Steffan T. Nawrocki

Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. Initially, survivin was described as an inhibitor of caspase-9. However, over the last years, research studies have shown that the role of survivin in cancer pathogenesis is not limited to apoptosis inhibition but also involves the regulation of the mitotic spindle checkpoint and the promotion of angiogenesis and chemoresistance. Survivin gene expression is transcriptionally repressed by wild-type p53 and can be deregulated in cancer by several mechanisms, including gene amplification, hypomethylation, increased promoter activity, and loss of p53 function. This article reviews the multiple functions of survivin in the regulation of apoptosis, the promotion of tumorigenesis, and the development of survivin inhibitors as a novel anticancer therapeutic strategy.

Novel therapeutic strategies are needed to address the emerging problem of imatinib resistance. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for imatinib-resistant chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of autophagy and apoptosis. Considering that autophagy may promote cancer cell survival, we hypothesized that disrupting autophagy would augment the anticancer activity of SAHA. Here we report that drugs that disrupt the autophagy pathway dramatically augment the antineoplastic effects of SAHA in CML cell lines and primary CML cells expressing wild-type and imatinib-resistant mutant forms of Bcr-Abl, including T315I. This regimen has selectivity for malignant cells and its efficacy was not diminished by impairing p53 function, another contributing factor in imatinib resistance. Disrupting autophagy by chloroquine treatment enhances SAHA-induced superoxide generation, triggers relocalization and marked increases in the lysosomal protease cathepsin D, and reduces the expression of the cathepsin-D substrate thioredoxin. Finally, knockdown of cathepsin D diminishes the potency of this combination, demonstrating its role as a mediator of this therapeutic response. Our data suggest that, when combined with HDAC inhibitors, agents that disrupt autophagy are a promising new strategy to treat imatinib-refractory patients who fail conventional therapy.

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression by catalyzing the removal of acetyl groups, stimulating chromatin condensation and promoting transcriptional repression. Since aberrant epigenetic changes are a hallmark of cancer, HDACs are a promising target for pharmacological inhibition. HDAC inhibitors can induce cell-cycle arrest, promote differentiation, and stimulate tumor cell death. These properties have prompted numerous preclinical and clinical investigations evaluating the potential efficacy of HDAC inhibitors for a variety of malignancies. The preferential toxicity of HDAC inhibitors in transformed cells and their ability to synergistically enhance the anticancer activity of many chemotherapeutic agents has further generated interest in this novel class of drugs. Here we summarize the different mechanisms of HDAC inhibitor-induced apoptosis and discuss their use in combination with other anticancer agents.

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2alpha (eif2alpha), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2alpha phosphorylation, ubiquitylated protein accumulation, and proteotoxicity.

Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.

We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.

Abstract NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-κB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

Hematological malignancies express high levels of CD47 as a mechanism of immune evasion. CD47-SIRPα triggers a cascade of events that inhibit phagocytosis. Preclinical research supports several models of antibody-mediated blockade of CD47-SIRPα resulting in cell death signaling, phagocytosis of cells bearing stress signals, and priming of tumor-specific T cell responses. Four different antibody molecules designed to target the CD47-SIRPα interaction in malignancy are currently being studied in clinical trials: Hu5F9-G4, CC-90002, TTI-621, and ALX-148. Hu5F9-G4, a humanized anti-CD47 blocking antibody is currently being studied in four different Phase I trials. These studies may lay the groundwork for therapeutic bispecific antibodies. Bispecific antibody (CD20-CD47SL) fusion of anti-CD20 (Rituximab) and anti-CD47 also demonstrated a synergistic effect against lymphoma in preclinical models. This review summarizes the large body of preclinical evidence and emerging clinical data supporting the use of antibodies designed to target the CD47-SIRPα interaction in leukemia, lymphoma and multiple myeloma.

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins ("aggresomes") in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer.

Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin-conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ-induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ-induced aggresome formation, but promoted CQ-mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR-mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR-induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ-induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ-mediated aggregate formation, superoxide generation and apoptosis.

Introduction: The small ubiquitin-like molecule NEDD8 has been identified as an essential regulator of the activity of the cullin-RING E3 ubiquitin ligases (CRLs), which control the turnover of multiple proteins with fundamental roles in cancer biology. The aberrant function of the NEDD8 cascade within the context of malignancy makes it an attractive target for the development of novel anticancer agents. MLN4924 is a first-in-class inhibitor of the proximal regulator of the NEDD8 system (NEDD8-activating enzyme, NAE) that has entered Phase-I trials for cancer therapy and has established that significant therapeutic benefit can be achieved by antagonizing NEDD8-mediated protein degradation.

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.

Autophagy is an evolutionarily conserved lysosomal degradation pathway that eliminates cytosolic proteins, macromolecules, organelles, and protein aggregates.Activation of autophagy may function as a tumor suppressor by degrading defective organelles and other cellular components.However, this pathway may also be exploited by cancer cells to generate nutrients and energy during periods of starvation, hypoxia, and stress induced by chemotherapy.Therefore, induction of autophagy has emerged as a drug resistance mechanism that promotes cancer cell survival via self-digestion.Numerous preclinical studies have demonstrated that inhibition of autophagy enhances the activity of a broad array of anticancer agents.Thus, targeting autophagy may be a global anticancer strategy that may improve the efficacy of many standard of care agents.These results have led to multiple clinical trials to evaluate autophagy inhibition in combination with conventional chemotherapy.In this review, we summarize the anticancer agents that have been reported to modulate autophagy and discuss new developments in autophagy inhibition as an anticancer strategy.

Abstract Purpose: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure and novel therapeutic strategies are urgently needed. MLN4924 is a NEDDylation inhibitor currently under investigation in multiple phase I studies. We investigated its anticancer activity in cisplatin-sensitive and -resistant ovarian cancer models. Experimental Design: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, clonogenic survival, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference established natural born killer/bcl-2–interacting killer (NBK/BIK) as a regulator of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamics were assessed in cisplatin-sensitive and -resistant xenograft models. Results: MLN4924 possessed significant activity against both cisplatin-sensitive and -resistant ovarian cancer cells and provoked the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells, suggesting that aberrant NEDDylation may contribute to drug resistance. MLN4924 and cisplatin cooperated to induce DNA damage, oxidative stress, and increased expression of the BH3-only protein NBK/BIK. Targeted NBK/BIK knockdown diminished the proapoptotic effects of the MLN4924/cisplatin combination. Administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and -resistant tumors. Conclusions: Our collective data provide a rationale for the clinical investigation of NEDD8-activating enzyme (NAE) inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies. Clin Cancer Res; 19(13); 3577–90. ©2013 AACR.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

Abstract A hallmark feature of tumorigenesis is uncontrolled cell division. Autophagy is regulated by more than 30 genes and it is one of several mechanisms by which cells maintain homeostasis. Autophagy promotes cancer progression and drug resistance. Several genes play important roles in autophagy‐induced tumorigenesis and drug resistance including Beclin‐1, MIF, HMGB1, p53, PTEN, p62, RAC3, SRC3, NF‐2, MEG3, LAPTM4B, mTOR, BRAF and c‐MYC . These genes alter cell growth, cellular microenvironment and cell division. Mechanisms involved in tumorigenesis and drug resistance include microdeletions, genetic mutations, loss of heterozygosity, hypermethylation, microsatellite instability and translational modifications at a molecular level. Disrupted or altered autophagy has been reported in hematological malignancies like lymphoma, leukemia and myeloma as well as multiple solid organ tumors like colorectal, hepatocellular, gall bladder, pancreatic, gastric and cholangiocarcinoma among many other malignancies. In addition, defects in autophagy also play a role in drug resistance in cancers like osteosarcoma, ovarian and lung carcinomas following treatment with drugs such as doxorubicin, paclitaxel, cisplatin, gemcitabine and etoposide. Therapeutic approaches that modulate autophagy are a novel future direction for cancer drug development that may help to prevent issues with disease progression and overcome drug resistance.

