 or worse nonhematologic toxicity occurred in 26% of patients and consisted mainly of fatigue (17%), dehydration (6%), and hypernatremia (5%); grade 3 or worse hematologic toxicity occurred in 26% of patients and consisted mainly of thrombocytopenia (22%). Pharmacokinetic analysis showed lower vorinostat maximum concentration and area under the curve (0 to 24 hours) values in patients treated with enzyme-inducing anticonvulsants, although this did not reach statistical significance. The trial met the prospectively defined primary efficacy end point, with nine of the first 52 patients being progression-free at 6 months. Median overall survival from study entry was 5.7 months (range, 0.7 to 28+ months). Immunohistochemical analysis performed in paired baseline and post-vorinostat treatment samples in a separate surgical subgroup of five patients with recurrent GBM showed post treatment increase in acetylation of histones H2B and H4 (four of five patients) and of histone H3 (three of five patients). Microarray RNA analysis in the same samples showed changes in genes regulated by vorinostat, such as upregulation of E-cadherin (P = .02). Conclusion Vorinostat monotherapy is well tolerated in patients with recurrent GBM and has modest single-agent activity. Histone acetylation analysis and RNA expression profiling indicate that vorinostat in this dose and schedule affects target pathways in GBM. Additional testing of vorinostat in combination regimens is warranted.

See also: Commentaire de travail de E. Dekker et al., pp. 216Endoscopy 2007; 39(03): 265-265DOI: 10.1055/s-0032-1306917

The ROS1 tyrosine kinase is activated through ROS1 gene rearrangements in 1-2% of non-small cell lung cancer (NSCLC), conferring sensitivity to treatment with the ALK/ROS1/MET inhibitor crizotinib. Currently, insights into patterns of metastatic spread and mechanisms of crizotinib resistance among ROS1-positive patients are limited.We reviewed clinical and radiographic imaging data of patients with ROS1- and ALK-positive NSCLC in order to compare patterns of metastatic spread at initial metastatic diagnosis. To determine molecular mechanisms of crizotinib resistance, we also analyzed repeat biopsies from a cohort of ROS1-positive patients progressing on crizotinib.We identified 39 and 196 patients with advanced ROS1- and ALK-positive NSCLC, respectively. ROS1-positive patients had significantly lower rates of extrathoracic metastases (ROS1 59.0%, ALK 83.2%, P=0.002), including lower rates of brain metastases (ROS1 19.4%, ALK 39.1%; P = 0.033), at initial metastatic diagnosis. Despite similar overall survival between ALK- and ROS1-positive patients treated with crizotinib (median 3.0 versus 2.5 years, respectively; P=0.786), ROS1-positive patients also had a significantly lower cumulative incidence of brain metastases (34% vs. 73% at 5 years; P<0.0001). Additionally, we identified 16 patients who underwent a total of 17 repeat biopsies following progression on crizotinib. ROS1 resistance mutations were identified in 53% of specimens, including 9/14 (64%) non-brain metastasis specimens. ROS1 mutations included: G2032R (41%), D2033N (6%), and S1986F (6%).Compared to ALK rearrangements, ROS1 rearrangements are associated with lower rates of extrathoracic metastases, including fewer brain metastases, at initial metastatic diagnosis. ROS1 resistance mutations, particularly G2032R, appear to be the predominant mechanism of resistance to crizotinib, underscoring the need to develop novel ROS1 inhibitors with activity against these resistant mutants.

Gastric cancer (GC) is the second most common cause of cancer-related deaths. It is known to be a heterogeneous disease with several molecular and histological subtypes. Here we perform whole-genome sequencing of 49 GCs with diffuse (N=31) and intestinal (N=18) histological subtypes and identify three mutational signatures, impacting TpT, CpG and TpCp[A/T] nucleotides. The diffuse-type GCs show significantly lower clonality and smaller numbers of somatic and structural variants compared with intestinal subtype. We further divide the diffuse subtype into one with infrequent genetic changes/low clonality and another with relatively higher clonality and mutations impacting TpT dinucleotide. Notably, we discover frequent and exclusive mutations in Ephrins and SLIT/ROBO signalling pathway genes. Overall, this study delivers new insights into the mutational heterogeneity underlying distinct histologic subtypes of GC that could have important implications for future research in the diagnosis and treatment of GC. Gastric cancer has two distinct morphologic subtypes, intestinal and diffuse, that differ in genetic composition and clinical manifestation. Here, the authors carry out whole-genome sequencing of diffuse and intestinal gastric cancer samples and characterize the mutational landscape of these different subtypes.

Purpose Addition of aprepitant, a neurokinin-1 receptor antagonist (NK1RA), to an ondansetron and dexamethasone regimen improves prevention of chemotherapy-induced nausea/vomiting (CINV), particularly during the delayed phase (DP; 25 to 120 hours). Therefore, recommended antiemetic regimens include multiple-day NK1RA administration. Preliminary data suggested that single-dose aprepitant before chemotherapy could provide CINV protection throughout the overall risk phase (OP; 0 to 120 hours). This study compared a 3-day oral aprepitant schedule to a regimen containing a single dose of the intravenous NK1RA fosaprepitant. Patients and Methods A randomized, double-blind, active-control design was used to test whether fosaprepitant is noninferior to aprepitant. Patients receiving cisplatin ≥ 70 mg/m 2 for the first time received ondansetron and dexamethasone with a standard aprepitant regimen (125 mg on day 1, 80 mg on day 2, 80 mg on day 3) or a single-dose fosaprepitant regimen (150 mg on day 1). The primary end point was complete response (CR; no vomiting, no rescue medication) during OP. Secondary end points were CR during DP and no vomiting during OP. Accrual of 1,113 evaluable patients per treatment arm was planned to confirm noninferiority with expected CR of 67.7% and noninferiority margin of minus 7 percentage points. Results A total of 2,322 patients were randomly assigned, and 2,247 were evaluable for efficacy. Antiemetic protection with aprepitant and fosaprepitant was equivalent within predefined bounds for noninferiority. Both regimens were well tolerated, although more frequent infusion site pain/erythema/thrombophlebitis was seen with fosaprepitant relative to aprepitant (2.7% v 0.3%, respectively). Conclusion Given with ondansetron and dexamethasone, single-dose intravenous fosaprepitant (150 mg) was noninferior to standard 3-day oral aprepitant in preventing CINV during OP and DP.

Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells.

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett's esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPalpha, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine-cytokine receptor interactions and TGF-beta. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

Vorinostat is a potent histone deacetylase inhibitor that blocks the catalytic site of these enzymes. A large number of cellular proteins are modified post-translationally by acetylation, leading to altered structure and/or function. Many of these proteins, such as core nucleosomal histones and transcription factors, function in key cellular processes and signal transduction pathways that regulate cell growth, migration, and differentiation. At concentrations that are non-toxic to normal cells, vorinostat dramatically alters cellular acetylation patterns and causes growth arrest and death and in a wide range of transformed cells, both in vitro and in animal tumor models. Vorinostat has shown promising clinical activity against hematologic and solid tumors at doses that have been well tolerated by patients. Recent non-clinical experiments that explored the effects of vorinostat in combination with other chemotherapeutic agents have begun to illuminate potential mechanisms of action for this histone deacetylase inhibitor and are providing guidance for new avenues of clinical investigation.

Background & AimsThe incidence of esophageal and junctional adenocarcinoma has increased 6-fold in the past 30 years and 5-year survival remains approximately 20%. Current staging is limited in its ability to predict survival which has ramifications for treatment choices. The aim of this study was to generate and validate a molecular prognostic signature for esophageal adenocarcinoma.MethodsGene expression profiling was performed and the resulting 42,000 gene signatures correlated with clinical and pathologic features for 75 snap-frozen esophageal and junctional resection specimens. External validation of selected targets was performed on 371 independent cases using immunohistochemistry to maximize clinical applicability.ResultsA total of 119 genes were associated significantly with survival and 270 genes with the number of involved lymph nodes. Filtering of these lists resulted in a shortlist of 10 genes taken forward to validation. Four genes proved to be prognostic at the protein level (deoxycytidine kinase [DCK], 3′-phosphoadenosine 5′-phosphosulfate synthase 2 [PAPSS2], sirtuin 2 [SIRT2], and tripartite motif-containing 44 [TRIM44]) and were combined to create a molecular prognostic signature. This 4-gene signature was highly predictive of survival in the independent external validation cohort (0/4 genes dysregulated 5-year survival, 58%; 95% confidence interval [CI], 36%–80%; 1–2/4 genes dysregulated 5-year survival, 26%; 95% CI, 20%–32%; and 3–4/4 genes dysregulated 5-year survival, 14%; 95% CI, 4%–24% (P = .001). Furthermore, this 4-gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (P = .013).ConclusionsThis study has generated a clinically applicable prognostic gene signature that independently predicts survival in an external validation cohort and may inform management decisions. The incidence of esophageal and junctional adenocarcinoma has increased 6-fold in the past 30 years and 5-year survival remains approximately 20%. Current staging is limited in its ability to predict survival which has ramifications for treatment choices. The aim of this study was to generate and validate a molecular prognostic signature for esophageal adenocarcinoma. Gene expression profiling was performed and the resulting 42,000 gene signatures correlated with clinical and pathologic features for 75 snap-frozen esophageal and junctional resection specimens. External validation of selected targets was performed on 371 independent cases using immunohistochemistry to maximize clinical applicability. A total of 119 genes were associated significantly with survival and 270 genes with the number of involved lymph nodes. Filtering of these lists resulted in a shortlist of 10 genes taken forward to validation. Four genes proved to be prognostic at the protein level (deoxycytidine kinase [DCK], 3′-phosphoadenosine 5′-phosphosulfate synthase 2 [PAPSS2], sirtuin 2 [SIRT2], and tripartite motif-containing 44 [TRIM44]) and were combined to create a molecular prognostic signature. This 4-gene signature was highly predictive of survival in the independent external validation cohort (0/4 genes dysregulated 5-year survival, 58%; 95% confidence interval [CI], 36%–80%; 1–2/4 genes dysregulated 5-year survival, 26%; 95% CI, 20%–32%; and 3–4/4 genes dysregulated 5-year survival, 14%; 95% CI, 4%–24% (P = .001). Furthermore, this 4-gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (P = .013). This study has generated a clinically applicable prognostic gene signature that independently predicts survival in an external validation cohort and may inform management decisions.

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.

Insulin-like growth factor-1 receptor (IGF-1R) mediates cellular processes in cancer and has been proposed as a therapeutic target. Dalotuzumab (MK-0646) is a humanized IgG1 monoclonal antibody that binds to IGF-1R preventing receptor activation. This study was designed to evaluate the safety and tolerability of dalotuzumab, determine the pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and identify a recommended phase II dose.Patients with tumors expressing IGF-1R protein were allocated to dose-escalating cohorts of three or more patients each and received intravenous dalotuzumab weekly, every 2 or 3 weeks. Plasma was collected for PK analysis. Paired baseline and on-treatment skin and tumor biopsy samples were collected for PD analyses.Eighty patients with chemotherapy-refractory solid tumors were enrolled. One dose-limiting toxicity was noted, but a maximum-tolerated dose was not identified. Grade 1 to 3 hyperglycemia, responsive to metformin, occurred in 15 (19%) patients. At dose levels or more than 5 mg/kg, dalotuzumab mean terminal half-life was 95 hours or more, mean C(min) was more than 25 μg/mL, clearance was constant, and serum exposures were approximately dose proportional. Decreases in tumor IGF-1R, downstream receptor signaling, and Ki67 expression were observed. (18)F-Fluorodeoxy-glucose positron emission tomography metabolic responses occurred in three patients. One patient with Ewing's sarcoma showed a mixed radiologic response. The recommended phase II doses were 10, 20, and 30 mg/kg for the weekly, every other week, and every third week schedules, respectively.Dalotuzumab was generally well-tolerated, exhibited dose-proportional PK, inhibited IGF-1R pathway signaling and cell proliferation in treated tumors, and showed clinical activity. The low clearance rate and long terminal half-life support more extended dosing intervals.

Abstract Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P &amp;lt; 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR.

IntroductionThe incidence rate of lung adenocarcinoma (LUAD), the predominant histological subtype of lung cancer, is elevated in Asians, particularly in female nonsmokers. The mutation patterns in LUAD in Asians might be distinct from those in LUAD in whites.MethodsWe profiled 271 resected LUAD tumors (mainly stage I) to characterize the genomic landscape of LUAD in Asians with a focus on female nonsmokers.ResultsMutations in EGFR, KRAS, erb-b2 receptor tyrosine kinase 2 gene (ERBB2), and BRAF; gene fusions involving anaplastic lymphoma receptor tyrosine kinase gene (ALK), ROS1, and ret proto-oncogene (RET); and Met Proto-Oncogene Tyrosine Kinase (MET) exon 14 skipping were the major drivers in LUAD in Asians, exhibiting mutually exclusive and differing prevalence from those reported in studies of LUAD in non-Asians. In addition, we identified a novel mutational signature of XNX (the mutated base N in the middle flanked by two identical bases at the 5′ and 3′ positions) that was overrepresented in LUAD tumors in nonsmokers and negatively correlated with the overall mutational frequency.ConclusionsIn this cohort, approximately 85% of individuals have known driver mutations (EGFR 59.4%, KRAS 7.4%, ALK 7.4%, ERBB2 2.6%, ROS1 2.2%, RET 2.2%, MET 1.8%, BRAF 1.1%, and NRAS 0.4%). Seventy percent of smokers and 90% of nonsmokers had defined oncogenic drivers matching the U.S. Food and Drug Administration–approved targeted therapies.

gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy.Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours.Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis.Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Reinhard Dummer*a, Marc Beyer*b, Kenneth Hymesc, Mirjam T. Eppingd, Rene Bernardsd, Matthias Steinhoffb, Wolfram Sterryb, Helmut Kerle, Karl Heathf, Janet D. Ahernf, James S. Hardwickf, Jose Garcia-Vargasf, Katrin Baumanna, Syed Rizvif, Stanley R. Frankelf, Sean J. Whittakerg & Chalid Assafbha University Hospital Zürich, Switzerlandb Charitè-Universitätsmedizin, Berlin, Germanyc New York University Clinical Cancer Center, NY, USAd The Netherlands Cancer Institute, Amsterdam, The Netherlandse Medical University of Graz, Austriaf Columbia University, New York, NY, USAg King's College London, UKh Helios Klinikum, Krefeld, Germany

Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members. Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members. p56lck, a member of the Src family of non-receptor tyrosine protein kinases (1Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar,4Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and activation of mature T cells in the periphery by antigen (5Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. 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Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 12Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 44: 301-308Abstract Full Text PDF Scopus (1122) Google Scholar, 13Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar, 14Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 15Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 17Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar, 18Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar, 19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 23Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride. 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride.-394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar).We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 37Yurchak L.K. Hardwick J.S. Amrein K. Pierno K. Sefton B.M. J. Biol. Chem. 1996; 271: 12549-12554Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It is likely that the effects of exposing cells to H2O2 are mediated by global inhibition of tyrosine phosphatases (27Hecht D. Zick Y. Biochem. Biophys. Res. Commun. 1992; 188: 773-779Crossref PubMed Scopus (247) Google Scholar, 28Sullivan S.G. Chiu D.T. Errasfa M. Wang J.M. Qi J.S. Stern A. Free Radical Biol. Med. 1994; 16: 399-403Crossref PubMed Scopus (104) Google Scholar, 29Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 30Bevan A.P. Drake P.G. Yale J.F. Shaver A. Posner B.I. Mol. Cell. Biochem. 1995; 153: 49-58Crossref PubMed Scopus (113) Google Scholar, 31Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H2O2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H2O2-induced phosphorylation of Tyr-394 required dephosphorylation of Tyr-505. Thus, the extent to which the H2O2-activated population of Lck molecules was phosphorylated on Tyr-505 was unclear. Here we show that the population of Lck that is phosphorylated on Tyr-394 in response to H2O2 exposure is also phosphorylated at Tyr-505. Therefore, dephosphorylation of Tyr-505 and untethering of the SH2 domain is not a prerequisite for either phosphorylation of Lck at Tyr-394 or activation of the kinase by Tyr-394 phosphorylation. These results indicate that phosphorylation of Tyr-394 positively regulates Lck activity and is dominant over any negative regulation induced by phosphorylation of Tyr-505.DISCUSSIONThe catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505.Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region.Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. p56lck, a member of the Src family of non-receptor tyrosine protein kinases (1Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar,4Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and activation of mature T cells in the periphery by antigen (5Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (180) Google Scholar, 6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Lck stably associates with the inner surface of the plasma membrane as a result of myristoylation of Gly-2 and palmitoylation of Ser-3 and Ser-5 (7Marchildon G.A. Casnellie J.E. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7679-7682Crossref PubMed Scopus (67) Google Scholar, 8Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 9Kwong J. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 10Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). There it binds to the T-cell receptor-associated glycoproteins CD4 and CD8 as well as other proteins through its unique amino terminus (11Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 12Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 44: 301-308Abstract Full Text PDF Scopus (1122) Google Scholar, 13Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar, 14Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 15Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 17Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar, 18Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar, 19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 23Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride. 1The abbreviations used are: Tyr(P), phosphotyrosine; PVDF, polyvinylidene difluoride.-394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 37Yurchak L.K. Hardwick J.S. Amrein K. Pierno K. Sefton B.M. J. Biol. Chem. 1996; 271: 12549-12554Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It is likely that the effects of exposing cells to H2O2 are mediated by global inhibition of tyrosine phosphatases (27Hecht D. Zick Y. Biochem. Biophys. Res. Commun. 1992; 188: 773-779Crossref PubMed Scopus (247) Google Scholar, 28Sullivan S.G. Chiu D.T. Errasfa M. Wang J.M. Qi J.S. Stern A. Free Radical Biol. Med. 1994; 16: 399-403Crossref PubMed Scopus (104) Google Scholar, 29Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 30Bevan A.P. Drake P.G. Yale J.F. Shaver A. Posner B.I. Mol. Cell. Biochem. 1995; 153: 49-58Crossref PubMed Scopus (113) Google Scholar, 31Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H2O2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H2O2-induced phosphorylation of Tyr-394 required dephosphorylation of Tyr-505. Thus, the extent to which the H2O2-activated population of Lck molecules was phosphorylated on Tyr-505 was unclear. Here we show that the population of Lck that is phosphorylated on Tyr-394 in response to H2O2 exposure is also phosphorylated at Tyr-505. Therefore, dephosphorylation of Tyr-505 and untethering of the SH2 domain is not a prerequisite for either phosphorylation of Lck at Tyr-394 or activation of the kinase by Tyr-394 phosphorylation. These results indicate that phosphorylation of Tyr-394 positively regulates Lck activity and is dominant over any negative regulation induced by phosphorylation of Tyr-505. DISCUSSIONThe catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505.Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region.Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. The catalytic activity of Lck is greatly influenced by the phosphorylation state of Tyr-394 (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 38Shibuya H. Kohu K. Yamada K. Barsoumian E. Perlmutter R. Taniguchi T. Mol. Cell. Biol. 1994; 14: 5812-5819Crossref PubMed Scopus (35) Google Scholar, 39Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). Mutation of Tyr-394 to phenylalanine not only decreases Lck activity in unstimulated cells, but also prevents activation of Lck by oxidative stress. In addition, Lck that has been genetically activated by mutation of Tyr-505 to phenylalanine loses its transforming ability when Tyr-394 is also mutated to phenylalanine (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 40Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (80) Google Scholar). We previously showed that the extent of Tyr-505 phosphorylation in Lck from H2O2-stimulated cells was at least as great as that of Tyr-394. Thus, H2O2 activation appeared not to require Tyr-505 dephosphorylation. These observations suggested that Tyr-394 phosphorylation could override any negative regulation of Lck due to Tyr-505 phosphorylation. However, it was impossible to rule out the possibility that two differentially phosphorylated subpopulations of Lck existed in H2O2-stimulated cells, a relatively inactive population phosphorylated only on Tyr-505 and an activated population phosphorylated only on Tyr-394. Thus, it was formally possible that activation of Lck by Tyr-394 phosphorylation occurred only in the absence of phosphorylation of Tyr-505. Through the use of a phosphorylation state-specific antibody, we have now shown formally that Lck that is phosphorylated on Tyr-505 may be additionally phosphorylated on Tyr-394. If dephosphorylation of Tyr-505 were required for phosphorylation of Tyr-394, we would expect that Lck immunoprecipitated by α-Tyr(P)-416 sera would only be phosphorylated on Tyr-394. This was not seen. The observation that the Tyr(P)-505:Tyr(P)-394 ratio is approximately 1:1 suggests that the molecules phosphorylated on Tyr-394 are also phosphorylated on Tyr-505. Thus, the activating effects of Tyr-394 phosphorylation are dominant over the inhibitory effects of Tyr-505 phosphorylation. Apparently, even when Lck is in a “closed” conformation with Tyr(P)-505 bound to the SH2 domain and the SH3 domain bound to the polyproline type II helix in the linker region between the SH2 and catalytic domains (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar), Tyr-394 is still accessible as a substrate for phosphorylation. Comparison of the crystal structure of Lck phosphorylated at Tyr-394 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar) with that of Src and Hck lacking phosphorylation at this site (19Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar,20Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) suggests that phosphorylation of Tyr-394 allows the formation of hydrogen bonds between Tyr(P)-394 and Arg-387 and Arg-363. These interactions appear to induce the repositioning of Glu-288, Leu-385, and Arg-387 (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar), and this in turn allows Lys-273, Glu-288, Asp-364, Asn-369, and Asp-382, residues critical to ATP binding and phosphate transfer, to assume positions characteristic of an active catalytic site (24Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (420) Google Scholar, 25Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1439) Google Scholar, 26Madhusudan Trafny E.A. Xuong N.H. Adams J.A. Ten Eyck L.F. Taylor S.S. Sowadski J.M. Protein Sci. 1994; 3: 176-187Crossref PubMed Scopus (260) Google Scholar). Our data suggest that such repositioning of residues in the catalytic site of Lck can occur while the SH3 domain is still intramolecularly bound to the SH2-kinase linker region. Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). 3J. D. Bjorge and D. J. Fujita, personal communication. Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar,42Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (136) Google Scholar, 43Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar, 44Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). 4K. Pierno and B. M. Sefton, unpublished results. Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck. Consistent with this second possibility is our finding that H2O2 stimulates the activity of an as yet unidentified tyrosine kinase in Lck-deficient JCaM1.6 cells that can phosphorylate Lck on Tyr-394.2 Our results show that it is possible for the Src family member Lck to be phosphorylated at both the conserved tyrosine in the activation loop and the C-terminal tyrosine simultaneously. Because H2O2 activates Lck while inducing an increase of phosphorylation of both Tyr-394 and Tyr-505, these results suggest that Lck phosphorylated on Tyr-394 and Tyr-505 is catalytically activated. Thus, activation of Lck can occur in the absence of Tyr505 dephosphorylation or SH3 domain disassociation from the SH2-kinase linker. It is reasonable to predict that phosphorylation of the absolutely conserved tyrosine in the activation loop of other Src kinases will also activate them in the absence of dephosphorylation of the conserved carboxyl-terminal tyrosine. These experiments would not have been possible without the very generous gift of the α-Tyr(P)-416 serum from Dr. Michael Weber.