Abstract Although it displays promising activity in other tumor models, the effects of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on human pancreatic cancer cells have not been comprehensively explored. We report that a majority of human pancreatic cancer cell lines (seven of nine) underwent apoptosis when they were exposed to recombinant human TRAIL in vitro. Characterization of surface TRAIL receptors by fluorescence-activated cell sorting showed that TRAIL-resistant cells (Panc-1 and HS766T) expressed lower levels of DR4 and DR5 than did TRAIL-sensitive cells. The proteasome inhibitor bortezomib (PS-341, Velcade) further increased TRAIL responsiveness in the TRAIL-sensitive cells and synergized with TRAIL to reverse resistance in Panc-1 and HS776T cells. The effects of bortezomib were mimicked by transfection with a small interfering RNA construct specific for the p65 subunit of nuclear factor-κB (NF-κB) or exposure to a selective chemical inhibitor of IKK (PS-1145). Silencing IκBα prevented TRAIL sensitization by PS-1145, confirming that IκBα mediated the effects of PS-1145. NF-κB inhibition resulted in down-regulation of BCL-XL and XIAP, and silencing either restored TRAIL sensitivity in TRAIL-resistant cells. Finally, therapy with TRAIL plus PS-1145 reversed TRAIL resistance in vivo to produce synergistic growth inhibition in orthotopic Panc-1 tumors. Together, our results show that NF-κB inhibits TRAIL-induced apoptosis in human pancreatic cancer cells and suggest that combination therapy with TRAIL and NF-κB inhibitors, such as bortezomib, PS-1145, or curcumin, should be considered as a possible treatment strategy in patients with pancreatic cancer. [Mol Cancer Ther 2006;5(9):2251–60]

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome. We examined the antitumor activity of combination therapy with bortezomib + docetaxel in two human pancreatic cancer cell lines (MiaPaCa-2 and L3.6pl) selected for their divergent responses to bortezomib alone. Bortezomib blocked docetaxel-induced apoptosis in the MiaPaCa-2 cells and failed to enhance docetaxel-induced apoptosis in L3.6pl cells in vitro but did interact positively with docetaxel to inhibit clonogenic survival. These effects were associated with decreased accumulation of cells in M phase, stabilization of the cyclin-dependent kinase inhibitors, p21 and p27, and inhibition of cdk2 and cdc2 activities. In orthotopic xenografts, combination therapy produced significant reductions in tumor weight and volume in both models associated with accumulation of p21, inhibition of proliferation, and increased apoptosis. Combination therapy also reduced tumor microvessel densities, effects that were associated with reductions in tumor cell production of vascular endothelial growth factor and increased levels of apoptosis in tumor-associated endothelial cells. Together, our results suggest that bortezomib enhances the antitumoral activity of taxanes by enforcing cell growth arrest and inhibiting angiogenesis.

The histone deacetylase inhibitor SAHA enhances cell death stimulated by the proteasome inhibitor bortezomib (BZ) by disrupting BZ-induced aggresome formation. Here we report that Myc regulates the sensitivity of multiple myeloma (MM) cells to BZ + SAHA-induced cell death. In MM cells, Myc expression directly correlated with intracellular ER content, protein synthesis rates, the percentage of aggresome-positive cells, and the sensitivity to BZ + SAHA-induced cell death. Accordingly, Myc knockdown markedly reduced the sensitivity of MM cells to BZ + SAHA-mediated apoptosis. Furthermore, activation of Myc was sufficient to provoke aggresome formation and thus sensitivity to BZ + SAHA, and these responses required de novo protein synthesis. BZ + SAHA-mediated stimulation of apoptosis includes the induction of the proapoptotic BH3-only protein Noxa as well as endoplasmic reticular stress, a disruption of calcium homeostasis, and activation of capase-4. Finally, knockdown studies demonstrated that both caspase-4 and Noxa play significant roles in Myc-driven sensitivity to BZ + SAHA-induced apoptosis. Collectively, our results establish a mechanistic link between Myc activity, regulation of protein synthesis, increases in HDAC6 expression and aggresome formation, induction of Noxa, and sensitivity to BZ + SAHA-induced apoptosis. These data suggest that MM patients with elevated Myc activity may be particularly sensitive to the BZ + SAHA combination.

Autophagy is an ancient cell survival pathway that allows cells to recoup ATP and essential building blocks for biosynthesis when they are starved of nutrients or when they are exposed to hypoxia, which are hallmarks of the tumor microenvironment. This pathway involves the formation of double-membraned vesicles, coined autophagosomes, which envelop bulk cellular material and/or organelles and that subsequently fuse with lysosomes that degrade their cargo. Autophagy has been suggested to play important roles in chemoresistance of cancer to some therapeutic agents, which typically induce an apoptotic response. For example, the histone deacetylase inhibitor SAHA induces both apoptosis and autophagy, suggesting that agents that disrupt the autophagy pathway might augment its efficacy as a therapeutic agent. We tested this notion in a model of Imatinib-refractory chronic myelogenous leukemia (CML) and in imatinib-resistant primary CML cells from patients bearing mutations in Bcr-Abl, including the T315I mutation that causes resistance to currently utilized tyrosine kinase inhibitors and translates into a very poor clinical prognosis. Agents that disrupt autophagy were shown to synergize with SAHA in provoking apoptotic death of these refractory tumors. These findings support the use of agents that disrupt the autophagy pathway in settings of chemorefractory malignancies.