Preclinical studies have shown that targeted combination therapy consisting of vorinostat and bortezomib has antitumor activity in multiple myeloma (MM). We examined this drug combination in advanced relapsing and/or refractory MM patients (n = 34). Although the maximum tolerated dose was not reached, the study found this combination regimen generally well tolerated and clinically active in relapsed and/or refractory MM patients.Development of targeted therapies for MM has improved response rates and increased patient survival, but ultimately the disease becomes refractory and progresses. Vorinostat combined with bortezomib has demonstrated synergistic antiproliferative and proapoptotic activity in preclinical models of MM. The objectives of this study were to determine the maximum tolerated dose for vorinostat with bortezomib in patients with advanced MM and to evaluate the clinical benefit of this new drug combination.Patients ≥ 18 years old with relapsed and/or refractory MM were enrolled into escalating dose cohorts of vorinostat and bortezomib combination therapy. Thirty-four patients were enrolled and were evaluable for safety and efficacy analyses.All patients reported adverse events, 89% of which were mild to moderate in severity. Thirteen patients experienced 29 serious adverse events, 12 (41%) of which were considered drug-related. The maximum tolerated dose was not reached. Partial responses were observed in 9 (27%) patients. Minimal responses were observed in 2 additional patients (6%), and another 20 patients (59%) experienced disease stabilization.Vorinostat with bortezomib is generally well-tolerated and has clinical activity in patients with relapsed and/or refractory MM. Response rates were similar in patients previously exposed to bortezomib and patients who were naive to bortezomib therapy.

We did whole-transcriptome sequencing and whole-genome sequencing on nine pairs of Hepatocellular carcinoma (HCC) tumors and matched adjacent tissues to identify RNA editing events. We identified mean 26,982 editing sites with mean 89.5% canonical A→G edits in each sample using an improved bioinformatics pipeline. The editing rate was significantly higher in tumors than adjacent normal tissues. Comparing the difference between tumor and normal tissues of each patient, we found 7 non-synonymous tissue specific editing events including 4 tumor-specific edits and 3 normal-specific edits in the coding region, as well as 292 edits varying in editing degree. The significant expression changes of 150 genes associated with RNA editing were found in tumors, with 3 of the 4 most significant genes being cancer related. Our results show that editing might be related to higher gene expression. These findings indicate that RNA editing modification may play an important role in the development of HCC.

Vorinostat (suberoylanilide hydroxamic acid), a potent, oral histone deacetylase inhibitor, has demonstrated clinical activity in non-Japanese patients with various hematological and solid tumors. We sought to determine the maximum tolerated dose and a recommended phase II dose for 18 Japanese patients with solid tumors (median age, 58 years; range, 25-72 years) who failed standard therapy. Patients received vorinostat for 14 days followed by a 7-day rest. The initial dose was 100 mg twice daily escalating by 100 mg twice daily. Once-daily dosing was tested at 400 and 500 mg. A maximum tolerated dose could not be identified. Dose-limiting toxicities (thrombocytopenia, anorexia, and fatigue) were observed in two of six patients receiving 200 mg twice daily and in one of six patients receiving 500 mg once daily. In the 100-500 mg dose range, vorinostat area under the concentration-time curve increased in proportion to dose with a pharmacokinetic profile similar to that established in non-Japanese patients. Vorinostat doses of 200 mg twice daily or 500 mg once daily for 14 days followed by a 7-day rest were well tolerated and are candidate doses for phase II trials, although a maximum tolerated dose for vorinostat was not reached.