Abstract Purpose: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus has exhibited promising anticancer activity for the treatment of renal cell cancers (RCC). Survivin expression has been implicated in drug resistance and reducing its levels with the histone deacetylase (HDAC) inhibitor vorinostat may enhance the anticancer activity of temsirolimus. Experimental Design: The sensitivity of RCC cell lines to the combination of temsirolimus and vorinostat was determined by measuring cell viability, clonogenic survival, and apoptosis. The effects of this combination on survivin levels were determined in vitro and in vivo. Survivin expression was silenced using small interfering RNA to evaluate its role in determining sensitivity to temsirolimus and vorinostat. The effect of the combination on angiogenesis was also determined in RCC xenograft models. Results: Vorinostat synergistically improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in two xenograft models in vivo. While each single agent led to a modest decrease in survivin levels, the combination dramatically reduced its expression, which correlated with an induction of apoptosis. Silencing survivin levels induced apoptosis and significantly improved the efficacy of temsirolimus and vorinostat. In addition, the temsirolimus/vorinostat combination led to a strong reduction in angiogenesis. Conclusions: Vorinostat augmented the anticancer activity of temsirolimus in both in vitro and in vivo models of RCC. The effectiveness of the combination was due to a decrease in survivin levels and corresponding induction of apoptosis, and enhanced inhibition of angiogenesis. Targeting survivin may be a promising therapeutic strategy to improve RCC therapy. Clin Cancer Res; 16(1); 141–53

Oncolytic virotherapy with reovirus has demonstrated anti-cancer activity and minimal toxicity in clinical trials, but the mechanisms underlying these effects have not been fully elucidated. Reolysin, a proprietary formulation of reovirus for cancer therapy, stimulated selective viral replication and apoptosis in multiple myeloma (MM) cells. Reolysin-mediated apoptosis was associated with an induction of endoplasmic reticular (ER) stress-related gene expression, swelling of the endoplasmic reticulum, increases in intracellular calcium levels and a strong induction of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knockdown of NOXA expression by short hairpin RNA significantly reduced the pro-apoptotic effects of Reolysin. We next showed that co-administration of Reolysin and bortezomib resulted in the dual accumulation of viral and ubiquitinated proteins, which led to enhanced ER stress, NOXA induction and apoptosis. Importantly, the combination of reovirus infection and proteasomal inhibition significantly decreased tumor burden in a xenograft and syngeneic bone disease model of MM without exhibiting adverse side effects. Our study establishes ER stress stimulation and NOXA induction as novel mediators of reovirus-induced apoptosis. Furthermore, reovirus infection can be used as a promising approach to augment the anti-myeloma activity of bortezomib by promoting additional stress to the endoplasmic reticulum of MM cells.

Hydroxychloroquine (HCQ) enhances the anti-cancer activity of the histone deacetylase inhibitor, vorinostat (VOR), in pre-clinical models and early phase clinical studies of metastatic colorectal cancer (mCRC). Mechanisms could include autophagy inhibition, accumulation of ubiquitinated proteins, and subsequent tumor cell apoptosis. There is growing evidence that autophagy inhibition could lead to improved anti-cancer immunity. To date, effects of autophagy on immunity have not been reported in cancer patients. To address this, we expanded an ongoing clinical study to include patients with advanced, refractory mCRC to evaluate further the clinical efficacy and immune effects of VOR plus HCQ. Refractory mCRC patients received VOR 400 milligrams orally with HCQ 600 milligrams orally daily, in a 3-week cycle. The primary endpoint was median progression-free survival (mPFS). Secondary endpoints include median overall survival (mOS), adverse events (AE), pharmacodynamic of inhibition of autophagy in primary tumors, immune cell analyses, and cytokine levels. Twenty patients were enrolled (19 evaluable for survival) with a mPFS of 2.8 months and mOS of 6.7 months. Treatment-related grade 3-4 AEs occurred in 8 patients (40%), with fatigue, nausea/vomiting, and anemia being the most common. Treatment significantly reduced CD4+CD25hiFoxp3+ regulatory and PD-1+ (exhausted) CD4+ and CD8+ T cells and decreased CD45RO-CD62L+ (naive) T cells, consistent with improved anti-tumor immunity. On-study tumor biopsies showed increases in lysosomal protease cathepsin D and p62 accumulation, consistent with autophagy inhibition. Taken together, VOR plus HCQ is active, safe and well tolerated in refractory CRC patients, resulting in potentially improved anti-tumor immunity and inhibition of autophagy.

Purpose: Although autophagy plays important roles in malignant pathogenesis and drug resistance, there are few clinical agents that disrupt this pathway, and the potential therapeutic benefit of autophagy inhibition remains undetermined. We used medicinal chemistry approaches to generate a series of novel agents that inhibit autophagic degradation.Experimental Design: ROC-325 was selected as a lead compound for further evaluation. Comprehensive in vitro and in vivo studies were conducted to evaluate the selectivity, tolerability, and efficacy of ROC-325 in preclinical models of renal cell carcinoma (RCC) with HCQ serving as a comparator. Markers of autophagy inhibition and cell death were evaluated in tumor specimens.Results: ROC-325 exhibited superior in vitro anticancer effects compared with the existing autophagy inhibitor hydroxychloroquine (HCQ) in 12 different cancer cell lines with diverse genetic backgrounds. Focused studies of the mechanism of action and efficacy of ROC-325 in RCC cells showed that drug treatment induced hallmark characteristics of autophagy inhibition, including accumulation of autophagosomes with undegraded cargo, lysosomal deacidification, p62 stabilization, and disruption of autophagic flux. Subsequent experiments showed that ROC-325 antagonized RCC growth and survival in an ATG5/7-dependent manner, induced apoptosis, and exhibited favorable selectivity. Oral administration of ROC-325 to mice bearing 786-0 RCC xenografts was well tolerated, was significantly more effective at inhibiting tumor progression than HCQ, and inhibited autophagy in vivoConclusions: Our findings demonstrate that ROC-325 has superior preclinical anticancer activity compared with HCQ and support the clinical investigation of its safety and preliminary efficacy in patients with RCC and other autophagy-dependent malignancies. Clin Cancer Res; 23(11); 2869-79. ©2016 AACR.

Abstract Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.

Abstract Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region‐Abelson kinase (BCR‐ABL). Notably, this agent retained high activity against the T315I and E255K BCR‐ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose‐dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR‐ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib‐induced apoptosis, indicating that this is an important factor underlying MLN8237’s ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.

The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation. Keywords: Akt, cell signalling, drug development, kinase, mTOR, PI3 kinase, PIM kinase, cancer, targeted therapies, pharmacokinetics

Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.

Abstract Adaptive resistance mediated by inhibitory ligands such as programmed death 1 ligand (PD-L1) has emerged as an important mechanism of malignant cell survival. This has spurred the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. Analysis of patient specimens from clinical trials of novel immune checkpoint inhibitors indicates that high basal expression of PD-L1 on tumor cells may predict sensitivity to and be necessary to elicit significant clinical benefit from this drug class. Notably, many multiple myeloma (MM) cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefitting from immune checkpoint inhibitor therapy. These data suggest that strategies that transiently increase PD-L1 levels could potentially sensitize malignant cells with low PD-L1 expression to anti-PD-1/PD-L1 blockade. The oncolytic reovirus-based anticancer agent Reolysin is known to have significant immunomodulatory effects and has demonstrated promising preclinical efficacy in MM models and favorable safety and tolerability in early MM clinical trials. We demonstrated that Reolysin selectively replicates in MM cells and possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274 (PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. A comparison of the anti-MM effects achieved by live reovirus versus UV-inactivated reovirus demonstrated that live reovirus is required to decrease MM cell viability and upregulate PD-L1 expression. Our data demonstrate proof of concept that reovirus infection and replication in MM cells can efficiently and selectively upregulate PD-L1 levels in malignant cells with low target expression. We hypothesized that Reolysin treatment could be used as a precision priming strategy to potentiate the anti-MM efficacy of PD-1/PD-L1 targeted therapy by promoting myeloma immune recognition and PD-L1 upregulation. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 108 TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P Disclosures Kelly: Amgen: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Jannsen: Honoraria.

Abstract Purpose: New therapies are urgently needed for patients with acute myelogenous leukemia (AML). The novel NEDDylation inhibitor MLN4924 (pevonedistat) has demonstrated significant preclinical antileukemic activity and preliminary efficacy in patients with AML in a phase I trial. On the basis of its antimyeloid and DNA-damaging properties, we investigated the ability of MLN4924 to augment conventional cytarabine (ara-C) therapy. Experimental Design: The effects of MLN4924/ara-C on viability, clonogenic survival, apoptosis, DNA damage, and relevant pharmacodynamic targets were determined. The efficacy and pharmacodynamics of MLN4924/ara-C were assessed in an AML xenograft model. Results: Cotreatment of AML cell lines and primary patient specimens with MLN4924 and ara-C led to diminished clonogenic survival, increased apoptosis, and synergistic levels of DNA damage. RNAi demonstrated that stabilization of CDT-1, an event previously shown to mediate the DNA-damaging effects of MLN4924, was not a key regulator of sensitivity to the MLN4924/ara-C combination. Global metabolic profiling revealed that MLN4924 disrupts nucleotide metabolism and depletes intracellular nucleotide pools in AML cells. Subsequent experiments showed that MLN4924 promoted increased incorporation of ara-C into the DNA of AML cells. This effect as well as the therapeutic benefit of the MLN4924/ara-C combination was antagonized by supplementation with the nucleotide building block ribose. Coadministration of MLN4924 and ara-C to mice bearing FLT3-ITD+ AML xenografts stably inhibited disease progression and increased DNA damage in vivo. Conclusions: Our findings provide strong rationale for clinical investigation of the MLN4924/ara-C combination and establish a new link between therapeutic inhibition of NEDDylation and alterations in nucleotide metabolism. Clin Cancer Res; 21(2); 439–47. ©2014 AACR.

Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.

Kidney cancer is the 7th most prevalent form of cancer in the United States with the vast majority of cases being classified as renal cell carcinoma (RCC). Multiple targeted therapies have been developed to treat RCC, but efficacy and resistance remain a challenge. In recent years, the modulation of autophagy has been shown to augment the cytotoxicity of approved RCC therapeutics and overcome drug resistance. Inhibition of autophagy blocks a key nutrient recycling process that cancer cells utilize for cell survival following periods of stress including chemotherapeutic treatment. Classic autophagy inhibitors such as chloroquine and hydroxychloroquine have been introduced into phase I/II clinical trials, while more experimental compounds are moving forward in preclinical development. Here we examine the current state and future directions of targeting autophagy to improve the efficacy of RCC therapeutics.

Autophagy plays an important role in the pathogenesis of pulmonary hypertension (PH). ROC-325 is a novel small molecule lysosomal autophagy inhibitor that has more potent anticancer activity than the antimalarial drug hydroxychloroquine, the latter has been prevalently used to inhibit autophagy. Here, we sought to determine the therapeutic benefit and mechanism of action of ROC-325 in experimental PH models.Hemodynamics, echocardiography, and histology measurement showed that ROC-325 treatment prevented the development of PH, right ventricular hypertrophy, fibrosis, dysfunction, and vascular remodeling after monocrotaline and Sugen5416/hypoxia administration. ROC-325 attenuated high K+ or alveolar hypoxia-induced pulmonary vasoconstriction and enhanced endothelial-dependent relaxation in isolated pulmonary artery rings. ROC-325 treatment inhibited autophagy and enhanced endothelial nitric oxide synthase activity in lung tissues of monocrotaline-PH rats. In cultured human and rat pulmonary arterial smooth muscle cell and pulmonary arterial endothelial cell under hypoxia exposure, ROC-325 increased LC3B (light chain 3 beta) and p62 accumulation, endothelial cell nitric oxide production via phosphorylation of endothelial nitric oxide synthase (Ser1177) and dephosphorylation of endothelial nitric oxide synthase (Thr495) as well as decreased HIF (hypoxia-inducible factor)-1α and HIF-2α stabilization.These data indicate that ROC-325 is a promising novel agent for the treatment of PH that inhibits autophagy, downregulates HIF levels, and increases nitric oxide production.

Recent studies have shown that the transcription factor, nuclear factor kappaB (NF-kappaB), regulates critical survival pathways in a variety of different cell types, including human pancreatic cancer cells. The activation of NF-kappaB is controlled by proteasome-mediated degradation of its endogenous polypeptide inhibitor, inhibitor of nuclear factor kappaBalpha. We investigated the effects of PS-341, a peptide boronate inhibitor of the proteasome in human pancreatic cancer cells in vitro and in vivo. Comparison of PS-341's effects on the growth of eight different human pancreatic cancer cell lines revealed marked heterogeneity in drug responsiveness, ranging from highly resistant (IC50 > 10 microM; Panc-48, HS766T, and Mia-PaCa-2) to extremely sensitive (IC50 < 40 nM; L3.6pl, Hpaf2, and BxPC3). However, these effects did not correlate with differential inhibition of NF-kappaB activation. Direct quantification of apoptosis revealed that PS-341's effects on cell growth largely correlated with sensitivity to programmed cell death. Evaluation of PS-341's effects on established orthotopic tumor xenografts demonstrated that biweekly intravenous administration of the maximum-tolerated dose of the drug (1 mg/kg) led to significant reductions in the volumes of L3.6pl tumors but not Mia-PaCa-2 tumors. Laser scanning cytometer-mediated quantification of drug-induced apoptosis in the xenografts confirmed that PS-341 induced DNA fragmentation and activation of caspase-3 in L3.6pl tumors but not in Mia-PaCa-2 tumors. However, histological examination of drug-treated tumors revealed extensive central necrosis and reductions in microvessel density and VEGF expression in both tumor types. Taken together, our results demonstrate that PS-341 inhibits the growth of human pancreatic tumors via direct effects on tumor cells and indirect effects on the tumor vasculature.

Abstract Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21+/+ cells but not in the HCT116 p21−/− cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA–mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab. [Cancer Res 2007;67(14):6987–94]

Reovirus is an oncolytic virus that is not associated with significant disease in humans, but is selectively able to replicate in cancer cells through exploitation of abnormal Ras signaling. Pre-clinical studies have demonstrated that treatment with reovirus is associated with significant anticancer activity across a range of tumor types. Reolysin is a proprietary formulation of the human reovirus developed by Oncolytics Biotech. Clinical evaluation of reovirus therapy has shown that it is well tolerated when administered locally or systemically. Encouraging anticancer efficacy has been observed with single-agent treatment and in combination with chemotherapy and radiotherapy. Phase II studies are currently evaluating reovirus alone and in combination with standard therapy in an array of tumor types. While immune sensitization hinders the anticancer efficacy of reovirus, it is important in preventing systemic toxicity. Immunosuppressive strategies are being developed that reduce immune neutralization of the virus to allow for improved tumor penetration, but retain sufficient antibody levels to protect normal tissues. The lack of toxicity and promising efficacy of reovirus has raised hopes that it will become an established anticancer agent.