Many disparate biomarkers have been proposed as predictors of response to histone deacetylase inhibitors (HDI); however, all have failed when applied clinically. Rather than this being entirely an issue of reproducibility, response to the HDI vorinostat may be determined by the additive effect of multiple molecular factors, many of which have previously been demonstrated. We conducted a large-scale gene expression analysis using the Cancer Genome Project for discovery and generated another large independent cancer cell line dataset across different cancers for validation. We compared different approaches in terms of how accurately vorinostat response can be predicted on an independent out-of-batch set of samples and applied the polygenic marker prediction principles in a clinical trial. Using machine learning, the small effects that aggregate, resulting in sensitivity or resistance, can be recovered from gene expression data in a large panel of cancer cell lines. This approach can predict vorinostat response accurately, whereas single gene or pathway markers cannot. Our analyses recapitulated and contextualized many previous findings and suggest an important role for processes such as chromatin remodeling, autophagy, and apoptosis. As a proof of concept, we also discovered a novel causative role for CHD4, a helicase involved in the histone deacetylase complex that is associated with poor clinical outcome. As a clinical validation, we demonstrated that a common dose-limiting toxicity of vorinostat, thrombocytopenia, can be predicted (r = 0.55, P = .004) several days before it is detected clinically. Our work suggests a paradigm shift from single-gene/pathway evaluation to simultaneously evaluating multiple independent high-throughput gene expression datasets, which can be easily extended to other investigational compounds where similar issues are hampering clinical adoption.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip eitherin vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases. The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip eitherin vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases. Herpesvirus saimiri (HVS) 1The abbreviations HVSherpesvirus saimiriLBDLck-binding domainPIPES1,4-piperazinediethanesulfonic acidPCRpolymerase chain reactionGSTglutathione S-transferaseWTwild typeHIVhuman immunodeficiency virus 1The abbreviations HVSherpesvirus saimiriLBDLck-binding domainPIPES1,4-piperazinediethanesulfonic acidPCRpolymerase chain reactionGSTglutathione S-transferaseWTwild typeHIVhuman immunodeficiency virusstrain C induces T cell lymphomas and lymphoid leukemias in New World monkeys (1Meléndez L.V. Hunt R.D. Daniel M.D. Blake B.J. Garcia F.G. Science. 1971; 171: 1161-1163Crossref PubMed Scopus (54) Google Scholar) and can transform human T cells in vitro (2Biesinger B. Muller-Fleckenstein I. Simmer B. Lang G. Wittmann S. Platzer E. Desrosiers R.C. Fleckenstein B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3116-3119Crossref PubMed Scopus (275) Google Scholar, 3Medveczky M.M. Geck P. Sullivan J.L. Serbousek D. Djeu J.Y. Medveczky P.G. Virology. 1993; 196: 402-412Crossref PubMed Scopus (57) Google Scholar). A region of the HVS genome that is essential for T cell transformation (3Medveczky M.M. Geck P. Sullivan J.L. Serbousek D. Djeu J.Y. Medveczky P.G. Virology. 1993; 196: 402-412Crossref PubMed Scopus (57) Google Scholar, 4Koomey J.M. Mulder C. Burghoff R.L. Fleckenstein B. Desrosiers R.C. J. Virol. 1984; 50: 662-665Crossref PubMed Google Scholar, 5Desrosiers R.C. Bakker A. Kamine J. Falk L.A. Hunt R.D. King N.W. Science. 1985; 228: 184-187Crossref PubMed Scopus (91) Google Scholar) encodes two proteins, Tip and STP (6Murthy S.C. Trimble J.J. Desrosiers R.C. J. Virol. 1989; 63: 3307-3314Crossref PubMed Google Scholar). STP, a small, collagen-like membrane protein, has been reported to interact with the Ras protein (7Jung J.U. Desrosiers R.C. Mol. Cell. Biol. 1995; 15: 6506-6512Crossref PubMed Scopus (94) Google Scholar). Tip (atyrosine-protein kinase-interactingprotein) binds to and activates the Lck tyrosine-protein kinase dramatically (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 10Wiese N. Tsygankov A.Y. Klauenberg U. Bolen J.B. Fleischer B. Broker B.M. J. Biol. Chem. 1996; 271: 847-852Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 11Lund T. Medveczky M.M. Medveczky P.G. J. Virol. 1997; 71: 378-382Crossref PubMed Google Scholar, 12Lund T.C. Prator P.C. Medveczky M.M. Medveczky P.G. J. Virol. 1999; 73: 1689-1694Crossref PubMed Google Scholar).Lck is a lymphoid-specific member of the Src family of membrane-associated cytoplasmic tyrosine-protein kinases (13Sefton B.M. Campbell M.-A. Ann. Rev. Cell Biol. 1991; 7: 257-274Crossref PubMed Scopus (50) Google Scholar). It is essential for both the development of T cells in the thymus and for the response of T cells to signals arising from the antigen receptor (14Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar,15Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). It is likely that the interaction of Tip with Lck plays a role in the induction of T cell disease by HVS.Lck contains two protein interaction domains: an SH3 domain that has been shown to bind to poly-proline type II helices (16Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (870) Google Scholar), and an SH2 domain that binds to sites of tyrosine phosphorylation (17Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (441) Google Scholar). The activity of Lck is regulated by phosphorylation. Phosphorylation of Tyr505 inhibits its activity (18Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar, 19Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Crossref PubMed Scopus (412) Google Scholar). Biochemical analysis (20Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar) and structural studies of other closely related Src kinases (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar) suggest that phosphorylated Tyr505 sequesters the SH2 domain of Lck through an intramolecular interaction and that the resultant closed conformation limits activity. Additionally, it is likely that the binding of the SH3 domain to the linker between the SH2 domain and the catalytic domain stabilizes this closed conformation (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar).Lck is activated by phosphorylation of Tyr394 (24Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). This site is subject to autophosphorylation that is likely to occur in an intermolecular fashion and to phosphorylation by an as yet unidentified kinase (24Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar).Tip appears to be anchored to the cytoplasmic face of cellular membranes by a carboxyl-terminal transmembrane domain (25Lund T. Medveczky M.M. Geck P. Medveczky P.G. J. Virol. 1995; 69: 4495-4499Crossref PubMed Google Scholar). It contains two Lck-binding domains. One domain, residues 132–141, is a proline-rich SH3 domain ligand (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 27Jung J.U. Lang S.M. Friedrich U. Jun T. Roberts T.M. Desrosiers R.C. Biesinger B. J. Biol. Chem. 1995; 270: 20660-20667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) also referred to as the SH3B domain. We will refer to this domain as Lck-binding domain 1 (LBD1). The other binding domain (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 27Jung J.U. Lang S.M. Friedrich U. Jun T. Roberts T.M. Desrosiers R.C. Biesinger B. J. Biol. Chem. 1995; 270: 20660-20667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), residues 104–113, binds to an as yet unidentified site in the carboxyl-terminal half of Lck. 2D. A. Hartley, K. Amdjadi, T. C. Lund, P. G. Medveczky, and B. M. Sefton, submitted for publication. 2D. A. Hartley, K. Amdjadi, T. C. Lund, P. G. Medveczky, and B. M. Sefton, submitted for publication. Referred to previously as the CSKH domain, we will refer to this domain as Lck-binding domain 2 (LBD2). Tip has no other recognizable protein interaction domains or catalytic domains. We have examined here the mechanism by which Tip activates Lck.DISCUSSIONOur results show that Tip can activate Lck under conditions where no activating changes in tyrosine phosphorylation of Lck can occur. This suggests strongly that Tip can activate Lck, at least partially, through an induced conformational change. The population of Lck bound to Tip in vivo is, however, phosphorylated to a greater extent at Tyr394, the site of activating phosphorylation, than at Tyr505, the site of inhibitory phosphorylation. There appear to be two steps in the activation of Lck by Tip. First, the binding of Tip induces a conformational change that leads to greater catalytic activity. Second, the Lck that has been activated by the Tip-induced conformational change undergoes increased phosphorylation at Tyr394 and possibly decreased phosphorylation at Tyr505, and this leads to a further increase in catalytic activity.It is likely that Tip exerts some of its effect as a result of displacement of the SH3 domain of Lck. The SH3 domains of two Src kinases, c-Src and Hck, bind intramolecularly to polyproline type II helices in the linker between their SH2 domains and their catalytic domains (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). This interaction may make it difficult for the enzyme to assume a fully active conformation (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). The human immunodeficiency virus Nef protein, which contains an SH3 domain-binding motif, can both bind to the SH3 domain of Hck and activate Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). It has been proposed that the activation of Hck by Nef results from displacement of the SH3 domain from the SH2 catalytic domain linker, thereby allowing the catalytic domain to achieve a catalytically more favorable conformation.Tip contains two Lck-binding domains. LBD1 is a proline-rich domain that binds to the SH3 domain of Lck. LBD2 binds to an as yet unidentified site in the carboxyl-terminal half of Lck.2The binding of the proline-rich LBD1 of Tip to the Lck SH3 domain could displace the SH3 domain and activate Lck by removing conformational constraints, as has been inferred to occur when Nef binds to Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar,44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Consistent with this model are earlier observations that mutation of either the SH3 domain (45Reynolds P.J. Hurley T.R. Sefton B.M. Oncogene. 1992; 7: 1949-1955PubMed Google Scholar, 46Weil R. Veillette A. J. Biol. Chem. 1994; 269: 22830-22838Abstract Full Text PDF PubMed Google Scholar) or of the SH2 catalytic domain linker (47Wright D.D. Sefton B.M. Kamps M.P. Mol. Cell. Biol. 1994; 14: 2429-2437Crossref PubMed Scopus (59) Google Scholar, 48Gonfloni S. Williams J.C. Hattula K. Weijland A. Wierenga R.K. Superti-Furga G. EMBO J. 1997; 16: 7261-7271Crossref PubMed Scopus (126) Google Scholar) activates Lck partially.The mechanism of activation of Lck by Tip is not identical to that by which Nef activates Hck. Tip containing only a functional LBD1 can bind to Lck in vitro through the Lck SH3 domain, but this does not activate Lck detectably (Fig. 2). This suggests that the binding of LBD2 to Lck also participates in the activation of Lck. In that LBD2 binds to the carboxyl-terminal half of the protein that consists largely of the catalytic domain, it is possible that LBD2 alters the conformation of the catalytic domain directly. How this might occur is not yet known. LBD2 has sequence identity with the αI helix in the Lck catalytic domain (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It is possible that it displaces this helix and that such a displacement is activating. Alternatively, this binding domain could affect the positioning of the αC helix in the catalytic domain in a manner similar to that by which cyclin activates cyclin-dependent kinases (49Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1208) Google Scholar).Tip differs from other known regulatory proteins of Src kinases in its bimodal binding to Lck. The middle T antigen of polyoma virus binds only to the carboxyl termini of Src kinases (50Dunant N.M. Senften M. Ballmer-Hofer K. J. Virol. 1996; 70: 1323-1330Crossref PubMed Google Scholar) and activates them apparently by interfering with inhibitory phosphorylation (26Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar). The HIV Nef protein appears to function only as an SH3 domain ligand (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and is not known to interact directly with the catalytic domain of Hck. A question of particular interest is whether Tip has a cellular homologue that functions as a natural, endogenous activator of Lck in T cells. If so, this would suggest that tyrosine-protein kinases may normally be subject to an as yet unappreciated form of regulation and that cellular Tip-like activators of other tyrosine-protein kinases may exist. Herpesvirus saimiri (HVS) 1The abbreviations HVSherpesvirus saimiriLBDLck-binding domainPIPES1,4-piperazinediethanesulfonic acidPCRpolymerase chain reactionGSTglutathione S-transferaseWTwild typeHIVhuman immunodeficiency virus 1The abbreviations HVSherpesvirus saimiriLBDLck-binding domainPIPES1,4-piperazinediethanesulfonic acidPCRpolymerase chain reactionGSTglutathione S-transferaseWTwild typeHIVhuman immunodeficiency virusstrain C induces T cell lymphomas and lymphoid leukemias in New World monkeys (1Meléndez L.V. Hunt R.D. Daniel M.D. Blake B.J. Garcia F.G. Science. 1971; 171: 1161-1163Crossref PubMed Scopus (54) Google Scholar) and can transform human T cells in vitro (2Biesinger B. Muller-Fleckenstein I. Simmer B. Lang G. Wittmann S. Platzer E. Desrosiers R.C. Fleckenstein B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3116-3119Crossref PubMed Scopus (275) Google Scholar, 3Medveczky M.M. Geck P. Sullivan J.L. Serbousek D. Djeu J.Y. Medveczky P.G. Virology. 1993; 196: 402-412Crossref PubMed Scopus (57) Google Scholar). A region of the HVS genome that is essential for T cell transformation (3Medveczky M.M. Geck P. Sullivan J.L. Serbousek D. Djeu J.Y. Medveczky P.G. Virology. 1993; 196: 402-412Crossref PubMed Scopus (57) Google Scholar, 4Koomey J.M. Mulder C. Burghoff R.L. Fleckenstein B. Desrosiers R.C. J. Virol. 1984; 50: 662-665Crossref PubMed Google Scholar, 5Desrosiers R.C. Bakker A. Kamine J. Falk L.A. Hunt R.D. King N.W. Science. 1985; 228: 184-187Crossref PubMed Scopus (91) Google Scholar) encodes two proteins, Tip and STP (6Murthy S.C. Trimble J.J. Desrosiers R.C. J. Virol. 1989; 63: 3307-3314Crossref PubMed Google Scholar). STP, a small, collagen-like membrane protein, has been reported to interact with the Ras protein (7Jung J.U. Desrosiers R.C. Mol. Cell. Biol. 1995; 15: 6506-6512Crossref PubMed Scopus (94) Google Scholar). Tip (atyrosine-protein kinase-interactingprotein) binds to and activates the Lck tyrosine-protein kinase dramatically (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 10Wiese N. Tsygankov A.Y. Klauenberg U. Bolen J.B. Fleischer B. Broker B.M. J. Biol. Chem. 1996; 271: 847-852Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 11Lund T. Medveczky M.M. Medveczky P.G. J. Virol. 1997; 71: 378-382Crossref PubMed Google Scholar, 12Lund T.C. Prator P.C. Medveczky M.M. Medveczky P.G. J. Virol. 1999; 73: 1689-1694Crossref PubMed Google Scholar). herpesvirus saimiri Lck-binding domain 1,4-piperazinediethanesulfonic acid polymerase chain reaction glutathione S-transferase wild type human immunodeficiency virus herpesvirus saimiri Lck-binding domain 1,4-piperazinediethanesulfonic acid polymerase chain reaction glutathione S-transferase wild type human immunodeficiency virus Lck is a lymphoid-specific member of the Src family of membrane-associated cytoplasmic tyrosine-protein kinases (13Sefton B.M. Campbell M.-A. Ann. Rev. Cell Biol. 1991; 7: 257-274Crossref PubMed Scopus (50) Google Scholar). It is essential for both the development of T cells in the thymus and for the response of T cells to signals arising from the antigen receptor (14Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar,15Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). It is likely that the interaction of Tip with Lck plays a role in the induction of T cell disease by HVS. Lck contains two protein interaction domains: an SH3 domain that has been shown to bind to poly-proline type II helices (16Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (870) Google Scholar), and an SH2 domain that binds to sites of tyrosine phosphorylation (17Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (441) Google Scholar). The activity of Lck is regulated by phosphorylation. Phosphorylation of Tyr505 inhibits its activity (18Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (236) Google Scholar, 19Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Crossref PubMed Scopus (412) Google Scholar). Biochemical analysis (20Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar) and structural studies of other closely related Src kinases (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar) suggest that phosphorylated Tyr505 sequesters the SH2 domain of Lck through an intramolecular interaction and that the resultant closed conformation limits activity. Additionally, it is likely that the binding of the SH3 domain to the linker between the SH2 domain and the catalytic domain stabilizes this closed conformation (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). Lck is activated by phosphorylation of Tyr394 (24Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). This site is subject to autophosphorylation that is likely to occur in an intermolecular fashion and to phosphorylation by an as yet unidentified kinase (24Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). Tip appears to be anchored to the cytoplasmic face of cellular membranes by a carboxyl-terminal transmembrane domain (25Lund T. Medveczky M.M. Geck P. Medveczky P.G. J. Virol. 1995; 69: 4495-4499Crossref PubMed Google Scholar). It contains two Lck-binding domains. One domain, residues 132–141, is a proline-rich SH3 domain ligand (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 27Jung J.U. Lang S.M. Friedrich U. Jun T. Roberts T.M. Desrosiers R.C. Biesinger B. J. Biol. Chem. 1995; 270: 20660-20667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) also referred to as the SH3B domain. We will refer to this domain as Lck-binding domain 1 (LBD1). The other binding domain (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 9Lund T. Medveczky M.M. Neame P.J. Medveczky P.G. J. Virol. 1996; 70: 600-606Crossref PubMed Google Scholar, 27Jung J.U. Lang S.M. Friedrich U. Jun T. Roberts T.M. Desrosiers R.C. Biesinger B. J. Biol. Chem. 1995; 270: 20660-20667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), residues 104–113, binds to an as yet unidentified site in the carboxyl-terminal half of Lck. 2D. A. Hartley, K. Amdjadi, T. C. Lund, P. G. Medveczky, and B. M. Sefton, submitted for publication. 2D. A. Hartley, K. Amdjadi, T. C. Lund, P. G. Medveczky, and B. M. Sefton, submitted for publication. Referred to previously as the CSKH domain, we will refer to this domain as Lck-binding domain 2 (LBD2). Tip has no other recognizable protein interaction domains or catalytic domains. We have examined here the mechanism by which Tip activates Lck. DISCUSSIONOur results show that Tip can activate Lck under conditions where no activating changes in tyrosine phosphorylation of Lck can occur. This suggests strongly that Tip can activate Lck, at least partially, through an induced conformational change. The population of Lck bound to Tip in vivo is, however, phosphorylated to a greater extent at Tyr394, the site of activating phosphorylation, than at Tyr505, the site of inhibitory phosphorylation. There appear to be two steps in the activation of Lck by Tip. First, the binding of Tip induces a conformational change that leads to greater catalytic activity. Second, the Lck that has been activated by the Tip-induced conformational change undergoes increased phosphorylation at Tyr394 and possibly decreased phosphorylation at Tyr505, and this leads to a further increase in catalytic activity.It is likely that Tip exerts some of its effect as a result of displacement of the SH3 domain of Lck. The SH3 domains of two Src kinases, c-Src and Hck, bind intramolecularly to polyproline type II helices in the linker between their SH2 domains and their catalytic domains (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). This interaction may make it difficult for the enzyme to assume a fully active conformation (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). The human immunodeficiency virus Nef protein, which contains an SH3 domain-binding motif, can both bind to the SH3 domain of Hck and activate Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). It has been proposed that the activation of Hck by Nef results from displacement of the SH3 domain from the SH2 catalytic domain linker, thereby allowing the catalytic domain to achieve a catalytically more favorable conformation.Tip contains two Lck-binding domains. LBD1 is a proline-rich domain that binds to the SH3 domain of Lck. LBD2 binds to an as yet unidentified site in the carboxyl-terminal half of Lck.2The binding of the proline-rich LBD1 of Tip to the Lck SH3 domain could displace the SH3 domain and activate Lck by removing conformational constraints, as has been inferred to occur when Nef binds to Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar,44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Consistent with this model are earlier observations that mutation of either the SH3 domain (45Reynolds P.J. Hurley T.R. Sefton B.M. Oncogene. 1992; 7: 1949-1955PubMed Google Scholar, 46Weil R. Veillette A. J. Biol. Chem. 1994; 269: 22830-22838Abstract Full Text PDF PubMed Google Scholar) or of the SH2 catalytic domain linker (47Wright D.D. Sefton B.M. Kamps M.P. Mol. Cell. Biol. 1994; 14: 2429-2437Crossref PubMed Scopus (59) Google Scholar, 48Gonfloni S. Williams J.C. Hattula K. Weijland A. Wierenga R.K. Superti-Furga G. EMBO J. 1997; 16: 7261-7271Crossref PubMed Scopus (126) Google Scholar) activates Lck partially.The mechanism of activation of Lck by Tip is not identical to that by which Nef activates Hck. Tip containing only a functional LBD1 can bind to Lck in vitro through the Lck SH3 domain, but this does not activate Lck detectably (Fig. 2). This suggests that the binding of LBD2 to Lck also participates in the activation of Lck. In that LBD2 binds to the carboxyl-terminal half of the protein that consists largely of the catalytic domain, it is possible that LBD2 alters the conformation of the catalytic domain directly. How this might occur is not yet known. LBD2 has sequence identity with the αI helix in the Lck catalytic domain (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It is possible that it displaces this helix and that such a displacement is activating. Alternatively, this binding domain could affect the positioning of the αC helix in the catalytic domain in a manner similar to that by which cyclin activates cyclin-dependent kinases (49Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1208) Google Scholar).Tip differs from other known regulatory proteins of Src kinases in its bimodal binding to Lck. The middle T antigen of polyoma virus binds only to the carboxyl termini of Src kinases (50Dunant N.M. Senften M. Ballmer-Hofer K. J. Virol. 1996; 70: 1323-1330Crossref PubMed Google Scholar) and activates them apparently by interfering with inhibitory phosphorylation (26Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar). The HIV Nef protein appears to function only as an SH3 domain ligand (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and is not known to interact directly with the catalytic domain of Hck. A question of particular interest is whether Tip has a cellular homologue that functions as a natural, endogenous activator of Lck in T cells. If so, this would suggest that tyrosine-protein kinases may normally be subject to an as yet unappreciated form of regulation and that cellular Tip-like activators of other tyrosine-protein kinases may exist. Our results show that Tip can activate Lck under conditions where no activating changes in tyrosine phosphorylation of Lck can occur. This suggests strongly that Tip can activate Lck, at least partially, through an induced conformational change. The population of Lck bound to Tip in vivo is, however, phosphorylated to a greater extent at Tyr394, the site of activating phosphorylation, than at Tyr505, the site of inhibitory phosphorylation. There appear to be two steps in the activation of Lck by Tip. First, the binding of Tip induces a conformational change that leads to greater catalytic activity. Second, the Lck that has been activated by the Tip-induced conformational change undergoes increased phosphorylation at Tyr394 and possibly decreased phosphorylation at Tyr505, and this leads to a further increase in catalytic activity. It is likely that Tip exerts some of its effect as a result of displacement of the SH3 domain of Lck. The SH3 domains of two Src kinases, c-Src and Hck, bind intramolecularly to polyproline type II helices in the linker between their SH2 domains and their catalytic domains (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). This interaction may make it difficult for the enzyme to assume a fully active conformation (21Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 22Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar, 23Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). The human immunodeficiency virus Nef protein, which contains an SH3 domain-binding motif, can both bind to the SH3 domain of Hck and activate Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). It has been proposed that the activation of Hck by Nef results from displacement of the SH3 domain from the SH2 catalytic domain linker, thereby allowing the catalytic domain to achieve a catalytically more favorable conformation. Tip contains two Lck-binding domains. LBD1 is a proline-rich domain that binds to the SH3 domain of Lck. LBD2 binds to an as yet unidentified site in the carboxyl-terminal half of Lck.2The binding of the proline-rich LBD1 of Tip to the Lck SH3 domain could displace the SH3 domain and activate Lck by removing conformational constraints, as has been inferred to occur when Nef binds to Hck (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar,44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Consistent with this model are earlier observations that mutation of either the SH3 domain (45Reynolds P.J. Hurley T.R. Sefton B.M. Oncogene. 1992; 7: 1949-1955PubMed Google Scholar, 46Weil R. Veillette A. J. Biol. Chem. 1994; 269: 22830-22838Abstract Full Text PDF PubMed Google Scholar) or of the SH2 catalytic domain linker (47Wright D.D. Sefton B.M. Kamps M.P. Mol. Cell. Biol. 1994; 14: 2429-2437Crossref PubMed Scopus (59) Google Scholar, 48Gonfloni S. Williams J.C. Hattula K. Weijland A. Wierenga R.K. Superti-Furga G. EMBO J. 1997; 16: 7261-7271Crossref PubMed Scopus (126) Google Scholar) activates Lck partially. The mechanism of activation of Lck by Tip is not identical to that by which Nef activates Hck. Tip containing only a functional LBD1 can bind to Lck in vitro through the Lck SH3 domain, but this does not activate Lck detectably (Fig. 2). This suggests that the binding of LBD2 to Lck also participates in the activation of Lck. In that LBD2 binds to the carboxyl-terminal half of the protein that consists largely of the catalytic domain, it is possible that LBD2 alters the conformation of the catalytic domain directly. How this might occur is not yet known. LBD2 has sequence identity with the αI helix in the Lck catalytic domain (8Biesinger B. Tsygankov A.Y. Fickenscher H. Emmrich F. Fleckenstein B. Bolen J.B. Broker B.M. J. Biol. Chem. 1995; 270: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It is possible that it displaces this helix and that such a displacement is activating. Alternatively, this binding domain could affect the positioning of the αC helix in the catalytic domain in a manner similar to that by which cyclin activates cyclin-dependent kinases (49Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1208) Google Scholar). Tip differs from other known regulatory proteins of Src kinases in its bimodal binding to Lck. The middle T antigen of polyoma virus binds only to the carboxyl termini of Src kinases (50Dunant N.M. Senften M. Ballmer-Hofer K. J. Virol. 1996; 70: 1323-1330Crossref PubMed Google Scholar) and activates them apparently by interfering with inhibitory phosphorylation (26Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar). The HIV Nef protein appears to function only as an SH3 domain ligand (43Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar, 44Briggs S. Sharkey M. Stevenson M. Smithgall T. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and is not known to interact directly with the catalytic domain of Hck. A question of particular interest is whether Tip has a cellular homologue that functions as a natural, endogenous activator of Lck in T cells. If so, this would suggest that tyrosine-protein kinases may normally be subject to an as yet unappreciated form of regulation and that cellular Tip-like activators of other tyrosine-protein kinases may exist. We thank Bob Hyman for the SAKRTLS cells and Wei Jiang for helpful advice about insect cells.

Abstract Tumors use tryptophan-catabolizing enzymes such as Indoleamine 2,3-dioxygenase-1 (IDO-1) to induce an immunosuppressive microenvironment. IDO-1 expression is upregulated in many cancers and described to be a resistance mechanism to immune checkpoint therapies. IDO-1 is induced in response to inflammatory stimuli such as IFN-ã and promotes immune tolerance through the catabolism of tryptophan and accumulation of tryptophan catabolites including kynurenine. IDO-1 activity leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As such, IDO1 is a nexus for the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology. We have identified and characterized a new IDO-1 inhibitor. PF-06840003 is a highly selective orally bioavailable IDO-1 inhibitor. PF-06840003 reversed IDO-1-induced T-cell anergy in vitro. In vivo, PF-06840003 reduced intratumoral kynurenine levels in mice by &amp;gt;80% and inhibited tumor growth in multiple preclinical syngeneic models in mice, in combination with immune checkpoint inhibitors. PF-0684003 has favorable predicted human pharmacokinetic properties, including a predicted t1/2 of 16-19 hours. These studies highlight the strong potential of PF-06840003 as a clinical candidate in Immuno-Oncology. Citation Format: Joseph Tumang, Bruno Gomes, Martin Wythes, Stefano Crosignani, Patrick Bingham, Pauline Bottemanne, Hélène Cannelle, Sandra Cauwenberghs, Jenny Chaplin, Deepak Dalvie, Sofie Denies, Coraline De Maeseneire, Peter Folger, Kim Frederix, Jie Guo, James Hardwick, Ken Hook, Katti Jessen, Erick Kindt, Marie-Claire Letellier, Kai-Hsin Liao, Wenlin Li, Karen Maegley, Reece Marillier, Nichol Miller, Brion Murray, Romain Pirson, Julie Preillon, Virginie Rabolli, Chad Ray, Stephanie Scales, Jay Srirangam, Jim Solowiej, Nicole Streiner, Vince Torti, Konstantinos Tsaparikos, Paolo Vicini, Gregory Driessens, Manfred Kraus. PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4863.

Lck, a lymphocyte-specific tyrosine protein kinase, is bound to cellular membranes as the result of myristoylation and palmitoylation of its amino terminus. Its activity is inhibited by phosphorylation of tyrosine 505 and stimulated by phosphorylation of tyrosine 394. The Tyr-505 → Phe mutant of Lck (F505Lck) exhibits elevated biological activity and constitutive phosphorylation of Tyr-394 in vivo. Mutations at sites of fatty acylation that prevent F505Lck from associating with cellular membranes abolish the biological activity of the molecule in vivo, compromise its activity as a protein kinase in vivo and in vitro, and eliminate the phosphorylation of Tyr-394. Here, we show that exposure of cells expressing cytoplasmic or nuclear forms of F505Lck to H2O2, a general inhibitor of tyrosine protein phosphatases, restores the catalytic activity of these mutant proteins through stimulation of phosphorylation of Tyr-394. H2O2 treatment induced the phosphorylation of Tyr-394 on catalytically inactive forms of Lck regardless of cellular localization. Phosphorylation of Tyr-394 therefore need not occur by autophosphorylation. Thus, there appear to be two mechanisms through which the phosphorylation of Lck at Tyr-394 can occur. One is restricted to the plasma membrane and does not require the presence of oxidants. The other is operational in the nucleus as well as the cytosol and is responsive to oxidants. Lck, a lymphocyte-specific tyrosine protein kinase, is bound to cellular membranes as the result of myristoylation and palmitoylation of its amino terminus. Its activity is inhibited by phosphorylation of tyrosine 505 and stimulated by phosphorylation of tyrosine 394. The Tyr-505 → Phe mutant of Lck (F505Lck) exhibits elevated biological activity and constitutive phosphorylation of Tyr-394 in vivo. Mutations at sites of fatty acylation that prevent F505Lck from associating with cellular membranes abolish the biological activity of the molecule in vivo, compromise its activity as a protein kinase in vivo and in vitro, and eliminate the phosphorylation of Tyr-394. Here, we show that exposure of cells expressing cytoplasmic or nuclear forms of F505Lck to H2O2, a general inhibitor of tyrosine protein phosphatases, restores the catalytic activity of these mutant proteins through stimulation of phosphorylation of Tyr-394. H2O2 treatment induced the phosphorylation of Tyr-394 on catalytically inactive forms of Lck regardless of cellular localization. Phosphorylation of Tyr-394 therefore need not occur by autophosphorylation. Thus, there appear to be two mechanisms through which the phosphorylation of Lck at Tyr-394 can occur. One is restricted to the plasma membrane and does not require the presence of oxidants. The other is operational in the nucleus as well as the cytosol and is responsive to oxidants. INTRODUCTIONp56lck, a member of the Src family of tyrosine protein kinases(1.Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2.Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed exclusively in lymphoid cells and predominantly in T cells. Lck associates with the cytoplasmic domain of the T-cell receptor-associated glycoproteins CD4 and CD8(3.Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 4.Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1121) Google Scholar), membrane immunoglobulin in B cells(5.Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar), and glycosylphosphatidylinositol-anchored proteins (6.Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 7.Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar) through its unique amino terminus. p56lck is essential for both T-cell development (8.Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar, 9.Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and T-cell activation(10.Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (180) Google Scholar, 11.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The activity of Lck is regulated by the phosphorylation of tyrosine residues at positions 394 and 505. Phosphorylation of Tyr-505 inhibits the activity of Lck (12.Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 13.Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar). Mutation of Tyr-505 to phenylalanine results in a constitutively active protein that is able to transform fibroblasts (12.Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 13.Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar), enhance T-cell responsiveness to antigen(14.Abraham N. Miceli M.C. Parnes J.R. Veillette A. Nature. 1991; 350: 62-66Crossref PubMed Scopus (256) Google Scholar), and induce antigen-independent interleukin-2 production in T cells(15.Luo K. Sefton B.M. Mol. Cell. Biol. 1992; 12: 4724-4732Crossref PubMed Scopus (52) Google Scholar). In contrast, phosphorylation of Tyr-394, the single site of autophosphorylation in vitro, enhances the catalytic activity of Lck and is required for the activation of Lck by mutation of Tyr-505 to phenylalanine(16.Boulet I. Fagard R. Fischer S. Biochem. Biophys. Res. Commun. 1987; 149: 56-64Crossref PubMed Scopus (13) Google Scholar, 17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar).Lck associates with the inner face of the plasma membrane through its amino terminus. This interaction is mediated by both myristic acid and palmitic acid that are bound to the amino-terminal glycine and Cys-3 and/or Cys-5, respectively(19.Marchildon G.A. Casnellie J.E. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7679-7682Crossref PubMed Scopus (67) Google Scholar, 20.Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 21.Kwong J.A. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). Mutations in which the amino-terminal glycine is mutated to alanine result in a protein that is neither myristoylated nor palmitoylated and is unable to associate with the plasma membrane(17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar). Mutation of both Cys-3 and Cys-5 to serine yields a protein that is myristoylated but not palmitoylated (21.Kwong J.A. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar, 23.Rodgers W. Crise B. Rose J.K. Mol. Cell. Biol. 1994; 14: 5384-5391Crossref PubMed Scopus (224) Google Scholar). The resulting Lck protein is completely cytoplasmic, indicating that myristoylation is not sufficient for anchoring Lck to the plasma membrane(22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar).Hydrogen peroxide is a general inhibitor of tyrosine protein phosphatases(24.Heffetz D. Bushkin I. Dror R. Zick Y. J. Biol. Chem. 1990; 265: 2896-2902Abstract Full Text PDF PubMed Google Scholar). Exposure of cells to H2O2 induces rapid tyrosine phosphorylation of numerous cellular proteins in vivo(24.Heffetz D. Bushkin I. Dror R. Zick Y. J. Biol. Chem. 1990; 265: 2896-2902Abstract Full Text PDF PubMed Google Scholar, 25.Schieven G.L. Kirihara J.M. Gilliland L.K. Uckun F.M. Ledbetter J.A. Mol. Biol. Cell. 1993; 4: 523-530Crossref PubMed Scopus (54) Google Scholar, 26.O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (141) Google Scholar, 27.Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 28.Nakamura K. Hori T. Sato N. Sugie K. Kawakami T. Yodoi J. Oncogene. 1993; 8: 3133-3139PubMed Google Scholar) and has been shown to be a potent activator of Lck(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). Activation of Lck by H2O2 is due to an increase in the phosphorylation of Tyr-394(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). In the current study, we examined the effect of H2O2 on three different non-membrane-bound forms of genetically activated Lck (F505Lck). Non-myristoylated, myristoylated but non-palmitoylated, and nuclear forms of F505Lck all exhibit severely reduced activity in vivo and in vitro. H2O2 is able to restore the in vitro catalytic activity of all three non-membrane-bound forms of Lck. This increase in activity correlates with, and in all probability is due to, an increase in the phosphorylation of Tyr-394. Apparently, phosphorylation of Tyr-394 occurs inefficiently when Lck is located at sites other than cellular membranes but can be induced by elevated levels of oxidants.EXPERIMENTAL PROCEDURESConstruction of lck MutantsAll mutations were introduced into murine lck cDNA by oligonucleotide-directed mutagenesis using the M13 bacteriophage except for lckS3/5F505, which was constructed by polymerase chain reaction mutagenesis. The construction of lckF505 and lckS3/5F505 has been described(12.Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). lckA2F505 was made from lckF505 by site-directed mutagenesis of glycine at position 2 to alanine using the oligonucleotide 5′-GATCATGGCATGCGTCTGC-3′. The lcknuc and lcknucF505 mutants were assembled by cloning a 33-base pair synthetic SphI fragment (5′-TCCTAAGAAGAAGCGCAAGGTCGAAGATCCATG-3′) encoding the nuclear localization signal (PKKKRKVEDPC) from SV40 large T antigen (29.Roberts B.L. Richardson W.D. Smith A.E. Cell. 1987; 50: 465-475Abstract Full Text PDF PubMed Scopus (138) Google Scholar) into a SphI site in genes encoding lckA2 or lckA2F505 that was generated by changing the codon for Cys-3 (TGT) to (TGC). All mutations were confirmed by sequencing. A BamHI-EcoRI restriction fragment of the murine lck cDNA containing the Lys-273 → Arg mutation (18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar) was used to replace the corresponding BamHI-EcoRI fragment in genes encoding lcknuc or lckA2 to create lcknucR273 and lckA2R273, respectively. All mutant lck cDNAs were subcloned into the retroviral expression vector LXSN(30.Miller A.D. Rosman G.F. BioTechniques. 1989; 7: 980-990PubMed Google Scholar). Recombinant retroviruses were produced by cotransfecting the LXSN constructs and the viral helper plasmid SV-ψ−-E-MLV (31.Landau N.R. Littman D.R. J. Virol. 1992; 66: 5110-5113Crossref PubMed Google Scholar) into COSm-6 cells(32.Muller A.J. Young J.C. Pendergast A.-M. Pondel M. Landau N.R. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar). 48 h after cotransfection, supernatant containing recombinant virus particles was collected and used to infect 208F cells as described previously(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar).Cell Lines208F rat fibroblasts expressing Lck were maintained in Dulbecco-Vogt modified Eagle's medium (DMEM) 1The abbreviations used are: DMEMDulbecco-Vogt modified Eagle's mediumPIPES1,4-piperazinediethanesulfonic acidPBSphosphate-buffered saline. (Mediatech, Washington, D. C.) supplemented with 10% calf serum (Gemini, Calabasas, CA) and 600 μg/ml G418 (Geneticin; Life Technologies, Inc.). COSm-6 cells were maintained in DMEM supplemented with 10% calf serum.Preparation of Cell Lysates and ImmunoprecipitationCells were lysed in RIPA buffer (150 mM NaCl, 10 mM sodium phosphate, pH 7.2, 1% (w/v) sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 200 μM Na3VO4, 50 mM NaF, 2 mM EDTA, 100 kallikrein inhibitor units/ml aprotinin) for 20 min at 4°C at a concentration of 2 × 106 cells/ml. 30 μl of Staphylococcus aureus cells (Pansorbin cells; Calbiochem) were added to the lysate, which was then clarified by centrifugation at 35,000 × g at 4°C for 45 min. Lck was isolated by the addition of rabbit polyclonal antisera specific for Lck (33.Hurley T.R. Sefton B.M. Oncogene. 1989; 4: 265-272PubMed Google Scholar) prebound to S. aureus cells. After adsorption for 30 min, the immune complexes were washed in RIPA buffer three times and once in TN buffer (50 mM Tris, pH 7.5, 150 mM NaCl). Whole cell lysates were prepared by lysing cells directly in reducing SDS-gel sample buffer at a concentration of 4 × 106 cells/ml.ImmunoblottingImmunoprecipitated Lck or whole cell lysates were fractionated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). Western blotting was performed with polyclonal rabbit anti-phosphotyrosine (34.Kamps M.P. Sefton B.M. Oncogene. 1988; 2: 305-315PubMed Google Scholar) or anti-Lck (33.Hurley T.R. Sefton B.M. Oncogene. 1989; 4: 265-272PubMed Google Scholar) antibodies and 125I-protein A (ICN, Costa Mesa, CA) as described previously(34.Kamps M.P. Sefton B.M. Oncogene. 1988; 2: 305-315PubMed Google Scholar, 35.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44696) Google Scholar).In Vivo Labeling and Peptide Mapping208F cells expressing Lck (approximately 2 × 106 per 10-cm dish) were washed twice with phosphate-free DMEM and incubated with 0.5 mCi/ml 32Pi (H332PO4, carrier-free; ICN) in 4 ml of phosphate-free DMEM supplemented with 10% phosphate-free fetal bovine serum for 5 h at 37°C. Labeled Lck was isolated by immunoprecipitation, subjected to gel electrophoresis, and transferred to a nitrocellulose membrane (Schleicher and Schuell). The region of the membrane containing Lck was identified by autoradiography, excised, and incubated with tosylphenylalanyl chloromethyl ketone trypsin as described(36.Luo K. Hurley T.R. Sefton B.M. Oncogene. 1990; 5: 921-924PubMed Google Scholar). Lck that had been autophosphorylated in vitro with [γ-32P]ATP and similarly digested with trypsin was used as a source of labeled tryptic peptide containing Tyr-394. Two-dimensional tryptic peptide mapping was carried out on cellulose thin-layer chromatography plates (EM Science, Gibbstown, NJ) by electrophoresis at pH 8.9 in the first dimension followed by ascending chromatography in phosphochromatography buffer as described (37.Hunter T. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1311-1315Crossref PubMed Scopus (1543) Google Scholar, 38.Collins T.L. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11885-11889Crossref PubMed Scopus (30) Google Scholar). Labeled peptides were visualized by autoradiography.In Vitro Kinase AssaysLck immunoprecipitates were incubated with 5 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham Corp.) and 2 mM Val5-angiotensin II (Star Biochemicals, Torrance, CA) in 20 μl of kinase buffer (40 mM PIPES, pH 7.0, 10 mM MnCl2) at room temperature. The reactions were stopped by the addition of 5% trichloroacetic acid, and the angiotensin was adsorbed onto Whatman p81 phosphocellulose paper (Whatman, Fairfield, NJ). The paper was then washed with 0.5% phosphoric acid to remove unincorporated [γ-32P]ATP. Phosphate incorporation into angiotensin was measured by liquid scintillation counting of the p81 paper.Hydrogen Peroxide Treatment of Cells2 × 106 208F cells expressing Lck were seeded in 10 ml of DMEM supplemented with 10% calf serum onto a 10-cm plate and allowed to recover for 18 h. H2O2 was added directly to the cell medium to a final concentration of 5 mM. The cells were incubated at 37°C for 15 min, washed once with ice-cold isotonic Tris-buffered saline, and lysed directly on the tissue culture dish in 1 ml of RIPA buffer. For experiments in which cells were biosynthetically labeled with 32Pi, the 5-h labeling period (see above) preceded the addition of H2O2 to the media.ImmunofluorescenceApproximately 2 × 106 208F cells expressing Lck were seeded on 25 × 25-mm glass coverslips placed in 35-mm Petri dishes. After 18 h of recovery at 37°C, the cells were washed once with phosphate-buffered saline (PBS) and fixed for 30 min in PBS containing 3% paraformaldehyde and 25 μM NaOH. Fixing and all subsequent manipulations were performed at room temperature. The cells were then permeabilized with rinse solution (PBS containing 10 mM glycine and 1% Triton X-100) for 10 min, incubated with rabbit anti-Lck antibodies (diluted 1:200 in fetal bovine serum (Intergen, Purchase, NY) containing 1% Triton X-100) for 30 min, washed four times with rinse solution, incubated with goat anti-rabbit IgG conjugated to fluorescein (Jackson Laboratories) (diluted 1:50 in fetal bovine serum), and washed an additional four times in rinse solution. The coverslips were mounted on slides with Vectashield mounting medium (Vector, Burlingame, CA). Antibody staining was visualized by fluorescence microscopy.RESULTSA Form of Activated Lck Localized to the Nucleus Does Not Induce an Increase in Tyrosine Phosphorylation of Cellular SubstrateTo determine if Lck, which had been both activated by mutation (F505) and retargeted to the nucleus, was able to transform fibroblasts, we constructed a form of Lck in which the nuclear localization signal from SV40 large T antigen was attached to the amino terminus, Gly-2 was mutated to alanine, and Tyr-505 was mutated to phenylalanine. This protein (nucF505Lck) was concentrated in the nucleus when expressed in fibroblasts that express no endogenous Lck (Fig. 1E). In contrast to cells expressing F505Lck, which were highly transformed, cells expressing the nuclear form of activated Lck were not transformed and were indistinguishable from uninfected fibroblasts (data not shown). To determine why nucF505Lck had no transforming activity, we examined the tyrosine phosphorylation of proteins in cells that expressed the mutant protein. Tyrosine phosphorylation in cells expressing nucF505Lck was unchanged from the level observed in uninfected cells (Fig. 2A). This is similar to what is observed in cells expressing A2F505Lck, a non-myristoylated form of activated Lck (17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar) or S3/5F505Lck, a form of activated Lck. which is myristoylated but not palmitoylated(22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar), two other non-membrane-bound forms of Lck, and contrasted strikingly with the elevated phosphotyrosine levels observed in cells expressing F505Lck, which is associated with the cell membrane (Fig. 2A).Figure 2:Tyrosine protein phosphorylation in cells expressing non-membrane-associated F505Lck. A, anti-phosphotyrosine Western blot. Lysates of fibroblasts expressing mutant Lcks were prepared and fractionated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane and incubated with antibodies against phosphotyrosine and 125I-protein A. Lane 1, uninfected 208F cells; lane 2, wild-type Lck (WTLck); lane 3, F505Lck; lane 4, nucF505Lck; lane 5, S3/5F505Lck; lane 6, A2F505Lck. B, anti-p56lck Western blot. Lck immunoprecipitates from fibroblasts expressing mutant Lcks were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with anti-Lck antibodies and 125I-protein A. The identity of the lanes is the same as in A. Non-membrane-bound forms of Lck have reduced electrophoretic mobility and typically exhibit multiple apparent molecular weights.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Non-membrane-associated Forms of Activated Lck Have Reduced Catalytic Activity in VitroTo determine whether the inability of nucF505Lck to phosphorylate cellular proteins was due to reduced enzymatic activity or due to a lack of access to substrates, nucF505Lck was isolated by immunoprecipitation and assayed for its ability to phosphorylate the exogenous substrate Val5-angiotensin II in vitro. The mutant forms of Lck are expressed at slightly different levels in the 208F cell lines examined here (Fig. 2B). Therefore, the amount of Lck in the immunoprecipitates used for measuring catalytic activity was measured by Western blotting with antibodies to Lck. The data in the figure were normalized to account for differences in Lck levels in the immunoprecipitates. We found that the catalytic activity of nucF505Lck was reduced significantly relative to wild-type Lck and membrane-bound F505Lck (Fig. 3A). This is similar to what has been observed with two other forms of activated Lck not associated with membrane, A2F505Lck and S3/5F505Lck (17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar) (Fig. 3A). The low enzymatic activity of non-membrane-bound forms of Lck almost certainly plays some role in the ability of these proteins to phosphorylate cellular proteins on tyrosine in vivo.Figure 3:In vitro protein kinase activity of non-membrane-bound forms of F505Lck. Mutant Lcks were isolated by immunoprecipitation and assayed in vitro for their ability to phosphorylate an exogenous substrate, Val5-angiotensin II. Assay results are expressed as the rate of labeled phosphate incorporation by the substrate per arbitrary unit of Lck. For normalization, Lck protein levels in the samples were measured by Western blotting of a fraction of the immunoprecipitates with antibodies to Lck and 125I-protein A. A, relative activities of wild-type (WT) Lck, F505Lck, nucF505Lck, S3/5Lck, and A2F505Lck from unstimulated cells. B, activities of non-membrane-bound forms of F505Lck before and after exposure to H2O2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Non-membrane-associated Forms of Activated Lck Are Not Phosphorylated on Tyr-394Phosphorylation of Tyr-394 in Lck stimulates the activity of Lck considerably(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). In contrast to membrane-bound F505Lck, which is highly phosphorylated at this residue, A2F505Lck is poorly phosphorylated at Tyr-394(17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar). To determine whether the phosphorylation of nucF505Lck and S3/5F505Lck at Tyr-394 was similarly impaired, cells expressing the mutant Lck proteins were labeled biosynthetically with 32Pi, and Lck was immunoprecipitated and analyzed by two-dimensional tryptic peptide mapping. The total labeling of S3/5F505Lck and nucF505Lck with 32P was much less than labeling seen in F505Lck, and phosphorylation of Tyr-394 was barely detectable (Fig. 4). nucF505Lck contained several labeled peptides not seen in A2F505Lck or S3/5F505Lck that reflect an increased level of serine and threonine in phosphorylation of Lck that presumably occurs in the nucleus (data not shown). The low activity of non-membrane-bound forms of activated Lck may result from reduced phosphorylation at Tyr-394.Figure 4:Analysis of Lck phosphorylation by two-dimensional tryptic peptide mapping. Lck was isolated by immunoprecipitation from cells that had been labeled biosynthetically with 32Pi. Lck was purified by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and digested with trypsin. The resulting peptides were separated horizontally by electrophoresis at pH 8.9 and then vertically by ascending chromatography. The peptides containing Tyr-394 are marked with open arrowheads. The sample origins are indicated by filled arrowheads. A, nucF505Lck; B, S3/5F505Lck; C, A2F505Lck; D, nucF505Lck +H2O2; E, S3/5F505Lck +H2O2; F, A2F505Lck +H2O2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exposure of Cells to H2O2 Activates Non-membrane-associated Forms of LckH2O2 is a potent activator of Lck that stimulates the catalytic activity of Lck by inducing the phosphorylation of Tyr-394(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). To examine whether H2O2 could increase the phosphorylation of Tyr-394 and possibly restore activity to non-membrane-bound forms of Lck, nucF505Lck, S3/5F505Lck, and A2F505Lck were immunoprecipitated from cells before and after exposure to H2O2 and assayed for catalytic activity in vitro. In addition, they were labeled biosynthetically with 32Pi and subjected to two-dimensional tryptic peptide mapping. H2O2 exposure resulted in a dramatic increase in the catalytic activity of each mutant (Fig. 3B). Thus, nucF505Lck, S3/5F505Lck, and A2F505Lck have neither unintended intrinsic defects that occurred during mutagenesis nor an inability to be activated. In each case, biosynthetic labeling showed that H2O2 induced the appearance of an additional labeled peptide that comigrated precisely with the peptide containing phosphorylated Tyr-394 (Fig. 4). Therefore, as is the case with membrane-bound Lck, H2O2 appears to activate non-membrane-bound forms by stimulating phosphorylation of Tyr-394.Phosphorylation of Non-membrane-associated Forms of Lck at Tyr-394 Need Not Occur through AutophosphorylationTo determine whether phosphorylation of non-membrane-associated Lck at Tyr-394 following exposure to H2O2 was due only to increased autophosphorylation, we asked whether catalytically inactive forms of Lck that were localized to the cytoplasm or nucleus became phosphorylated at Tyr-394 following exposure to H2O2. Mutation of a conserved lysine residue in the ATP phosphate anchor at position 273 to arginine results in a greater than 95% reduction of Lck catalytic activity in vitro(8, 38). ( (2)K. Pierno, unpublished results.) Fibroblasts expressing introduced nucR273Lck or A2R273Lck, but no endogenous Lck, were labeled biosynthetically with 32Pi and then exposed to H2O2. Lck was isolated and subjected to two-dimensional tryptic peptide mapping. Both forms of catalytically inactive Lck were phosphorylated at Tyr-505 in untreated cells, but phosphorylation of Tyr-394 was not detectable (Fig. 5, panels A and C). Both mutant proteins became phosphorylated on Tyr-394 following H2O2 treatment as indicated by the appearance of a labeled peptide that comigrated with a marker peptide containing labeled Tyr-394 (Fig. 5, panels B and D, and data not shown).Figure 5:The effect of H2O2 treatment on the phosphorylation of catalytically inactive, non-membrane-associated Lck. Cells were labeled biosynthetically with 32Pi and then exposed to H2O2 as described. Two-dimensional tryptic peptide mapping was performed as in Fig. 4. Sample origins are indicated by arrowheads. A, nucR273Lck from unstimulated fibroblasts; B, nucR273Lck from fibroblasts stimulated with H2O2; C, A2R273Lck from unstimulated fibroblasts; D, A2R273Lck from fibroblasts stimulated with H2O2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)H2O2 Does Not Induce Reorganization of the Cellular Location of Non-myristoylated F505LckThe data presented above are consistent with the phosphorylation of non-membrane-bound Lck at Tyr-394 being mediated by an as yet unidentified kinase in the cytosol and nucleus. However, it was possible that H2O2 induced non-membrane-associated Lck to relocalize to the plasma membrane where it was phosphorylated on Tyr-394 and activated. To determine if such a relocalization occurred, we examined the cellular location of F505Lck and A2F505Lck by immunofluorescence microscopy before and after H2O2 treatment (Fig. 1, panels F-I). The transformed cells expressing F505Lck were rounded up and displayed a much more three-dimensional morphology than the cells expressing A2F505Lck, which were flattened and had a normal fibroblastic morphology. Unlike F505Lck, which showed a pattern of staining typical of a membrane-associated protein in untreated cells, A2F505Lck was present throughout the cell, including the nucleus (Fig. 1, panels F and H). Following H2O2 treatment, the locations of F505Lck and A2F505Lck in the cell were not altered detectably (Fig. 1, panels G and I).DISCUSSIONThe lack of in vivo catalytic and biological activity of A2F505Lck, S3/5F505Lck, and nucF505Lck, none of which can bind stably to cellular membranes, shows that F505Lck must be able to associate with membranes to be active. All of these non-membrane-associated forms of F505Lck differ from F505Lck in that they lack detectable phosphorylation on Tyr-394, a residue at which phosphorylation positively regulates Lck and that is highly phosphorylated in membrane-bound F505Lck. It is likely that this lack of phosphorylation in vivo and in vitro is responsible for their low activity(17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 39.Veillette A. Fournel M. Oncogene. 1990; 5: 1455-1462PubMed Google Scholar). This implies that there is some protein or cofactor localized to membranes that is normally required for the activation of F505Lck through phosphorylation of Tyr-394. Phosphorylation of Tyr-394, however, can occur at intracellular sites other than the cellular membranes. When cells expressing cytoplasmic or nuclear F505Lck are exposed to H2O2, both the phosphorylation of Tyr-394 and the in vitro catalytic activity of the mutant Lcks are stimulated markedly. This rules out the possibility that intrinsic defects in the mutagenized genes encoding nucF505Lck, S3/5F505Lck, and A2F505Lck are responsible for the lack of activity in these proteins in unstimulated cells. However, the mechanism by which phosphorylation of Tyr-394 in the cytosol and nucleus occurs is unclear. It is not a result of relocalization to the plasma membrane. Immunofluorescence microscopy (Fig. 1, panels H and I) as well as cell fractionation experiments (data not shown) show clearly that the bulk of A2F505Lck remains cytosolic in H2O2-treated cells. While a small population of A2F505Lck may relocalize to the plasma membrane in the presence of H2O2, the extensive phosphorylation of A2F505Lck on Tyr-394 indicates that reorganization of cellular location is not the mechanism by which the kinase is activated.The phosphorylation of A2F505Lck and nucF505Lck is also not necessarily the result of autophosphorylation since catalytically inactive A2Lck and nucLck become phosphorylated at Tyr-394 when cells lacking active Lck are treated with H2O2. There must, therefore, be at least one tyrosine kinase in the cytoplasm and nucleus that has the ability to phosphorylate Lck at Tyr-394. This unidentified kinase may normally be subject to regulation by oxidant levels within the cell. In addition, apparently undiminished phosphorylation of Tyr-505 was observed in cytosolic and nuclear-targeted forms of kinase-inactive Lck. The tyrosine protein kinase CSK phosphorylates the carboxyl-terminal tyrosine of Src family members, including Tyr-505 of Lck, and negatively regulates the activities of these proteins(40.Bergman M. Mustelin T. Oetken C. Partanen J. Flint N.A. Amrein K.E. Autero M. Burn P. Alitalo K. EMBO J. 1992; 11: 2919-2924Crossref PubMed Scopus (273) Google Scholar). Our results demonstrate that Csk is able to phosphorylate Tyr-505 of non-membrane-bound, kinase-inactive Lck. The fact that Tyr-505 was phosphorylated in non-membrane-bound forms of kinase-inactive Lck suggests that either CSK itself or a CSK-like activity is present in the nucleus as well as the cytosol.S3/5F505Lck and A2F505Lck, which cannot be phosphorylated at position 505, are less active as tyrosine kinases in vitro than S3/5Lck or A2Lck, which contain phosphorylated Tyr-505 (data not shown). This difference may result from the fact that the SH2 domain of A2F505Lck and S3/5F505Lck does not have a phosphorylated Tyr-505 with which to form an intramolecular complex. In wild-type Lck, the SH2 domain binds to phosphorylated Tyr-505 (41.Sieh M. Bolen J. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (185) Google Scholar) and apparently yields a conformation that exhibits in vitro activity in the absence of extensive phosphorylation at Tyr-394. In contrast, the untethered SH2 domain of F505Lck may induce a conformation that requires phosphorylation of Tyr-394 for catalytic activity.The properties of nucF505Lck, S3/5F505Lck, and A2F505Lck differ from those of analogous cytosolic forms of the activated Src kinase. A2v-Src and A2F527c-Src, although nontransforming when expressed in fibroblasts, both retain the ability to phosphorylate most cellular substrates in vivo, and A2v-Src exhibits undiminished activity to phosphorylate exogenous substrates in vitro(42.Reynolds A.B. Roesel D.J. Kanner S.B. Parsons J.T. Mol. Cell. Biol. 1989; 9: 629-638Crossref PubMed Scopus (286) Google Scholar, 43.Buss J.E. Kamps M.P. Gould K. Sefton B.M. J. Virol. 1986; 4: 2696-2704Google Scholar). The reason for the differences between the cytosolic, non-membrane-bound forms of activated Lck and Src is not clear.It appears that there are at least two activities that can phosphorylate Tyr-394. One is restricted to the plasma membrane and does not require the presence of oxidants for activity. Most likely, this is the mechanism that is responsible for the high level of Tyr-394 phosphorylation and increased activity of F505Lck at the plasma membrane. This membrane-restricted activity could be due to Lck itself or another Src family member. Additionally, there are one or more activities in the cytosol and nucleus that can phosphorylate Tyr-394, but these kinases require activation by oxidants. It is unlikely that a Src family kinase is responsible for this phosphorylation since Src family proteins are generally associated with cellular membranes. In the absence of oxidants, the lack of phosphorylation at Tyr-394 in forms of F505Lck not tethered to membranes could result from simple lack of phosphorylation of this site or a more rapid rate of dephosphorylation than phosphorylation of the site. Since H2O2 will almost certainly inhibit the phosphatase responsible for dephosphorylating Tyr-394 in the cytosol, we cannot determine whether the phosphorylation of Tyr-394 induced by H2O2 results from activation of the kinase phosphorylating the site or simply from inhibition of the phosphatase dephosphorylating the site. Nevertheless, it is clear that the steady-state phosphorylation of Tyr-394 in F505Lck is greatly reduced when the protein is removed from membranes and that there must exist one or more enzymes or functions restricted to membranes that facilitate constitutive phosphorylation of Tyr-394. An important question is the identity of the normal substrates of the oxidant-regulated tyrosine protein kinases in the cytosol and nucleus and the role of these tyrosine protein kinases in normal signal transduction. INTRODUCTIONp56lck, a member of the Src family of tyrosine protein kinases(1.Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 2.Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar), is expressed exclusively in lymphoid cells and predominantly in T cells. Lck associates with the cytoplasmic domain of the T-cell receptor-associated glycoproteins CD4 and CD8(3.Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 4.Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1121) Google Scholar), membrane immunoglobulin in B cells(5.Campbell M.-A. Sefton B.M. EMBO J. 1990; 9: 2125-2131Crossref PubMed Scopus (161) Google Scholar), and glycosylphosphatidylinositol-anchored proteins (6.Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. Science. 1991; 254: 1016-1019Crossref PubMed Scopus (762) Google Scholar, 7.Shenoy-Scaria A.M. Timson Gauen L.K. Kwong J. Shaw A.S. Lublin L.M. Mol. Cell. Biol. 1993; 13: 6385-6392Crossref PubMed Scopus (225) Google Scholar) through its unique amino terminus. p56lck is essential for both T-cell development (8.Allen J.M. Forbush K.A. Perlmutter R.M. Mol. Cell. Biol. 1992; 12: 2758-2768Crossref PubMed Scopus (105) Google Scholar, 9.Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) and T-cell activation(10.Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (180) Google Scholar, 11.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). The activity of Lck is regulated by the phosphorylation of tyrosine residues at positions 394 and 505. Phosphorylation of Tyr-505 inhibits the activity of Lck (12.Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 13.Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar). Mutation of Tyr-505 to phenylalanine results in a constitutively active protein that is able to transform fibroblasts (12.Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 13.Marth J.D. Cooper J.A. King C.S. Ziegler S.F. Tinker D.A. Overell R.W. Krebs E.G. Perlmutter R.M. Mol. Cell. Biol. 1988; 8: 540-550Crossref PubMed Scopus (182) Google Scholar), enhance T-cell responsiveness to antigen(14.Abraham N. Miceli M.C. Parnes J.R. Veillette A. Nature. 1991; 350: 62-66Crossref PubMed Scopus (256) Google Scholar), and induce antigen-independent interleukin-2 production in T cells(15.Luo K. Sefton B.M. Mol. Cell. Biol. 1992; 12: 4724-4732Crossref PubMed Scopus (52) Google Scholar). In contrast, phosphorylation of Tyr-394, the single site of autophosphorylation in vitro, enhances the catalytic activity of Lck and is required for the activation of Lck by mutation of Tyr-505 to phenylalanine(16.Boulet I. Fagard R. Fischer S. Biochem. Biophys. Res. Commun. 1987; 149: 56-64Crossref PubMed Scopus (13) Google Scholar, 17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar).Lck associates with the inner face of the plasma membrane through its amino terminus. This interaction is mediated by both myristic acid and palmitic acid that are bound to the amino-terminal glycine and Cys-3 and/or Cys-5, respectively(19.Marchildon G.A. Casnellie J.E. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7679-7682Crossref PubMed Scopus (67) Google Scholar, 20.Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 21.Kwong J.A. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). Mutations in which the amino-terminal glycine is mutated to alanine result in a protein that is neither myristoylated nor palmitoylated and is unable to associate with the plasma membrane(17.Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar). Mutation of both Cys-3 and Cys-5 to serine yields a protein that is myristoylated but not palmitoylated (21.Kwong J.A. Lublin D. Biochem. Biophys. Res. Commun. 1995; 207: 868-876Crossref PubMed Scopus (46) Google Scholar, 22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar, 23.Rodgers W. Crise B. Rose J.K. Mol. Cell. Biol. 1994; 14: 5384-5391Crossref PubMed Scopus (224) Google Scholar). The resulting Lck protein is completely cytoplasmic, indicating that myristoylation is not sufficient for anchoring Lck to the plasma membrane(22.Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar).Hydrogen peroxide is a general inhibitor of tyrosine protein phosphatases(24.Heffetz D. Bushkin I. Dror R. Zick Y. J. Biol. Chem. 1990; 265: 2896-2902Abstract Full Text PDF PubMed Google Scholar). Exposure of cells to H2O2 induces rapid tyrosine phosphorylation of numerous cellular proteins in vivo(24.Heffetz D. Bushkin I. Dror R. Zick Y. J. Biol. Chem. 1990; 265: 2896-2902Abstract Full Text PDF PubMed Google Scholar, 25.Schieven G.L. Kirihara J.M. Gilliland L.K. Uckun F.M. Ledbetter J.A. Mol. Biol. Cell. 1993; 4: 523-530Crossref PubMed Scopus (54) Google Scholar, 26.O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (141) Google Scholar, 27.Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 28.Nakamura K. Hori T. Sato N. Sugie K. Kawakami T. Yodoi J. Oncogene. 1993; 8: 3133-3139PubMed Google Scholar) and has been shown to be a potent activator of Lck(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). Activation of Lck by H2O2 is due to an increase in the phosphorylation of Tyr-394(18.Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar). In the current study, we examined the effect of H2O2 on three different non-membrane-bound forms of genetically activated Lck (F505Lck). Non-myristoylated, myristoylated but non-palmitoylated, and nuclear forms of F505Lck all exhibit severely reduced activity in vivo and in vitro. H2O2 is able to restore the in vitro catalytic activity of all three non-membrane-bound forms of Lck. This increase in activity correlates with, and in all probability is due to, an increase in the phosphorylation of Tyr-394. Apparently, phosphorylation of Tyr-394 occurs inefficiently when Lck is located at sites other than cellular membranes but can be induced by elevated levels of oxidants.