Abstract Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A link between Aurora A expression and sensitivity to ara‐C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara‐C. Accordingly, alisertib significantly potentiated the antileukemic activity of ara‐C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro‐apoptotic effects of the alisertib/ara‐C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara‐C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara‐C that warrants further investigation.

The Aurora family of serine/threonine kinases is essential for chromosome alignment, segregation, centrosomal maturation, mitotic spindle formation, and cytokinesis during mitosis. Their fundamental role in cell cycle regulation and aberrant expression in a broad range of malignancies prompted the development of small molecules that selectively inhibit their activity. Recent studies have revealed new insights into the cellular effects of Aurora kinase inhibition. Moreover, early phase clinical studies have shown that these agents have therapeutic efficacy. In this review, we will outline the functions of Aurora kinases in normal cell division and in malignancy. We will focus on recent preclinical and clinical studies that have explored the mechanism of action and clinical effect of Aurora inhibitors in cancer treatment.

Among the solid tumors, human pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis. Gemcitabine is the standard first line of therapy for pancreatic cancer but has limited efficacy due to inherent or rapid development of resistance and combining EGFR inhibitors with this regimen results in only a modest clinical benefit. The goal of this study was to identify molecular targets that are activated during gemcitabine therapy alone or in combination with an EGFR inhibitor.PDAC cell lines were used to determine molecular changes and rates of growth after treatment with gemcitabine or an EGFR inhibitor, AG1478, by Western blot analysis and MTT assays respectively. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after gemcitabine treatment. ShRNA was used to knockdown STAT3. An in vivo orthotopic animal model was used to evaluate STAT3 as a target. Immunohistochemical analysis was performed to analyze Ki67 and STAT3 expression in tumors.Treatment with gemcitabine increased the levels of EGFRTyr1068 and ERK phosphorylation in the PDAC cell lines tested. The constitutive STAT3Tyr705 phosphorylation observed in PDAC cell lines was not altered by treatment with gemcitabine. Treatment of cells with gemcitabine or AG1478 resulted in differential rate of growth inhibition. AG1478 efficiently blocked the phosphorylation of EGFRTyr1068 and inhibited the phosphorylation of down-stream effectors AKT and ERKs, while STAT3Tyr705 phosphorylation remained unchanged. Combining these two agents neither induced synergistic growth suppression nor inhibited STAT3Tyr705 phosphorylation, thus prompting further studies to assess whether targeting STAT3 improves the response to gemcitabine or AG1478. Indeed, knockdown of STAT3 increased sensitivity to gemcitabine by inducing pro-apoptotic signals and by increasing G1 cell cycle arrest. However, knockdown of STAT3 did not enhance the growth inhibitory potential of AG1478. In vivo orthotopic animal model results show that knockdown of STAT3 caused a significant reduction in tumor burden and delayed tumor progression with increased response to gemcitabine associated with a decrease in the Ki-67 positive cells.This study suggests that STAT3 should be considered an important molecular target for therapy of PDAC for enhancing the response to gemcitabine.

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6 pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6 pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6 pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX-2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6 pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.

Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms

Although macroautophagy/autophagy is a key contributor to malignant pathogenesis and therapeutic resistance, there are few FDA-approved agents that significantly affect this pathway. We used medicinal chemistry strategies to develop ROC-325, an orally available novel inhibitor of lysosomal-mediated autophagy. Detailed in vitro and in vivo studies in preclinical models of renal cell carcinoma demonstrated that ROC-325 triggered the hallmark features of lysosomal autophagy inhibition, was very well tolerated, and exhibited significant superiority with respect to autophagy inhibition and anticancer activity over hydroxychloroquine. Our findings support the clinical investigation of the safety and preliminary efficacy of ROC-325 in patients with autophagy-dependent malignancies and other disorders where aberrant autophagy contributes to disease pathogenesis.

The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-γ-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1α, HIF-2α and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.

Constitutive activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogenic event in certain types of human cancer and is associated with poor patient survival. Small molecule signaling inhibitors have improved the clinical outcomes of patients with various cancer types but attempts to target KRAS have been unsuccessful. Plinabulin represents a novel class of agents that inhibit tubulin polymerization with a favorable safety profile in clinical trials. In the present study, the potency of plinabulin to inhibit tubulin polymerization and growth of KRAS‑driven cancer cells was characterized. In vivo efficacy of plinabulin was tested in two different mouse models; one being the RCAS/t‑va gene transfer system and the other being a xenograft model. In vitro cell culture tubulin polymerization assays were used to complement the mouse models. There was improved survival in a KRAS‑driven mouse gene transfer glioma model, but lack of benefit in a similar model, without constitutively active KRAS, which supports the notion of a KRAS‑specific effect. This survival benefit was mediated, at least in part, by the ability of plinabulin to inhibit tubulin polymerization and disrupt endosomal recycling. It was proposed a mechanism of compromised endosomal recycling of displaced KRAS through targeting microtubules that yields inhibition of protein kinase B, but not extracellular signal regulated kinase (ERK) signaling, therefore lending rationale to combination treatments of tubulin‑ and ERK‑targeting agents in KRAS‑driven cancer.

Abstract Inhibition of bromodomain and extra terminal (BET) protein family members, including BRD4, decreases the expression of c-MYC and other key oncogenic factors and also significantly induces histone deacetylase 6 (HDAC6) expression. On the basis of the role of HDAC6 in malignant pathogenesis, we hypothesized that rational cotargeting of HDAC6 and BET family proteins may represent a novel approach that yields synergistic antimyeloma activity. We used genetic and pharmacologic approaches to selectively impair HDAC6 and BET function and evaluated the consequential impact on myeloma pathogenesis. These studies identified HDAC6 upregulation as an efficacy reducing mechanism for BET inhibitors because antagonizing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of multiple myeloma (MM) cell lines and primary CD138+ cells obtained from patients with MM. The synergy of this therapeutic combination was linked to significant reductions in c-MYC expression and increases in apoptosis induction. Administration of the clinical HDAC6 inhibitor ricolinostat was very well tolerated and significantly augmented the in vivo antimyeloma activity of JQ1. Ex vivo pharmacodynamic analyses demonstrated that the combination of JQ1 and ricolinostat led to significantly lower MM cell proliferation and increased apoptosis and diminished expression of c-MYC and BCL-2. These data demonstrate that cotargeting of HDAC6 and BET family members is a novel and clinically actionable approach to augment the efficacy of both classes of agents that warrants further investigation.

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches.

Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib. Immunoblotting, qRT–PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo. Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated. These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC.