Ultra-deep next-generation sequencing of circulating tumor DNA (ctDNA) holds great promise as a tool for the early detection of cancer and for monitoring disease progression and therapeutic responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection.To achieve high accuracy of variant calling via better distinguishing low-frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. The results on both simulated data and real data from healthy subjects demonstrate that the proposed algorithm consistently outperforms a current, state-of-the-art, position-specific error polishing model, particularly when the sample size of healthy subjects is small.TNER significantly enhances the specificity of downstream ctDNA mutation detection without sacrificing sensitivity. The tool is publicly available at https://github.com/ctDNA/TNER .

Abstract Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.

9021^ Background: Addition of a neurokinin-1 antagonist (NK1A) to a standard ondansetron (O) and dexamethasone (D) antiemetic regimen improves prevention of cisplatin-induced nausea/vomiting (CINV), particularly during the delayed period (25-120 hours) (DP). Therefore approved regimens include multiple day NK1A administration extending into the DP. Preliminary data (Grunberg, Supp Care Cancer 17:589, 2009) suggested that a single NK1A dose prior to chemotherapy could give excellent CINV protection throughout the overall period of risk (0-120 hours) (OP). The present study compared the approved schedule of oral NK1A aprepitant (Emend) (A) to single-dose intravenous NK1A fosaprepitant (Emend for Injection) (F). Methods: A randomized double-blind active-control design was used to test whether F is non-inferior to A. Patients (pts) receiving cisplatin ≥70 mg/m2 for the first time received O and D with a standard A regimen (125 mg day 1, 80 mg day 2, 80 mg day 3) or an abbreviated F regimen (150 mg day 1). Primary endpoint was Complete Response (no vomiting, no rescue medication) (CR) during OP. Secondary endpoints were CR during DP and no vomiting (NV) during OP. Accrual of 1,113 evaluable pts per arm was required to confirm non-inferiority with expected CR of 67.7% and non-inferiority margin of 7 percentage points. Adverse events were observed and recorded. Results: 2,322 pts were entered, with 2,247 considered evaluable for efficacy (1,138 receiving A and 1,109 receiving F). Antiemetic protection was similar between A and F within predefined bounds in terms of CR OP (72.3% vs. 71.9%), CR DP (74.2% vs. 74.3%) and NV OP (74.6% vs. 72.9%). Both regimens were well tolerated, although more frequent infusion site pain/erythema/phlebitis was seen with F (0.6% vs. 2.4%). Conclusions: Given with O and D, a single-day regimen of F was non-inferior to a standard 3-day regimen of A in preventing CINV during OP and DP. Equivalence of these regimens allows more convenient administration of an NK1A but also raises question as to whether the key variable in NK1A antiemetic protection during DP is serum drug exposure, duration of receptor occupancy, or timing of antiemetic administration (prior to the emetogenic challenge). Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Merck Merck Merck Merck In compliance with the guidelines established by the ASCO Conflict of Interest Policy (J Clin Oncol. 2006 Jan 20;24[3]:519-521) and the Accreditation Council for Continuing Medical Education (ACCME), ASCO strives to promote balance, independence, objectivity, and scientific rigor through disclosure of financial and other interests, and identification and management of potential conflicts. According to the ASCO Conflict of Interest Policy, the following financial and other relationships must be disclosed: employment or leadership position, consultant or advisory role, stock ownership, honoraria, research funding, expert testimony, and other remuneration (J Clin Oncol. 2006 Jan 20;24[3]:520). The ASCO Conflict of Interest Policy disclosure requirements apply to all authors who submit abstracts to the Annual Meeting. For clinical trials that began accrual on or after April 29, 2004, ASCO's Policy places some restrictions on the financial relationships of principal investigators (J Clin Oncol. 2006 Jan 20;24[3]:521). If a principal investigator holds any restricted relationships, his or her abstract will be ineligible for placement in the 2010 Annual Meeting unless the ASCO Ethics Committee grants an exception. Among the circumstances that might justify an exception are that the principal investigator (1) is a widely acknowledged expert in a particular therapeutic area; (2) is the inventor of a unique technology or treatment being evaluated in the clinical trial; or (3) is involved in international clinical oncology research and has acted consistently with recognized international standards of ethics in the conduct of clinical research. NIH-sponsored trials are exempt from the Policy restrictions. Abstracts for which authors requested and have been granted an exception in accordance with ASCO's Policy are designated with a caret symbol (^) in the Annual Meeting Proceedings. For more information about the ASCO Conflict of Interest Policy and the exceptions process, please visit www.asco.org/conflictofinterest.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTChemical Genetic and Genomic Approaches Reveal a Role for Copper in Specific Gene ActivationBrent R. Stockwell, James S. Hardwick, Jeffrey K. Tong, and Stuart L. SchreiberView Author Information Howard Hughes Medical Institute Department of Chemistry & Chemical Biology Harvard Institute of Chemistry & Cell Biology and Center for Genomics Research, Harvard University 12 Oxford Street, Cambridge, Massachusetts 02138 Cite this: J. Am. Chem. Soc. 1999, 121, 45, 10662–10663Publication Date (Web):October 30, 1999Publication History Received22 June 1999Published online30 October 1999Published inissue 1 November 1999https://doi.org/10.1021/ja9921221Copyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views257Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (42 KB) Get e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Copper,Genetics,Ions,Metals,Small molecules Get e-Alerts

2004 Background: Vorinostat is a histone deacetylase inhibitor that represents a rational therapeutic target in GBM. Methods: Recurrent GBM patients (pts) having received = 1 prior chemo regimen for progressive disease were eligible. Vorinostat dose was 200 mg bid x 14 days q 3 weeks. We applied a phase II design which had 90% power to declare the regimen active if the study PFS6 rate was 25%, a 15% rate increase over PFS6 in historical controls (10%). Results: 68 patients were treated. Grade 3+ non-hematologic toxicity consisting mainly of fatigue, diarrhea and dehydration occurred in 23% of pts; Grade 3+ hematologic toxicity consisting mainly of thrombocytopenia occurred in 25% of pts. Intra-patient dose escalation to 300 mg bid resulted in higher incidence of grade 3+ thrombocytopenia (45%) and was aborted. Pts receiving enzyme-inducing anticonvulsants (EIAC) had significantly less grade 3 + non-hematologic toxicity (p=0.01) and grade 3/4 thrombocytopenia (p=0.04). Pharmacokinetic analysis showed lower vorninostat C-max and AUC (0–24h) values and higher vorinostat-glucuronide C-max and AUC (0–24h) values in EIAC pts, although this did not reach statistical significance. The trial met the prospectively defined primary efficacy endpoint at the planned interim analysis with 5 of the first 22 patients (23%) being progression-free at 6 mo. RNA array analysis, performed in paired baseline and post-vorinostat treatment samples in a separate subgroup of 5 surgical recurrent GBM pts, who received vorinostat for 6 doses prior to surgery, showed upregulation of E-cadherin (p=0.02), thus indicating a biologic effect of the HDAC inhibitor on the glioblastoma tumors. Histone acetylation, Akt, phospho-Akt, P21 Waf1/Cip1 and P27 Kip1 expression analysis in all patients and correlation with outcome is ongoing. Conclusions: Vorinostat is well tolerated in recurrent GBM patients. EIAC patients have less toxicity, likely due to increased vorinostat metabolism via glucuronidation. Interim efficacy analysis is indicative of antitumor activity. Final efficacy analysis, RNA array data and correlative tissue analysis will be reported. No significant financial relationships to disclose.

A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining.NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells.The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119.Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

Patients with familial adenomatous polyposis (FAP) have a 5%-10% lifetime risk of developing duodenal cancer. In severe duodenal polyposis, pancreaticoduodenectomy according to Whipple has been considered the only way to cure duodenal polyposis. However, polyps recur even after surgery. We describe a patient with severe adenomatosis of the small bowel in the afferent loop of a Roux-en-Y anastomosis after a Whipple procedure, detected by double balloon endoscopy (DBE). This is the first description of the use of DBE for this indication, and emphasizes the need for surveillance of the small bowel after surgery, especially in the area of the biliary anastomosis.

Abstract Crizotinib (PF-02341066) is a small molecule tyrosine kinase inhibitor of ALK, ROS1 and c-MET that is approved in over 70 countries for the treatment of ALK fusion positive non-small cell lung cancer (ALK+ NSCLC). Crizotinib achieved robust objective response rates of approximately 60% in ALK+ NSCLC and significantly improved progression free survival compared to chemotherapy. The emergence of secondary mutations within the ALK kinase domain or the activation of compensatory signaling pathways in crizotinib and other ALKi refractory tumors prompted searches for next generation of ALKi active against resistance mutations as single agents or in combination with other treatments. Such effort led to our recent discovery of PF-06463922, an ALK/ROS1 inhibitor with greatly improved ALK potency, brain penetration, and broad spectrum activity against all known clinical ALKi-resistant mutations. PF-06463922 is being tested in a Phase I clinical trial in both ALK+ and ROS1 fusion positive NSCLC in treatment naive or ALKi relapsed patients. In our current preclinical study, we explored rational combination strategies to further improve the efficacy of PF-06463922 in ALKi resistant cells or tumors. Our results show that compared to PF-06463922 alone, the combination of this compound with PI3K pathway inhibitors, such as PF-05212384 (PI3K/mTOR), GDC0941 (pan-PI3K) or GDC0032 (beta-sparing) leads to more robust anti-proliferative activity in vitro and greater duration of efficacy in vivo in the ALKi resistant models. These PI3K pathway inhibitors also partially overcome EGF or HGF ligand-induced resistance to PF-06463922. Interestingly, in addition to AKT signaling, both compounds inhibit ERK signaling as well, which may be essential for their enhancement of PF-06463922 cell activity or tumor efficacy in combination settings. Studies are ongoing to identify optimal partners for PF-06463922 combination using isoform selective PI3Ki, AKTi and mTORi. We are also exploring the breadth of efficacy of this combination in overcoming resistance to crizotinib, PF-06463922 or other ALKi. The findings provide important evidence that will help define the clinical development path for PF-06463922. This research effort may ultimately lead to more effective approaches to treat ALKi refractory patients in the clinic. Citation Format: Ping Wei, Ming Qiu, Nathan Lee, Joan Cao, Hui Wang, Konstantinos Tsaparikos, Conglin Fan, Timothy Sargis, Justine Lam, Maruja E. Lira, Goldie Lui, James Hardwick, Valeria Fantin, Paul Rejto, Tod Smeal. Rational combination of PF-06463922 (next-generation ALK inhibitor) with PI3K pathway inhibitors overcomes ALKi resistance in EML4-ALK+ NSCLC models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 764. doi:10.1158/1538-7445.AM2015-764

Abstract Purpose: Aprepitant was shown previously to be effective for prevention of CINV associated with MEC in breast cancer patients. A recent study (PN130) assessed aprepitant in patients with a variety of tumor types receiving a broad range of MEC regimens. A post-hoc subgroup analysis of patients with breast cancer was performed and results (nominal p-values) are reported.Methods: This randomized, gender-stratified, double-blind trial enrolled patients with confirmed malignancies, naïve to HEC or MEC agents, who received at least one MEC agent. Patients received an aprepitant triple-therapy regimen (aprepitant 125 mg PO day 1 and 80 mg PO days 2-3, ondansetron 8 mg PO bid day 1, and dexamethasone 12 mg PO day 1) or an active control regimen (ondansetron 8 mg PO bid days 1-3 and dexamethasone 20 mg PO day 1). Primary and key secondary efficacy endpoints were proportions of patients with No Vomiting and Complete Response (no vomiting/no rescue medication use) respectively, during the overall phase (120 hours post-chemotherapy). Treatment group comparisons were based on a logistic regression model with terms for treatment, region, and gender.Results: Of 832 patients in the modified intent to treat population analyzed, 53% (n=439) had breast cancer. In the overall phase, significantly more patients in the aprepitant group achieved No Vomiting and Complete Response both overall and in the subgroup of patients with breast cancer (Table 1). The magnitude of the treatment group differences were similar to the differences observed in the entire study population. Similar trends were seen in the acute and delayed phase in both the overall population and the subgroup of patients with breast cancer. Results for additional endpoints are consistent with the data presented here. Adverse events were generally similar in the aprepitant and control groups. Aprepitant, n/m (%)Active control, n/m (%)No vomitingAll tumors324/425 (76.2)*252/406 (62.1) Breast cancer152/219 (69.4)*116/220 (52.7)Complete responseAll tumors292/425 (68.7)*229/407 (56.3) Breast cancer139/219 (63.5)*102/220 (46.4)n/m = patients with favorable response/patients included in subgroup; * p-value &amp;lt; 0.001Conclusions: The aprepitant regimen provided superior efficacy over the control regimen for prevention of CINV for all patients receiving MEC. The benefit of aprepitant triple therapy in patients with breast cancer in this trial is in agreement with the results of a previous large-scale trial of aprepitant in this population. Control of CINV in breast cancer patients in this trial was consistently lower than in the overall population for all endpoints, demonstrating the importance of CINV prophylaxis in women with breast cancer undergoing MEC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1116.

Abstract Introduction: Gamma-secretase inhibitors (GSIs) inhibit Notch pathway signaling and have potential as cancer therapeutics. Clinical development of GSIs has been complicated by mechanism-related goblet cell hyperplasia and dose-limiting diarrhea. A non-invasive biomarker of Notch pathway inhibition would facilitate the identification of a well-tolerated dose/schedule with maximal biological effect. In this study, a transcriptional biomarker of Notch pathway inhibition was developed from whole genome profiling of human plucked hair follicles (PHFs) for use in demonstrating target engagement and defining a PK/PD relationship. Procedures: This randomized, placebo-controlled, 3-period crossover study evaluated the effects of 350mg or 1000mg of the oral GSI MK-0752 on PHF gene expression in 30 healthy male subjects. Pooled PHFs, single anagen hairs, and whole blood samples were collected at various timepoints for pharmacokinetic and pharmacodynamic (mRNA profiling) analysis. Results: There was a significant decrease in a pre-specified 15-gene Notch signature score (NSS) at 8.5, 28.5, 48 and 96 hours in response to either 350mg or 1000mg MK-0752, compared to placebo, at the nominal 1-sided 0.05 level. The maximum response in pooled PHFs was seen following administration 1000mg MK-0752, which resulted in a significant (p &amp;lt;0.001) decrease from baseline in the NSS at 8.5 hours post-treatment, with an observed effect size of 2.34. A smaller, but significant, decrease from baseline in the NSS was seen in single anagen hairs at 8.5h post-treatment for 1000mg MK-0752 compared to placebo (effect size 1.40, p&amp;lt;0.001). Changes in the hair follicle NSS were driven primarily by 5 Notch pathway genes. A linear concentration-effect relationship was shown to be consistent across both dose levels using PHFs. In addition to Notch pathway changes, transcriptional signatures of PI3K signaling and cell proliferation were shown to be suppressed at 8.5h and 28.5h post-dose, respectively. Treatment with MK-0752 also resulted in various late (48h post-dose) transcriptional changes in hair follicles including the upregulation of other signaling pathways including Wnt and receptor tyrosine kinases. Conclusions: The results of this study demonstrate the development of a compound-specific biomarker in a novel surrogate tissue. This is the first known report of whole genome mRNA profiling of human hair follicles in response to a GSI, and demonstrates that PHFs contain cells with intact Notch signaling pathways responsive to MK-0752. A 1000mg dose of MK-0752, which is smaller than the clinically-tolerated dose of 1800mg/week, decreases expression of canonical Notch pathway genes for up to 96 hours. In addition, treatment with a GSI results in various biological changes in PHFs, including early downregulation of PI3K pathway and proliferation genes, and a late (48h) upregulation of other signaling pathways that are potentially relevant to cancer cell proliferation. This novel plucked hair follicle biomarker provides important insights into the effect of GSIs on cell signaling, and also facilitates the development of gamma secretase targeted drugs for oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B41.

Abstract Abstract 1710 Poster Board I-736 Introduction Treatment regimens for cancer are typically based on cytotoxic chemotherapy, which is poorly tolerated. There is an unmet medical need for new therapies that retain efficacy, but combine this with an improved safety and tolerability profile. Vorinostat is a histone deacetylase (HDAC) inhibitor, approved in the United States for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma (CTCL) in patients who have progressive, persistent, or recurrent disease on or following 2 systemic therapies. Vorinostat is also being investigated as a treatment for various other solid and hematologic malignancies, in which HDACs are further implicated as key regulators of transcription. Herein we present an overview of the safety and tolerability profile of vorinostat, gathered from prior clinical experience. Methods Safety and tolerability data, including adverse events (AEs), QTc interval data and incidence of thromboembolic events (TEE), were collated from patients who received vorinostat, administered as monotherapy or in combination therapy for solid or hematologic malignancies. Results (adverse events) In Phase I and II clinical trials, 498 patients who received vorinostat were analyzed. A total of 341 patients received vorinostat monotherapy (107 with CTCL, 105 other hematologic malignancies, 129 solid tumors) and the most common drug-related AEs in this group were: fatigue (61.9%), nausea (55.7%), diarrhea (49.3%), anorexia (48.1%), and vomiting (32.8%); Grade 3/4 AEs included fatigue (12.0%) and thrombocytopenia (10.6%), and 3 drug-related deaths (ischemic stroke, tumor hemorrhage, unspecified) occurred. Thirty-eight patients (11.1%) discontinued due to drug-related AEs, 71 patients (20.8%) required dose modifications, and 1 patient (0.3%) discontinued due to Grade 2 chest pain. The remaining 157 patients received vorinostat combination therapy (with pemetrexed/cisplatin for advanced cancer [n=46], bortezomib for multiple myeloma [n=34], bexarotene for CTCL [n=23], and erlotinib [n=30], gemcitabine/platin [n=21] or carboplatin/paclitaxel [n=3] for non-small-cell lung cancer). The most common drug-related AEs in this group were: nausea (48.4%), diarrhea (40.8%), fatigue (34.4%), and vomiting (31.2%); the most common Grade 3/4 AE was fatigue (13.4%), and 1 drug-related death (hemoptysis) occurred. Thirty-one patients (19.7%) discontinued due to drug-related AEs and 27 patients (17.2%) required dose modifications. Results (QTcF interval) A trial of 24 patients with advanced cancer was undertaken for rigorous assessment of QTcF interval. In this trial, a single supratherapeutic 800 mg dose of vorinostat did not prolong QTcF interval (monitored over 24 hours). The upper limit of the 90% confidence interval for the placebo-adjusted mean change-from-baseline of vorinostat was &lt;10 msec at every timepoint, no patient had a QTcF change-from-baseline value &gt;30 msec, and 1 patient had a QTcF interval &gt;450 msec (after both vorinostat and placebo administration). The most common drug-related AE in this trial was nausea. There were no serious clinical or laboratory AEs, no discontinuations due to an AE and no patients experienced a cardiac-related AE. Results (incidence of TEE) A review of vorinostat clinical trials, published literature and post-marketing surveillance reports was conducted by a committee of independent academic experts to determine the incidence of TEE in cancer patients who had received vorinostat. In &gt;1845 patients reviewed through November 3, 2008, 107 patients (&lt;5.8%) reported TEE as a serious AE (SAE), 47 (&lt;2.6%) of which were recorded as being related to vorinostat, and 4/47 (&lt;0.3%) TEE SAEs were fatal. Conclusions In this review, the majority of observed AEs were 'Grade 2, there was no observed prolongation of the QTcF interval, and the incidence of TEE with vorinostat was similar to reported rates of TEE in advanced cancer patients. Vorinostat is generally well tolerated when administered as monotherapy or in a combination regimen in cancer patients. Disclosures Siegel: Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celegne: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rubin:Merck: Employment, Equity Ownership. Iwamoto:Merck: Employment, Equity Ownership. Hussein:Celgene: Employment. Belani:Merck: Consultancy. Hardwick:Merck: Employment, Equity Ownership. Rizvi:Merck: Employment, Equity Ownership.

&lt;div&gt;Abstract&lt;p&gt;NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in &lt;i&gt;NOTCH1&lt;/i&gt;. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the &lt;i&gt;NOTCH1&lt;/i&gt; mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19&lt;sup&gt;INK4d&lt;/sup&gt;) and CDKN1B (p27&lt;sup&gt;Kip1&lt;/sup&gt;), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL. [Cancer Res 2009;69(7):3060–8]&lt;/p&gt;&lt;/div&gt;

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

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Supplementary Table S1 from Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

Supplementary Table S1 from Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Insulin-like growth factor-1 receptor (IGF-1R) mediates cellular processes in cancer and has been proposed as a therapeutic target. Dalotuzumab (MK-0646) is a humanized IgG1 monoclonal antibody that binds to IGF-1R preventing receptor activation. This study was designed to evaluate the safety and tolerability of dalotuzumab, determine the pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and identify a recommended phase II dose.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Patients with tumors expressing IGF-1R protein were allocated to dose-escalating cohorts of three or more patients each and received intravenous dalotuzumab weekly, every 2 or 3 weeks. Plasma was collected for PK analysis. Paired baseline and on-treatment skin and tumor biopsy samples were collected for PD analyses.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Eighty patients with chemotherapy-refractory solid tumors were enrolled. One dose-limiting toxicity was noted, but a maximum-tolerated dose was not identified. Grade 1 to 3 hyperglycemia, responsive to metformin, occurred in 15 (19%) patients. At dose levels or more than 5 mg/kg, dalotuzumab mean terminal half-life was 95 hours or more, mean &lt;i&gt;C&lt;/i&gt;&lt;sub&gt;min&lt;/sub&gt; was more than 25 μg/mL, clearance was constant, and serum exposures were approximately dose proportional. Decreases in tumor IGF-1R, downstream receptor signaling, and Ki67 expression were observed. &lt;sup&gt;18&lt;/sup&gt;F-Fluorodeoxy-glucose positron emission tomography metabolic responses occurred in three patients. One patient with Ewing's sarcoma showed a mixed radiologic response. The recommended phase II doses were 10, 20, and 30 mg/kg for the weekly, every other week, and every third week schedules, respectively.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Dalotuzumab was generally well-tolerated, exhibited dose-proportional PK, inhibited IGF-1R pathway signaling and cell proliferation in treated tumors, and showed clinical activity. The low clearance rate and long terminal half-life support more extended dosing intervals. &lt;i&gt;Clin Cancer Res; 17(19); 6304–12. ©2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Insulin-like growth factor-1 receptor (IGF-1R) mediates cellular processes in cancer and has been proposed as a therapeutic target. Dalotuzumab (MK-0646) is a humanized IgG1 monoclonal antibody that binds to IGF-1R preventing receptor activation. This study was designed to evaluate the safety and tolerability of dalotuzumab, determine the pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and identify a recommended phase II dose.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Patients with tumors expressing IGF-1R protein were allocated to dose-escalating cohorts of three or more patients each and received intravenous dalotuzumab weekly, every 2 or 3 weeks. Plasma was collected for PK analysis. Paired baseline and on-treatment skin and tumor biopsy samples were collected for PD analyses.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Eighty patients with chemotherapy-refractory solid tumors were enrolled. One dose-limiting toxicity was noted, but a maximum-tolerated dose was not identified. Grade 1 to 3 hyperglycemia, responsive to metformin, occurred in 15 (19%) patients. At dose levels or more than 5 mg/kg, dalotuzumab mean terminal half-life was 95 hours or more, mean &lt;i&gt;C&lt;/i&gt;&lt;sub&gt;min&lt;/sub&gt; was more than 25 μg/mL, clearance was constant, and serum exposures were approximately dose proportional. Decreases in tumor IGF-1R, downstream receptor signaling, and Ki67 expression were observed. &lt;sup&gt;18&lt;/sup&gt;F-Fluorodeoxy-glucose positron emission tomography metabolic responses occurred in three patients. One patient with Ewing's sarcoma showed a mixed radiologic response. The recommended phase II doses were 10, 20, and 30 mg/kg for the weekly, every other week, and every third week schedules, respectively.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Dalotuzumab was generally well-tolerated, exhibited dose-proportional PK, inhibited IGF-1R pathway signaling and cell proliferation in treated tumors, and showed clinical activity. The low clearance rate and long terminal half-life support more extended dosing intervals. &lt;i&gt;Clin Cancer Res; 17(19); 6304–12. ©2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

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&lt;p&gt;Allred proportion (ranging from 0-5) and intensity (ranging from 0-3) scores for each tumor. The median score among triplicate TMA cores and reviewers was utilized. Most MCC tumors with an Allred score of 7 continued to express MHC class I in all tumor cells, however the intensity of expression was significantly reduced as compared to those with an Allred score of 8.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total &lt;i&gt;n&lt;/i&gt; = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (&lt;i&gt;P&lt;/i&gt; &lt; 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression &lt;i&gt;in vitro&lt;/i&gt;, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. &lt;i&gt;Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Allred proportion (ranging from 0-5) and intensity (ranging from 0-3) scores for each tumor. The median score among triplicate TMA cores and reviewers was utilized. Most MCC tumors with an Allred score of 7 continued to express MHC class I in all tumor cells, however the intensity of expression was significantly reduced as compared to those with an Allred score of 8.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in &lt;i&gt;NOTCH1&lt;/i&gt;. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the &lt;i&gt;NOTCH1&lt;/i&gt; mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19&lt;sup&gt;INK4d&lt;/sup&gt;) and CDKN1B (p27&lt;sup&gt;Kip1&lt;/sup&gt;), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL. [Cancer Res 2009;69(7):3060–8]&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total &lt;i&gt;n&lt;/i&gt; = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (&lt;i&gt;P&lt;/i&gt; &lt; 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression &lt;i&gt;in vitro&lt;/i&gt;, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. &lt;i&gt;Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Abstract Background: The Hedgehog (Hh) signaling pathway is key to development, differentiation, and stem cell maintenance. In cancer, dysregulation of Hh signaling is associated with solid tumors and hematological malignancies. Hh signaling in AML and MDS is thought to promote the renewal and maintenance of leukemic stem cells, which may lead to chemotherapy resistance and recurrence. Glasdegib is a potent and selective oral inhibitor of Smoothened, a transmembrane protein that stimulates Hh signaling. Here we report the results of an exploratory analysis to profile mutations in 118 bone marrow samples and 77 peripheral blood samples of patients with previously untreated AML or high-risk MDS treated with chemotherapy with or without glasdegib in phase I/II clinical trials. Materials and Methods: We used targeted resequencing to survey 109 genes, and a polymerase chain reaction assay to detect FLT3 internal tandem duplications. Resequencing and response data were obtained from two arms, 1) an intensive group (n=61) treated with a combination of cytarabine/daunorubicin plus glasdegib, and 2) a non-intensive group (n=73) treated with low-dose cytarabine (LDAC) plus glasdegib (n=44) or LDAC alone (n=19). We assessed significantly mutated genes by Fisher's Exact Test at p ≤ 0.05. Results: In the combined dataset we found that mutations in NF1 (P=0.006), KRAS (P=0.028), or a combination of KRAS, NRAS, and NF1 (P=0.013) were associated with a negative response. Mutations in NPM1 (P=0.006), MPL (P=0.041), RB1 (P=0.041), or SF3B1 (P=0.041) were associated with a positive response. When split by arm, in the intensive group mutations in TP53 (P=0.001), NF1 (P=0.035), or CREBBP (P=0.041) were associated with negative response, whereas mutation in NPM1 was associated with positive response (P=0.046). In the non-intensive group, mutations in RSPH10B2 (P=0.036) or a combination of KRAS, NRAS, and NF1 (P=0.031) were associated with negative response, whereas mutation in CREBBP (P=0.007) was associated with positive response. We discuss the implications of these mutations in the context of Smoothened inhibition and the leukemic stem cell hypothesis. Conclusions: Mutational profiling of patients with AML or high-risk MDS treated with glasdegib highlights alterations in genes associated with response and the potential role of Hh signaling in these malignancies. Citation Format: Keith A. Ching, Donghui Huang, Kai Wang, Mark Ozeck, Paul Lira, Jingjin Gao, Jadwiga Bienkowska, Paul Rejto, James Hardwick, Thomas O'Brien, Geoffrey Chan. Analysis of mutations associated with response to glasdegib in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-215.