Comprehensive quantitative proteomic profiling of the pharmacodynamic changes induced by MLN4924 in acute myeloid leukemia cells establishes rationale for its combination with azacitidine

Although recent treatment advances have improved outcomes for patients with multiple myeloma (MM), the disease frequently becomes refractory to current therapies. MM thus remains incurable for most patients and new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics that provide tumor-targeted therapy by specifically infecting and replicating within cancerous cells. Oncolytic therapy yields results from both direct killing of malignant cells and induction of an anti-tumor immune response. In this review, we will describe oncolytic viruses that are being tested for MM therapy with a focus on those agents that have advanced into clinical trials.

Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the in vitro and in vivo antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH(2)-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy.

Tumor necrosis factor-related apoptosis-inducing ligand or Apo2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) superfamily that induces apoptosis upon binding to its death domain-containing transmembrane receptors. The preferential toxicity of TRAIL to cancer cells and the sparing of normal cells make it an ideal cancer therapeutic agent. TRAIL induces apoptosis via the extrinsic death receptor apoptotic pathway and activates the JNK, ERK, Akt and NF-κB signaling cascades. However, not all cancer cells are sensitive to TRAIL therapy. This may limit its efficacy in the clinic, although ways have already been identified to overcome resistance by combining TRAIL with chemotherapeutic and other biological agents. This review focuses on TRAIL receptor-targeting as anticancer therapy, the apoptotic signaling pathways induced by TRAIL receptors, the prognostic implications of TRAIL receptor expression and modulation by combination therapies. The mechanisms of TRAIL resistance and strategies to overcome drug resistance will also be addressed. Finally, the progress of TRAIL and DR4/DR5-specific agonistic antibodies in clinical trials and the development of new receptor-selective TRAIL variants are discussed including future directions for apoptosis inducing therapy. Keywords: apoptosis, cancer, DR4, DR5, DcR1, TRAIL, clinical trials, p53, TNF, combination therapy

Activating mutations in RAS are present in the majority of pancreatic cancer cases and represent an ideal therapeutic target. Reolysin is a proprietary formulation of oncolytic reovirus that is currently being evaluated in multiple clinical trials due to its ability to selectively replicate in cells harboring an activated RAS pathway. Here we report for the first time the presence of reovirus replication and induction of endoplasmic reticular (ER) stress in a primary tumor specimen collected from a pancreatic cancer patient receiving intravenous Reolysin and gemcitabine.We describe the case of a 54-year old patient diagnosed with pancreatic adenocarcinoma in February 2012. Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012. Stable disease was achieved with significant improvement in cancer-related pain. Following 25 cycles of treatment over 23 months, a second biopsy was collected and immunohistochemical analyses revealed the presence of reovirus replication and induction of the ER stress-related gene GRP78/BIP and the pro-apoptotic protein NOXA. Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.This is the first report of reoviral protein detection in primary tumor biopsies taken from a pancreatic cancer patient receiving intravenous Reolysin therapy. The accumulation of reoviral protein was associated with ER stress induction and caspase-3 processing suggesting that Reolysin and gemcitabine treatment exhibited direct pro-apoptotic activity against the tumor.

Elevated expression of the antiapoptotic factor survivin has been implicated in cancer cell survival and disease progression. However, its specific contribution to renal cell carcinoma (RCC) pathogenesis is not well defined. We investigated the roles of survivin in RCC tumor progression, resistance to mTOR inhibitors, and evaluated the therapeutic activity of the survivin suppressant YM155 in RCC models. Here, we report that survivin expression levels were significantly higher in RCC cell lines compared with normal renal cells. Stable targeted knockdown of survivin completely abrogated the ability of 786-O RCC tumors to grow in mice, thus demonstrating its importance as a regulator of RCC tumorigenesis. We next explored multiple strategies to therapeutically inhibit survivin function in RCC. Treatment with the mTOR inhibitor temsirolimus partially diminished survivin levels and this effect was augmented by the addition of YM155. Further analyses revealed that, in accordance with their combined anti-survivin effects, YM155 significantly improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in xenograft models in vivo. Similar to pharmacologic inhibition of survivin, shRNA-mediated silencing of survivin expression not only inhibited RCC tumor growth, but also significantly sensitized RCC cells to temsirolimus therapy. Subsequent experiments demonstrated that the effectiveness of this dual survivin/mTOR inhibition strategy was mediated by a potent decrease in survivin levels and corresponding induction of apoptosis. Our findings establish survivin inhibition as a novel approach to improve RCC therapy that warrants further investigation.

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated β-galactosidase (SA-β-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-β-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