Abstract Vorinostat is a histone deacetylase inhibitor with potent in vitro and in vivo anti-leukemia activity. Several gene expression signatures were developed from preclinical models as biomarkers for clinical response to vorinostat. The signatures were evaluated in peripheral blood mononuclear cell (PBMC) samples from a Phase I study of oral vorinostat in patients with advanced leukemias or myelodysplastic syndromes (MDS). Of 41 registered patients, 31 had acute myeloid leukemia (AML), 4 had chronic lymphocytic leukemia, 3 had MDS, 2 had acute lymphoblastic leukemia, and 1 had chronic myeloid leukemia. Seven of 41 patients showed improvement with treatment, 2 complete responses, 2 complete responses that were not maintained for 28 days, and 3 with &gt;50% decreases in blast counts without count recovery. All responses occurred in patients with AML at or below the MTD. The following three clinical gene expression signatures were shown to be predictive of clinical response in PBMC samples of AML patients. First, a positive prediction trend was observed when a preclinical 40-gene resistance prediction signature was evaluated in baseline (pre-dose and first day of dosing) AML PBMC samples (8 responder and 21 non-responder samples) or all PBMC samples (14 responder and 56 non-responder samples, including non-AML patients). If this trend is maintained in pretreatment samples of a larger patient population, it may allow for exclusion of up to 50% of non-responders from future clinical trials. Second, a similar prediction of resistance was obtained based on expression levels of known reactive oxygen species scavengers that were upregulated in non-responders relative to responders, indicating that cellular ability to withstand oxidative stress may be protective with respect to vorinostat exposure. This supports the hypotheses that oxidative stress pathways play an important role in the response to vorinostat. Finally, overexpression of p21 and several p53-responsive genes was also associated with resistance, consistent with preclinical observations. These results are being validated at the protein level in a tissue array platform. Post-dose downregulation of genes associated with cellular proliferation was also observed in responders but not in non-responder PBMC samples. This suggests that proliferation may be used as a post-dose efficacy biomarker. Further evaluation of vorinostat in patients with leukemias and MDS, including analysis of a potentially predictive gene expression signature for response to vorinostat, is warranted.

Abstract Liquid biopsies have the potential to revolutionize the way physicians select personalized anti-cancer therapies, monitor patient responses to treatment, and characterize acquired resistance to cancer drugs. New tests that use a simple peripheral blood draw offer snapshots of a patient‘s total tumor DNA mutation profile and are attractive because of their minimally-invasive modality and because they integrate information from both primary and metastatic disease. Currently, most plasma cell-free DNA (cfDNA) mutation detection tests used in clinical research detect known hotspot mutations in a limited number of genes. Technologies that interrogate multi-gene panels in cfDNA are advancing, but commercially-available options suitable for clinical use are limited, come at a high cost, and are not customizable. We designed and developed a customized, next generation sequencing-based, liquid biopsy assay capable of detecting somatic mutations in 87 breast cancer genes involved in cell cycle and estrogen receptor signaling. Targeted regions (147 Kb) were enriched using hybrid capture resulting in an average capture specificity and uniformity of 65.93% and 96.38%, respectively. When tested on cfDNA from healthy donors (n=14), we demonstrated a level of specificity &amp;gt;99.99%. Analytical sensitivity of 0.1% was established on HapMap and reference mutant cell line DNA. Using a pool of HapMap genomic DNA (n=10), 96% (48/50) of SNPs with expected allele frequency of 0.1% were detected. In reference mutant cell line DNA with 1% or 0.1% mutant allele frequencies, we were able to reliably detect all mutations present at 1% and mutations at 0.1% in 50% of the cases. Assay validation on plasma cfDNA with matching tumor from ER+, HER2- breast cancer patients will be presented. In conclusion, we developed a highly sensitive and specific liquid biopsy assay to interrogate 87 breast cancer-related genes. The high level of specificity and sensitivity makes the test ideal not only for detecting known cancer gene hotspot mutations but also for detection of novel gene mutations that may arise during treatment as a result of acquired drug resistance. Citation Format: Maruja E. Lira, Tao Xie, Shibing Deng, Jennifer Kinong, Jingjin Gao, Zhou Zhu, Nathan Lee, Paul Rejto, Jadwiga Bienkowska, James Hardwick, Kai Wang, Stephen Huang. Liquid biopsy testing allows highly-sensitive detection of plasma cfDNA mutations in 87 breast cancer-related genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2017-2749

Abstract The use of ultra-deep, next generation sequencing of circulating tumor DNA (ctDNA) holds great promise for early detection of cancer as well as a tool for monitoring disease progression and therapeutic responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection. To achieve high accuracy of variant calling via better distinguishing low frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. It significantly enhances the specificity for downstream ctDNA mutation detection without sacrificing sensitivity. Results on both simulated and real healthy subjects’ data demonstrate that the proposed algorithm consistently outperforms a current, state of the art, position-specific error polishing model, particularly when the sample size of healthy subjects is small. TNER is publicly available at https://github.com/ctDNA/TNER .

The transcriptional profile of Saccharomyces cerevisiae exposed to rapamycin mimics the profile induced by amino acid starvation

Abstract Vorinostat is a histone deacetylase inhibitor currently under evaluation in numerous oncology clinical trials. In a Phase IIb trial, oral vorinostat resulted in a 29.7% overall objective response rate in patients (pts) with advanced cutaneous T-cell lymphoma (CTCL) and had an acceptable safety profile. These results prompted efforts to identify gene expression patterns that could elucidate the molecular mechanism of action (MOA), assess exposure to vorinostat and enrich for pts who are likely to respond. In the Phase IIb trial, gene expression profiles were obtained from 24 predose and 30 postdose (2 hr postdose on Day 15) PBMC samples. The gene expression associated with Sezary burden was easily identified in predose samples and consistent with published results. Although the power of this dataset was limited for development of a predose predictor of response, we identified three biologically-relevant pathways that correlated with response and deserve further validation. First, we found a coherent cluster of proliferation/cell cycle genes to be associated with resistance to therapy. This may imply that tumor aggressiveness is an important factor for clinical response. Second, a set of antioxidant genes was upregulated in non-responders. The generation of reactive oxygen species (ROS) is a component of the vorinostat MOA and increased ROS scavenging ability may confer resistance. Finally, cytotoxic cell markers were upregulated in responders and may represent another factor associated with contribution of T and NK cells to response. Each of these 3 patterns, if confirmed, would allow for 20–50% responder enrichment. We observed robust postdose gene expression changes in which ~942 genes exhibited significant regulation (fold-change&amp;gt;2, P&amp;lt;0.01 by paired t-test between predose and postdose samples) regardless of clinical outcome. Treated samples were discriminated from untreated with 87.5% accuracy based on leave one-out-cross-validation (LOOCV) using penalized analysis of microarrays (PAM). To understand the biology, we projected the preclinical postdose signatures derived from acute postdose changes in a panel of human lymphoid cell lines. Overall, 85% of genes significantly regulated by vorinostat in lymphoid cell lines were also regulated in the same direction in PBMC samples from CTCL pts. Thus, most of the observed postdose changes result from acute vorinostat effects on gene expression. The average preclinical postdose signature can be used to predict proximal vorinostat exposure with 90% accuracy. Among the gene expression signatures observed in clinical samples but not in cell lines, two deserve special attention. First, proliferation-associated genes are downregulated postdose and are differentially expressed between responders and non-responders. It may serve as an efficacy biomarker and would allow for 80% accurate discrimination of responders from non-responders in postdose samples based on LOOCV using PAM. Second, cytokines and genes associated with the humoral immune response were downregulated at the same time genes and cytokines associated with a cytotoxic immune response were upregulated. Such changes in the Th1-Th2 balance may reflect part of the MOA for vorinostat, and may be particularly relevant to CTCL, a disease caused by Th2 type skin-homing lymphocytes. Further evaluation of vorinostat in CTCL, including additional validation of gene expression signatures that may predict response, is warranted.

Abstract Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC). Despite recent development of effective targeted therapeutic agents for lung adenocarcinoma, patients with SqCLC often receive conventional cytotoxic chemotherapy as this cancer subtype lacks genomic alterations that can be targeted by personalized medicine. Hence, novel approaches that enhance the efficacy of chemotherapy will benefit treatment outcomes in this patient population. CDK inhibitors comprise a class of drugs that targets the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial data, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. In this preclinical study, we explored the effect of palbociclib on several chemotherapies (taxanes, platins, and antimetabolites) in preclinical models of SqCLC. Because the activity of chemotherapy generally requires cell cycle progression, careful combination/sequencing of this class of drugs with CDK inhibitors may be important to achieve synergy as well as avoid potential antagonism. To obtain optimal activity of palbociclib and chemotherapy combinations, we investigated several combination/sequencing regimens (concurrent, chemotherapy followed by palbociclib or the reverse sequence) in several SqCLC cell lines. We did not encounter antagonism of chemotherapy-mediated cytotoxicity by palbociclib in any of the tested regimens. Rather, we observed robust combinatorial anti-cancer cell activity in all settings. Combination of palbociclib with chemotherapy was associated with reduction of RB phosphorylation and FOXM1 protein levels, and the induction of p21. Our studies demonstrated that, while palbociclib partially antagonized chemotherapy-induced apoptosis, it significantly synergized with chemotherapy to induce cell cycle arrest as well as a senescence-like phenotype. Cells pretreated with palbociclib plus cisplatin or palbociclib plus docetaxel displayed less cell growth upon drug removal compared to those treated with monotherapies. Finally, palbociclib treatment that followed docetaxel, nab-paclitaxel or cisplatin treatment significantly enhanced the antitumor activity of the chemotherapies in several cell line-derived or patient-derived xenograft models. Our results suggest that treatment with optimal palbociclib and chemotherapy combination/sequencing could lead to better clinical outcomes for SqCLC patients. Citation Format: Ping Wei, Joan Cao, Goldie Lui, Hui Wang, Konstantinos Tsaparikos, David Shields, Kim Arndt, Paul Rejto, Todd VanArsdale, James Hardwick. RB pathway disruption by the CDK4/6 inhibitor palbociclib enhances responses to chemotherapy in squamous cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A43.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a 5 year survival rate of less than 5%. Deaths caused by pancreatic cancer are projected to exceed the number from colorectal carcinoma by 2020, making PDAC the second leading cause of cancer-related death in the United States, behind only NSCLC. At the molecular level, PDAC is enriched for a number of genetic events central to CDK4/6:CyclinD1 control of cell cycle progression - 90% of tumors harbor oncogenic KRAS mutations, which are synthetic lethal with CDK4/6 inhibition, while the majority of PDAC cases also feature loss of p16INK4A, the endogenous inhibitor of CDK4/6. Rb loss is uncommon in PDAC and phosphorylation of Rb, the canonical CDK4/6 substrate, is detectable at high frequencies, suggesting that aberrant CDK4/6 signaling may be central to loss of cell cycle control in pancreatic cancer. To determine the significance of CDK4/6 activity in pancreatic cancer, patient-derived xenograft models of early and late stage disease were used to evaluate the selective CDK4/6 inhibitor, palbociclib. Combinatorial efficacy of palbociclib with the standard of care agents, Gemcitabine and nab-paclitaxel (Gem/nab-Pac) was also assessed. The majority of models display greater than 50% tumor growth inhibition following treatment with single agent palbociclib, which is comparable to the response to Gem/nab-Pac in those models. Addition of palbociclib to Gem/nab-Pac confers further benefit in most models by increasing the degree of tumor response on therapy and/or maintaining tumor response after drug removal. To dissect the driver activities in the triplet combination, palbociclib was assessed in combination with either Gem or nab-Pac in a series of tumor growth inhibition/delay studies. Palbociclib and nab-Pac are the dominant agents in the combination - palbociclib/nab-Pac doublet activity surpasses the anti-tumor effects of Gem/nab-Pac in the majority of models, while palbociclib/nab-Pac is equivalent or superior to the triple combination in all models. In contrast, the addition of palbociclib to Gem yields little signs of positive combinatorial activity. Based on these data, the palbociclib/nab-Pac combination will be evaluated in an upcoming clinical trial for PDAC patients. Ongoing pre-clinical studies are focused on the mechanistic and molecular basis for the robust activity of the palbociclib/nab-Pac combination in pancreatic cancer. Citation Format: Manuel Hidalgo, Camino Menendez, Jing Yuan, Beatriz Salvador, Tao Xie, John Chionis, Pedro Lopez-Casas, Xianxian Zheng, James Hardwick, Paul Rejto, Peter Olson, Todd VanArsdale, David J. Shields. Palbociclib potentiates nab-paclitaxel efficacy in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A42.

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy with a high propensity for early metastasis. Response rates to first-line chemotherapy are typically high, but short lived. We describe an epigenetic-based mechanism for targeting SCLC using inhibitors for the LSD1 histone lysine demethylase. Mechanistically reversible and irreversible LSD1 inhibitors (GSK690 and OG-86) demonstrate induction of either cell cycle arrest or apoptosis in 54% of SCLC cell lines (12/22) that becomes apparent upon continuous treatment for 7-to-10 days. Maximal rates of growth inhibition in sensitive cell lines vary from 50-to-95% and plateaus between 18-to-21 days of LSD1 inhibitor treatment. Thus, heterogeneous sensitivity to LSD1 inhibitors exists between cell line models as well as within subpopulations of cells in the same cell line. To understand the mechanisms underlying LSD1 inhibitor activity in SCLC we have performed RNA-seq and ChIP-seq experiments coupled with bioinformatic analysis of expression signatures in sensitive and resistant models. Our data indicate that although LSD1 is over-expressed in SCLC cell lines and patient samples relative to non-small cell lung cancers, high LSD1 expression does not predict sensitivity to LSD1 inhibitors. Pathway analysis demonstrates that LSD1 inhibition modulates the expression of genes involved in cell adhesion and axon guidance including members of the Ephrin and Semaphorins families. At LSD1 target genes, we demonstrate site-specific H3K4me2 histone methylation changes overlapping LSD1 binding sites, however no global changes in H3K4me2 were observed. Interestingly we find morphological and cell adhesion changes in sensitive cell lines that coincide with expression changes in markers of neuroendocrine differentiation of SCLC such as GRP, NCAM, and NEUROD1. Based on these data, we propose a model that LSD1 inhibition modulates the neuroendocrine differentiation program of SCLC cells promoting tumor inhibition in sensitive SCLC models. Citation Format: Thomas A. Paul, Shikhar Sharma, Jill Hallin, Tao Xie, Timothy Nichols, Mike Greig, James Hardwick, Martin Wythes, Dominique Verhelle. Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2015-3518

Abstract Patient-Derived Xenograft (PDX) provides important preclinical model for pharmacological testing of oncology drug candidates. Molecular profiling of PDX tumors contributes to many areas of drug discovery from target discovery to development of clinical biomarker hypotheses and clinical trial design. We established a work flow to perform genomic and histopathology analyses of large numbers of PDX tumor models being made available for Pfizer internal research. To date we have generated whole-genome sequencing (WGS), whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-Seq) data on PDX models spanning six cancer types including colon, pancreatic and breast cancers. Bioinformatics pipelines were developed to quantify gene expression and detect genetic alterations including mutation, copy number variations and gene fusions. A controlled evaluation study demonstrated that in silico classification of NGS reads into human/mouse origins is more effective than laboratory-based methods for removing mouse tissue contamination. Comparative analyses of molecular profiles from PDX and primary tumors of the same cancer origins suggest that important patterns of gene expression are retained by PDX models. An integrative genomic classifier was developed using the random forest algorithm, trained on primary tumor data, and shown to identify PDX cancer subtypes with high accuracy. Citation Format: Zhengyan Kan, Edward Rosfjord, James Hardwick, Ying Ding, Xianxian Zheng, Julio Fernandez, Stephanie Shi, Mark Ozeck, Hui Wang, Gabriel Troche, Eric Upeslacis, Amy Jackson-Fisher, Keith Ching, Shibing Deng, Xie Tao, John Chionis, Maruja Lira, Xiaorong Li, Konstantinos Tsaparikos, Patrick Lappin, Pamela Vizcarra, David Shields, Judy Lucas, Paul Rejto. Molecular profiling of patient-derived xenograft models across cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-33.

Abstract Cellular proliferation is dependent on an orderly movement through the various phases of cell cycle. Progression through the G1 phase in particular requires phosphorylation of the retinoblastoma (Rb) protein which in turn releases E2F transcription factors resulting in transcriptional activation of response gene necessary for progression into S-phase. The cyclin D-CDK4/6 signaling pathway represent a critical regulatory pathway controlling transition from G1 into S-phase and greater than 90% of human tumors have mutations in this pathway. Palbociclib is a potent, selective, and orally bioavailable inhibitor of Cdk4/6. In human tumor xenograft models, Palbociclib has significant antitumor activities. The clinical activities of Palbociclib have also be demonstrated in the phase II PALOMA-1 trial for dramatic efficacy of postmenopausal patients with locally advanced or newly diagnosed estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer in combination with letrozole. To better understand the molecular mechanisms of Palbociclib response, we have identified a common set of gene signatures for ER+ BC as well as melanoma models. The physiological role of these genes regulated by Palbociclib is associated with DNA replication and repair, cell cycle, signal transduction, and Mitosis. Many of these genes have been previously identified as E2F signatures. In this study, we are aiming to expand this analysis to include two additional indications including Head and Neck squamous cell carcinoma (HNSCC) as well as squamous cell lung carcinoma (Sq lung) using Illumina RNASeq technology platform. Results from these studies will be presented and core Palbo-response signature will be discussed. In addition, we will also present the pharmacological effects of Letrozole on the gene expression changes for these core signatures in ER+ BC models. Citation Format: Xianxian Zheng, Mark Ozeck, Zhou Zhu, Keith Ching, David Shields, James Hardwick, Paul Rejto, Todd VanArsdale. Identification of Palbociclib response signature across indications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 940. doi:10.1158/1538-7445.AM2015-940

Abstract Clinical development of cancer therapies is associated with attrition rates as high as 80-95%. This high attrition suggests that standard preclinical pharmacology models do not accurately reflect clinical responses. The development of more predictive preclinical models requires several considerations; the relevance of the in vivo model, the administration of test agent, and the interpretation of efficacy data. PDX are cancer models developed from the direct transfer of patient tumor tissue into immunocompromised mice. A collection of PDX models, by retaining the genetic and histologic characteristics of the patients from which they were derived, represents the complexity and heterogeneity of human cancer. To minimize the clinical attrition rates of oncology compounds, we are developing hundreds of PDX models in seven major cancer indications. The collection is being molecularly profiled by RNAseq, WES, and proteomics. Profiling has identified models with robust expression of target proteins or mutant oncogenes that are likely to respond in preclinical efficacy tests. Conversely, the PDX models may provide an understanding of resistance, for example evaluating models with good target expression that fail to respond to therapy. Patient and tumor information, if known, has been collected for each PDX model including age, sex, cancer stage and grade, diagnosis, primary or metastatic site, and prior treatments. In addition to the improvements provided by the PDX models, a preclinical paradigm shift away from treatment with maximally tolerated dose towards clinically relevant dose (CRD), taking into consideration such aspects as exposure, formulation, route and schedule, is critical when attempting to predict clinical outcome from preclinical data. Also essential is the incorporation of clinically meaningful endpoints (regression) when assessing preclinical activity. We have initiated studies on cohorts of non small cell lung and breast PDX models to predict the likely clinical efficacy of candidate compounds for clinical development and to determine the CRD for standard of care (SOC) regimens required to define the most promising Phase II/III combination therapies. Anti-tumor activities were characterized using RECIST criteria of progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR). Target expression was evaluated by RNA, proteomics and immunohistochemistry. Preliminary results demonstrate a spectrum of responses against experimental therapeutics, including Phase I ADCs and are defining the CRD required for combination treatments with SOC. Identification of the most critical parameters of PDX models predicting clinical outcome will help in validating the utility of ‘n of 1′ studies with the PDX collection, inform patient enrollment strategies, guide combination therapies, and provide insight for identifying new tumor indications. Citation Format: Edward Rosfjord, Xin Han, Danielle Leahy, Erik Upeslacis, Justin Lucas, Jonathon Golas, Andrea Hooper, Fred Immermann, Bingwen Lu, Jeremy Myers, Zhengyan Kan, James Hardwick, Eric Powell, Puja Sapra, Paul Rejto, Hans-Peter Gerber, Judy Lucas. Patient derived xenograft (PDX) models: improving predictability of experimental cancer therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2015-1469

Abstract Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole genome sequencing (WGS) study of 88 matched HCC tumour/normal pairs, 81 of which are HBV positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find β-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumour suppressor (35.2%). The Wnt/β-catenin pathway, altered in 62.5% of cases, is likely to act as the major oncogenic driver in HCC. TP53 alterations appear to cause increased levels of genomic arrangement and chromosomal instability. We identified chromothripsis in 5 HCC genomes (5.7%) recurrently affecting chromosomal arms 1q and 8q. We also identified recurrent HBV integration events at the known and putative cancer-related genes such as TERT, MLL4 and CCNE1, which showed upregulated gene expression in tumour versus normal tissue. The frequently altered genes and pathways in HCC reflect classical cancer hallmarks. This study identified several prevalent and actionable mutations that provide a path towards therapeutic intervention of the disease. Citation Format: Mao Mao, Hancheng Zheng, Zhengyan Kan, Jiangchun Xu, Xiao Liu, Shuyu Li, Thomas Barber, Zhuolin Gong, Huan Gao, Ke Hao, Melinda Willard, Robert Hauptschein, Paul Rejto, Julio Fernandez, Guan Wang, Qinghui Zhang, Bo Wang, Ronghua Chen, Jian Wang, Nikki Lee, Wei Zhou, Zhao Lin, Zhiyu Peng, Kang Yi, Shengpei Chen, Lin Li, Xiaomei Fan, Jie Yang, Rui Ye, Jia Ju, Kai Wang, Heather Estrella, Shibing Deng, Ping Wei, Ming Qiu, Isabella Wulur, Jiangang Liu, Mariam Ehsani, Chunsheng Zhang, Andrey Loboda, Wing Kin Sung, Amit Aggarwal, Ronnie Poon, Sheung Tat Fan, Jun Wang, James Hardwick, Christoph Reinhard, Hongyue Dai, Yingrui Li, John Luk. Whole genome sequencing reveals genetic landscape of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2013-LB-229

Abstract There is a large effort in the public domain to systematically perform DNA and RNA sequencing on large numbers of tumor samples. These efforts will bring us to a greater understanding of tumor biology and lead to identification of new tumor drivers. However, these efforts may fall short in allowing us to understand the progression of tumor resistance, relapse and metastasis, factors which make tumors difficult to treat and increase mortality rates. We are currently performing targeted DNA sequencing on 4000 tumor samples. We have selected 1321 genes, 5 GB of the genome to sequence. Genes were selected through a thorough review of the literature, mutation databases and key pathways in tumorigenesis such as growth factor signaling, DNA damage, p53 signaling, cell cycle and apoptosis. Our effort not only includes a diverse panel of breast, colon, ovarian, and kidney tumors but we have also sequenced pancreatic, liver, esophageal, cervix, endometrial, melanoma, CLL, DLBCL and carcinoid tumors. Our dataset also includes approximately 300 matched tumor-metastatic pairs and an additional 550 metastatic samples. Clinical phenotypes, treatment information, gene expression profiling and copy number variation data is available for all samples. To achieve the next level of care for cancer patients we must understand the biology of the tumors we are trying to treat, which are often metastatic and standard of care resistant. We believe that increasing our efforts in targeted DNA sequencing of key tumor and metastatic samples will allow us to achieve these goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 435. doi:1538-7445.AM2012-435

Introduction The incidence of oesophageal and junctional adenocarcinoma has increased sixfold in the last 30 years and 5-year survival remains Methods Gene expression profiling was performed and the resulting 42 000 gene signatures correlated with clinical features for 91 snap frozen oesophageal and junctional resection specimens. External validation of selected targets was carried out on 371 independent cases using immunohistochemistry to maximise clinical applicability. Results Gene expression profiles were obtained from 75/91 of the samples (82%). 119 genes were significantly associated with survival and 270 genes with the number of involved lymph nodes. Ten of these genes were taken forward to external validation at the protein level. Three genes were prognostic in the external validation data set (TRIM44, SIRT2 and PAPSS2). Patients with dysregulation of none of these three genes had a significantly better outcome (5-year survival 48%) than those with dysregulation of one gene (5-year survival 29%), who in turn did better than those with two or more genes dysregulated (5-year survival 15%) (p=0.004). The three gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (p=0.003). Conclusion We have generated and externally validated an immunohistochemical prognostic gene signature which is independently highly prognostic in an external validation data set. In addition these three genes are possible therapeutic targets raising the possibility of personalised therapy based on a patient9s molecular prognostic signature. Our three gene signature is suitable for application in routine clinical practice and this study may provide a framework for similar future projects.

Abstract Introduction: Gastro-esophageal adenocarcinomas are rapidly increasing in incidence and have a five year survival of less than 14%. Analysis of signaling pathways in gastro-esophageal adenocarcinomas can be used as a route to identifying possible therapeutic targets. Aims and methods: We had already identified MAPK and Wnt pathways as important in this disease. The aim of this work was to identify biomarkers of pathway activation for future application to primary tumors for selection of targeted therapies. Three cell lines (CP-B, CP-C, CP-D) without constitutive activation were stimulated with exogenous ligands for the Wnt or MAPK or pathways. The TGFβ pathway was also stimulated as a negative control to test biomarker specificity. The expression of potential biomarkers of transcriptional activity was then profiled using real time PCR. To determine applicability of these markers to human tissue the expression of these markers was assessed in an expression array data set consisting of 75 gastro-esophageal adenocarcinomas. Results: Out of 8 (MAPK) and 13 (Wnt) potential markers, three markers were selected as specific for the MAPK pathway (EGR-1, JUN and FOSL1) or the Wnt pathway (BMP4, TCF7, LEF1). All markers had upregulation of expression of at least 2 fold that were specific to activation of the relevant pathway (Table 1).Table 1.Fold changes of expression upon activation by stimulation with exogenous ligandsMarkerEGF (MAPK pathway)LiCl (Wnt pathway)TGFβ (TGFβ pathway)P-value (ANOVA)EGR-1130.50.91.3&amp;lt; 10-4JUN3.31.21.0&amp;lt; 10-4FOSL111.02.60.6&amp;lt; 10-4BMP42.18.31.5&amp;lt; 10-4LEF10.523.30.5&amp;lt; 10-4TCF72.44.80.7&amp;lt; 10-4 Next we assessed the expression of selected markers in 75 gastro-esophageal adenocarcinomas for which expression profiling data was available. In these tumors 27 (36%) had activation of the MAPK pathway alone, 10 (13%) had activation of the Wnt pathway alone, and a further 12 (16%) had activation of both pathways. Conclusions: We have determined a set of markers which can be used to assess pathway activation of the MAPK and Wnt pathways in gastro-esophageal adenocarcinomas. External validation of these pathway activation markers is ongoing using multiplex PCR as a prelude to a clinical trial of targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 789.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are ubiquitous in the environment. Humans are exposed to NDL-PCBs mainly via food. Exposure to NDL-PCBs is suspected to be of concern to human health. This chapter discusses the NDL-PCB sources, their environmental transport and occurrence in food items. Human groups with high exposure are addressed. Furthermore, this chapter deals with toxicokinetics and the toxicity profile of NDL-PCBs. It also provides information about the regulatory situation of NDL-PCBs, which could be useful, both for academic and government scientists. This information will also be of value for the food industry, policy makers and consumers.

Abstract Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC) and is responsible for ~100,000 deaths in the US and EU. Most SqCLC patients receive chemotherapy as 1st line treatments and have a high un-met medical need for new therapies. Therapeutic approaches that enhance the efficacy of chemotherapy would therefore improve clinical outcomes for this patient population. CDK inhibitors comprise a class of drugs that target the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial results, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. Like hormone receptor positive breast cancer patients, the vast majority of SqCLC patients harbor wild type RB and thus may also benefit from palbociclib treatment. Previously, we reported robust cytotoxicity and antitumor effects of palbociclib plus taxanes, including nanoparticle albumin-bound paclitaxel (Nab-PTX) or docetaxel (DTX), in several preclinical models of SqCLC. In the present study, we extended our efficacy studies of this combination to additional RB+ SqCLC models with diverse molecular genetic backgrounds. In search of mechanisms of action underlying the observed combinatorial effects, we identified several novel mechanisms, including cell cycle checkpoint abrogation as well as reduction of hypoxia-inducible factor 1 alpha (HIF-1α). Decrease in HIF-1α protein led to strong modulation of downstream genes involved in angiogenesis, resulting in reduced blood vessel size in tumor vasculature. Our results suggest that palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in SqCLC. Citation Format: Joan Cao, Zhou Zhu, Hui Wang, Tim Nichols, Edward Rosfjord, Christine Hopf, Erik Upeslacis, Paul Rejto, Scott Weinrich, Todd Vanarsdale, James Hardwick, Ping Wei. Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2355. doi:10.1158/1538-7445.AM2017-2355

<h3>Introduction</h3> The incidence of oesophageal and junctional adenocarcinoma has increased sixfold in the last 30 years and 5-year survival remains &lt;14%. Most patients present with advanced disease and current staging is limited in its ability to predict survival. We aimed to generate and externally validate a molecular prognostic signature for oesophageal adenocarcinoma. <h3>Methods</h3> Gene expression profiling was performed and the resulting 42 000 gene signatures correlated with clinical features for 91 snap frozen oesophageal and junctional resection specimens. External validation of selected targets was carried out on 371 independent cases using immunohistochemistry to maximise clinical applicability. <h3>Results</h3> Gene expression profiles were obtained from 75/91 of the samples (82%). 119 genes were significantly associated with survival and 270 genes with the number of involved lymph nodes. Ten of these genes were taken forward to external validation at the protein level. Three genes were prognostic in the external validation data set (TRIM44, SIRT2 and PAPSS2). Patients with dysregulation of none of these three genes had a significantly better outcome (5-year survival 48%) than those with dysregulation of one gene (5-year survival 29%), who in turn did better than those with two or more genes dysregulated (5-year survival 15%) (p=0.004). The three gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (p=0.003). <h3>Conclusion</h3> We have generated and externally validated an immunohistochemical prognostic gene signature which is independently highly prognostic in an external validation data set. In addition these three genes are possible therapeutic targets raising the possibility of personalised therapy based on a patient9s molecular prognostic signature. Our three gene signature is suitable for application in routine clinical practice and this study may provide a framework for similar future projects.

APCMin/+ genotypes when compared to the APCMin/+ mice.However, upon haplo-reduction of KLF5 (KRASV12G/APCMin/+/KLF5+/-), both tumor numbers and size are significantly lowered compared to both KRASV12G/APCMin/+ and APCMin/+mice.These results, along with the immunohistochemical data, suggest a crucial role played by KLF5 in mediating intestinal tumorigenesis in KRASV12G/APCMin/+ mice.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 694 Vorinostat is a histone deacetylase (HDAC) inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo . Vorinostat has demonstrated antitumor activity in both hematological malignancies and solid tumors, including an overall response rate of approximately 30% in cutaneous manifestations of cutaneous T-cell lymphoma (CTCL) in patients with progressive, persistent, or recurrent disease on or following 2 systemic therapies. As not all clinical responses are permanent, we developed model systems to investigate the mechanisms of acquired resistance to vorinostat and used these results to identify rational combination strategies to overcome this clinical issue. Using a dose escalation protocol vorinostat resistant clones were derived from sensitive HCT116 colon carcinoma cells and A549 non-small cell lung cancer (NSCLC) cells. This method established cell lines that can be maintained in media containing vorinostat at 2 μM (A549-VR2), 3μM (A549-VR3), 5 μM (A549-VR5) and 8 μM (HCT116-VR8) for a prolonged period of time (3 weeks) without signs of apoptosis. Gene expression profiling was performed on the parental HCT116 cells and vorinostat-resistant cells. In addition to an unbiased analysis of the data, the status of several pathways possibly related to vorinostat's mechanism of action were examined. Preliminary analyses revealed alterations in the expression of genes that participate in the apoptotic cascade (e.g. Bcl-2, survivin), protein acetylation (CBP/p300), redox homeostasis (SOD2, metallothionein) and Wnt/ β-catenin signaling pathway (frizzle, β-catenin and cyclin D1). The expression of candidate genes from this analysis was assessed across the panel of vorinostat-resistant A549 clones. The results indicate that acquired resistance to vorinostat appears to comprise mechanisms that inhibit the induction of apoptosis, including an increase in antioxidants and in anti-apoptotic Bcl-2 levels. In particular, upregulation of antioxidant enzymes and thiol modulating proteins including SOD2 and metallothioneins have been associated with clinical resistance to cytotoxic drugs and radiation therapy as well as poor prognosis. Future studies will help to address the clinical relevance of these findings. One prediction from this analysis is that compounds that inhibit the glutathione system like buthionine sulfoximine or agents that downregulate or block Bcl-2 could be used to revert these mechanisms of resistance. Combination strategies with emerging treatment modalities may help to increase vorinostat efficacy in specific genetic contexts.

699 Vorinostat is an HDAC inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo , and is currently being tested in the clinic for a variety of indications. In particular, vorinostat has demonstrated an overall response rate of approximately 30% in cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers that predict response to vorinostat in CTCL using gene expression profiling in preclinical model systems. Herein we report the results of our evaluation of the STAT (signal transducer and activator of transcription) signaling pathway. The relative level of mRNA and protein expression as well as activation status of several members of this family has been evaluated in lymphoid cell lines with diverse vorinostat sensitivity (proliferation/ viability IC 50 range: 0.3-14 μM). Among them the more responsive CTCL cells lines HH and HUT102 exhibited an IC 50 ~ 0.8 μM. These cells underwent apoptosis in response to vorinostat as assessed by TUNEL assay, in contrast to Hut78 and MJ cells (IC 50 > 2 μM) that did not show signs of DNA fragmentation upon incubation in vorinostat-containing media for 48 h. Our results indicate that in lymphoma cell lines elevated protein levels and persistent activation of STAT1, 3 and 5 correlate with resistance to vorinostat. Simultaneous treatment with pan-JAK inhibitors resulted in synergistic anti-proliferative effects consistent with a survival, anti-apoptotic role for STAT proteins in the response to vorinostat treatment. Immunohistochemical analysis of STAT1 in skin biopsies isolated from CTCL patients (N=48) enrolled in the vorinostat Phase IIb clinical trial showed that malignant T cells were positively stained in approximately half of the samples and in those cases a relationship between nuclear accumulation of STAT1 and lack of response to treatment exists (p

408 Introduction: A specific and sensitive imaging biomarker to monitor immune activation and quantify pharmacodynamic (PD) responses would be a useful tool in drug development for immunomodulating anti-cancer agents. PF-07062119 is a T-cell engaging CD3 bispecific antibody targeting GUCY2C (GUCY2C-CD3) which is over expressed widely across colorectal cancer (CRC) and other gastrointestinal malignancies. Previous data has demonstrated the efficacy of PF-0762119 as a targeted immuno-therapeutic candidate in human CRC xenograft mouse models with adoptive transfer of human T cells (ref). 89Zr-Df-IAB22M2C, an 89Zr-labeled human CD8 specific minibody developed by ImaginAb, is currently under testing in a phase 2 clinical study (ClinicalTrials.gov Identifier: NCT03802123) in patients with metastatic solid tumors to measure CD8 T-cell tumor infiltrates. In this study we investigated the ability of 89Zr-Df-IAB22M2C to track responses to interventions with varying doses of PF-07062119 in a human CRC adoptive transfer mouse model, as well as, correlation of the radiotracer to CD8 immunohistochemical (IHC) staining. Materials & Method: NOD scid gamma (NSG) female mice (total 70; n = 5-12 per group) bearing human CRC LS1034 tumors were used in this study. Following a pilot study, animals were treated with PF-07062119 in four different dose groups (0.03 mg/kg, 0.06 mg/kg, 0.1 mg/kg or 1 mg/kg), or with a non-targeted CD3 bispecific control at a dose of 1 mg/kg, on study Day-0 and injected with activated T-cells on Day-1. Different imaging time points, Day-4, Day 6, or Day-9 were assessed with 89Zr-Df-IAB22M2C intravenously injected (200 µL, with radioactivity of approximately 65 µCi) on Day-3, Day-5, and Day-8, respectively. All animals underwent a 20-minute static PET scan at 22h post 89Zr-Df-IAB22M2C injection, followed by CT scan for anatomical registration. For Day-9 imaging group animals, a second treatment of PF-07062119 was conducted on Day-7. Following PET/CT imaging, mice were euthanized under anesthesia and dissected for ex vivo distribution analysis of tissues [whole blood, brain, heart, kidneys, large intestine (emptied), liver, lungs, skeletal muscle (quadriceps), skin, small intestine (emptied), spleen, tail, and tumor]. Tissues were collected, weighed and gamma counted for their radioactivity. Data analyzed and reported as %ID/g and SUV for both in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were pretreated with 10% neutral buffered formalin (NBF) prior to the gamma counting for upcoming tissue section and CD8 IHC staining. Results & Conclusions: The results demonstrated substantial mean uptake in treated tumors, up to 13.0 %ID/g (2.7 S.D.) in highest dosed animals (ex vivo gamma counting), with significant differences in comparison to isotype controls. The data also showed a dose-dependent response for both time and treatment: significantly higher radioactivity concentration was observed at the higher dose levels of PF-0762119 (0.1 and 1 mg/kg) on Day-9 compared to Day-4 (p