Drug resistance is a major cause of treatment failure for patients with acute myeloid leukaemia (AML) and novel strategies that circumvent resistance mechanisms are urgently needed (Swords et al, 2010). The PIM kinases (PIM1, PIM2, PIM3) are a small family of proto-oncogenes within the CAMK superfamily that are frequently overexpressed in many forms of cancer including AML. PIM kinases have essential roles in the regulation of signal transduction cascades that promote cell survival, proliferation, and drug resistance (Amaravadi & Thompson, 2005; Giles, 2005; Nawijn et al, 2011). However, the specific roles of PIM kinases as regulators of AML pathogenesis and of the sensitivity to standard agents utilized in AML therapy remain to be fully elucidated. SGI-1776 is novel small molecule inhibitor of PIM kinase activity that has demonstrated preclinical activity in cancer models and has entered Phase I clinical trials (Chen et al, 2009; Mumenthaler et al, 2009). Considering the roles of the PIM kinases in the regulation of cell survival and proliferation and their high basal expression in AML cells, we hypothesized that SGI-1776 would possess significant anti-leukaemic activity in AML models. We first investigated the in vitro efficacy of SGI-1776 in a panel of nine human AML cell lines (Fig 1A). Treatment of AML cells with SGI-1776 led to a dose-dependent reduction in viability, impaired clonogenic survival (Fig 1B), and apoptotic cell death (Fig 1C, D). These effects were associated with a significant reduction in the phosphorylation of the PIM kinase substrate and apoptotic regulator Bad (Ser112), an event that increases its pro-apoptotic function. The drug-related reduction in Bad phosphorylation did not appear to be due to alterations in AKT activity as SGI-1776 treatment did not significantly affect the phosphorylation of AKT (Thr308) in MV4-11 cells, which have constitutive AKT activity (Fig 1E). Inhibition of PIM kinase signalling disrupts AML cell survival. (A) SGI-1776 causes a dose-dependent reduction in AML cell viability. Nine human AML cell lines were treated with the indicated concentrations of SGI-1776 for 72 h. Cell viability was determined by MTT assay as previously described (Carew et al, 2007). n = 3 Mean ± SD. (B) SGI-1776 diminishes clonogenic survival. Human AML cell lines were treated with SGI-1776 for 24 h. The drug was washed away and cells were plated in MethoCult methylcellulose-containing medium. Colonies were scored 14 d later with the assistance of an Alpha Innotech imaging system (Alpha Innotech, Inc., San Leandro, CA, USA) as previously described (Carew et al, 2007). n = 3 Mean ± SD. (C) SGI-1776 induces apoptosis. Cells were treated with SGI-1776 for 48 h. Apoptosis was quantified by propidium iodide/fluorescence-activated cell sorting (PI/FACS) analysis as previously described (Carew et al, 2007). n = 3 Mean ± SD. (D) SGI-1776 activates caspase-3. Cells were exposed to SGI-1776 for 48 h. The percentages of cells expressing the active form of caspase-3 were determined using the BD Biosciences Active Caspase-3 Mab Apoptosis kit (BD Biosciences, Inc., San Jose, CA, USA) followed by flow cytometry. n = 3 Mean ± SD. (E) SGI-1776 abrogates phosphorylation of the BH3-only protein BAD (Ser112). MV4-11 cells were treated with SGI-1776 for 24 h. Protein lysates were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and the impact of drug treatment on the relative expression levels of phosphorylated BAD, total BAD, phosphorylated AKT, and total AKT were evaluated by immunoblotting. Tubulin served as a loading control. (F) FLT3 inhibition contributes to the efficacy of SGI-1776 in AML. MV4-11 cells with and without stable shRNA-mediated FLT3 knockdown (Swords et al, 2010) were treated with the indicated doses of SGI-1776 for 72 h and the consequential impact on cell viability was determined by MTT assay. n = 3 Mean ± SD, *P < 0·05. Approximately 30% of patients with AML have constitutive fms-like tyrosine kinase-3 (FLT3) activity due to internal tandem duplication (ITD) or activating mutations. Considering that in vitro kinase activity screens with SGI-1776 and other PIM kinase inhibitors have demonstrated some off-target inhibition of FLT3, we utilized MV4-11 cells with stable FLT3 knockdown to investigate whether these potential off-target effects were a critical factor underlying the anti-leukaemic activity of SGI-1776 (Swords et al, 2010). FLT3 knockdown caused a modest reduction in sensitivity to SGI-1776 (Fig 1F), indicating that FLT3 inhibition contributes to the efficacy of SGI-1776, but is not its primary mechanism of action in AML. Several recent studies have suggested a mechanistic link between aberrant expression of PIM kinases and reduced sensitivity to certain anticancer agents (Xie et al, 2006, 2010). To address this issue in a manner relevant to AML therapy, we evaluated the expression levels of PIM1, PIM2, and PIM3 in paired HL-60 cells that are sensitive and resistant to cytarabine (ara-C). Our results showed that the levels of PIM1 and PIM3, but not PIM2, were significantly higher in ara-C-resistant HL-60 cells (Fig 2A, B). Consistent with this observation, ara-C treatment led to increased PIM1 and PIM3 expression as assessed by immunoblotting (MOLM-13 cells, Fig 2C) and quantitative reverse transcription polymerase chain reaction (RT-PCR) (MOLM-13 cells and primary AML blasts, Fig 2D). We next investigated whether inhibiting PIM kinase signalling with SGI-1776 could augment the efficacy of ara-C. Treatment of AML cells with the combination of ara-C and SGI-1776 led to significantly greater diminished viability and inhibition of clonogenic survival over what was achieved by either single agent (Fig 2E, F). Propidium iodide/fluorescence-activated cell sorting (PI/FACS) analysis of the effects of SGI-1776, ara-C, and the combination of these agents on cell cycle distribution showed that SGI-1776 promoted the accumulation of cells with G1 DNA content (Fig 2G). Targeting PIM kinase activity significantly increases the efficacy of cytarabine (ara-C). (A) Ara-C resistance is linked to overexpression of PIM1 and PIM3. The relative expression levels of PIM1, 2, and 3 were evaluated in paired HL-60 cells that are sensitive and resistant to ara-C by immunoblotting. Tubulin documented equal loading. (B) Relative expression levels of PIM kinases in HL-60 ara-C sensitive and –resistant cells. Quantitative RT-PCR was used to assess PIM1, 2, and 3 expression levels, n = 3 Mean ± SD *P < 0·05. (C) Treatment with ara-C induces PIM-1 and PIM-3 expression. Cells were treated with ara-C for 24 h. Protein lysates were subjected to SDS-PAGE. Immunoblotting was utilized to quantify the levels of PIM1, 2, and 3. Tubulin documented equal loading. (D) Impact of ara-C treatment on PIM expression. Primary human AML cells were isolated from the bone marrow of AML patients after obtaining informed consent. MOLM-13 cells and primary blasts from two patients with AML were treated with ara-C for 24 h. Quantitative RT-PCR was used to assess PIM1, 2, and 3 expression levels, n = 3 Mean ± SD *P < 0·05. (E) SGI-1776 significantly increases the in vitro anticancer activity of ara-C. Cells were treated with SGI-1776, ara-C, or both for 72 h. Cell viability was determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. n = 3 Mean ± SD. *Indicates a significant difference compared to controls or **single agent treatment, P < 0·05. (F) SGI-1776 and ara-C cooperate to disrupt clonogenic survival. Cells were treated with SGI-1776, ara-C, or both for 24 h. Drug-containing medium was washed away and cells were plated in Methocult. Colonies were scored 14 d later with the assistance of an Alpha Innotech imaging system. Mean ± SD, n = 3. *Indicates a significant difference compared to controls or **single agent treatment, P < 0·05. (G) SGI-1776 and ara-C cooperate to disrupt cell cycle kinetics and induce apoptosis. MOLM-13 cells were treated with SGI-1776, ara-C, or the combination for 48 h. PI/FACS was used to evaluate drug-related effects on cell cycle distribution and apoptosis (cells with sub G0/G1 DNA content). Representative histograms are shown. (H) SGI-1776 partially restores the sensitivity of ara-C resistant cells to ara-C treatment. Paired HL-60 cells that are sensitive and resistant to ara-C were treated with SGI-1776, ara-C, or the combination for 72 h. The effects of drug treatment on cell viability were quantified. n = 3 Mean ± SD. (I) Effects of drug treatment on tumour growth. MOLM-13 AML cells were implanted subcutaneously into the flanks of nude mice as previously described (Carew et al, 2008). Mice with palpable tumours were randomized into groups of 10 and treated with vehicle, SGI-1776 (100 mg/kg orally administered 5 d per week for 3 weeks), ara-C (75 mg/kg intraperitoneal injection 3 d per week for 3 weeks), or both drugs for 21 d. Tumour volume was monitored with calliper measurements, *Indicates a significant difference compared to vehicle or **single agent treatment, P < 0·05. (J) Treatment with SGI-1776 and ara-C is well tolerated. Mouse weight (g) was monitored throughout the 21-d treatment regimen. (K) SGI-1776 and ara-C diminish BAD phosphorylation (Ser112), inhibit tumour cell proliferation, and activate apoptosis. Immunohistochemistry was utilized to assess the relative levels of phospho-BAD (p-BAD), total BAD, PCNA, and active caspase-3 (C-3) in tumour sections obtained from animals in all treatment groups. Images were captured using an Olympus microscope with a DP71 camera (Olympus America, Inc., Center Valley, PA, USA) and a 20× objective. (L) Quantification of the relative intra-tumoural expression levels of phospho-BAD, PCNA, and active caspase-3. The relative intensity of phospho-BAD was quantified using image-pro plus software 6.2.1. (Media Cybernetics, Inc., Bethesda, MD, USA) Mean ± SD, n = 5. The percentage of PCNA and active caspase-3 positive cells were scored manually. Mean ± SD, n = 5. *Indicates a significant difference compared to controls or **single agent treatment, P < 0·05. HL-60 ara-C sensitive and resistant cells were utilized to investigate whether targeting PIM kinase activity with SGI-1776 could be used as a strategy to overcome intrinsic ara-C resistance. Our results showed that SGI-1776 partially restored the sensitivity of ara-C resistant cells to ara-C (Fig 2H), indicating that ara-C resistance is a multifaceted problem with multiple underlying mechanisms including PIM overexpression (Fig 2A, B). Additionally, our findings show that abrogating PIM kinase activity could possibly be utilized as a novel approach to improve the therapeutic efficacy of ara-C including in circumstances of de novo ara-C resistance. In order to further investigate the therapeutic utility of this combination, we established AML xenografts in nude mice using the MOLM-13 AML cell line. Mice were randomized into groups of 10 and were administered vehicle, ara-C, SGI-1776, or ara-C and SGI-1776 for 21 d. Treatment with the combination of these two agents was well tolerated and significantly increased the efficacy of single agent ara-C therapy (Fig 2I, J). Immunohistochemical analyzes of tumours from mice revealed that SGI-1776 significantly diminished Bad phosphorylation and cooperated with ara-C in vivo to promote activation of caspase-3 and inhibit tumour cell proliferation (PCNA expression) (Fig 2K, L). Collectively, our data demonstrate that antagonizing PIM kinase activity is an effective treatment approach that represents a new strategy to augment the therapeutic efficacy of ara-C in AML. Further investigations aimed to define the role(s) of PIM kinases in AML pathogenesis and evaluate the therapeutic potential of PIM kinase inhibition as a strategy to circumvent drug resistance are warranted. The authors would like to thank Dr Kapil Bhalla for kindly providing cytarabine-resistant HL-60 cells. This work was supported by grants from LeukemiaTexas, the AT&T Foundation and the J.C. & Irene H. Heyser Myeloma Endowment. KRK was involved in all aspects of the study including experimental design, performing research, data analysis, and manuscript preparation; CME provided intellectual input regarding experimental design and data interpretation, performed research, and was involved in the preparation of the manuscript; PT, GC, and FJG provided intellectual input regarding experimental design and data interpretation; SP obtained primary patient specimens and provided intellectual input regarding experimental design, data interpretation, and manuscript preparation; STN provided intellectual input regarding experimental design, data interpretation, and manuscript preparation; JSC directed the study and was involved in all aspects of experimental design, data analysis/interpretation, and manuscript preparation. Pietro Taverna and Gavin Choy are employees of Supergen Inc.

Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC.ELR510444 decreased HIF-1α and HIF-2α levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis.ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascular disrupting and anti-angiogenic properties. Further investigation of ELR510444 for the therapy of RCC is warranted.

Multiple myeloma (MM) is a genetically complex disease. Identification of mutations and aberrant signaling pathways that contribute to the progression of MM and drug resistance has potential to lead to specific targets and personalized treatment. Aberrant signal pathways include RAS pathway activation due to RAS or BRAF mutations (targeted by vemurafenib alone or combined with cobimetinib), BCL-2 overexpression in t(11:14) (targeted by venetoclax), JAK2 pathway activation (targeted by ruxolitinib), NF-κB pathway activation (treated with DANFIN combined with bortezomib), MDM2 overexpression, and PI3K/mTOR pathway activation (targeted by BEZ235). Cyclin D1 (CCND1) and MYC are also emerging as key potential targets. In addition, histone deacetylase inhibitors are already in use for the treatment of MM in combination therapy, and targeted inhibition of FGFR3 (AZD4547) is effective in myeloma cells with t(4;14) translocation. Bromodomain and extra terminal (BET) protein antagonists decrease the expression of MYC and have displayed promising antimyeloma activity. A better understanding of the alterations in signaling pathways that promote MM progression will further inform the development of precision therapy for patients.

MLN4924 (pevonedistat) is a first-in-class NEDD8-activating enzyme (NAE) inhibitor in clinical trials for the treatment of solid tumors and hematologic malignancies. Despite the promising activity of MLN4924 observed in early trials, drug resistance has been noted in some patients. Identifying the underlying cause of treatment failure may help to better stratify patients that are most likely to benefit from this novel agent. Early preclinical studies revealed that the development of NAE mutations promotes resistance to MLN4924. However, these mutations have not been detected in patients that are relapsed/refractory to MLN4924, suggesting that other mechanisms are driving clinical resistance. To better understand the potential mechanisms of MLN4924 resistance, we generated MLN4924-resistant ovarian cancer cells. Interestingly, these cells did not develop mutations in NAE. Transcriptome analyses revealed that one of the most upregulated genes in resistant cells was ABCG2. This result was validated by quantitative real-time PCR and immunoblotting. Importantly, the sensitivity of MLN4924-resistant cells was restored by lentiviral short hairpin RNA (shRNA) targeting ABCG2. Further investigation using ABCG2-overexpressing NCI-H460/MX20 cells determined that these cells are resistant to the anticancer effects of MLN4924 and can be sensitized by co-treatment with the ABCG2 inhibitors YHO-13351 and fumitremorgin C. Finally, HEK293 models with overexpression of wild-type ABCG2 (R482) and variants (R482G and R482T) all demonstrated significant resistance to MLN4924 compared to wild-type cells. Overall, these findings define an important molecular resistance mechanism to MLN4924 and demonstrate that ABCG2 may be a useful clinical biomarker that predicts resistance to MLN4924 treatment.

Pancreatic cancer is the fourth leading cause of cancer-related death in the US. However, there is a growing belief that novel biological agents could improve survival of patients with this cancer. Gemcitabine-based chemotherapy remains the cornerstone treatment for advanced pancreatic cancers. So far, the current targeted agents that have been used in combination with gemcitabine have failed to improve clinical outcomes. This failure may stem from the heterogeneous molecular pathogenesis of pancreatic cancers, which involves several oncogenic pathways and defined genetic mutations.The aims of this review are: i) to define the existing treatments available at present for patients with pancreatic cancers in the neo-adjuvant, adjuvant, locally advanced and metastatic settings; ii) to highlight the molecular heterogeneity of the cancers and the rationale for targeting specific oncogenic pathways; iii) to give an overview of targeted agents that may potentially have an impact in the treatment of pancreatic cancers.Molecular pathogenesis of pancreatic cancer involves several pathways and defined genetic mutations. Targeting these complex molecular pathways with a combination of novel biological and chemotherapeutic agents could potentially improve patient outcome.

Abstract The use of combination drug regimens has dramatically improved the clinical outcome for patients with multiple myeloma. However, to date, combination treatments have been limited to approved drugs and a small number of emerging agents. Using a systematic approach to identify synergistic drug combinations, combination high-throughput screening (cHTS) technology, adenosine A2A and β-2 adrenergic receptor (β2AR) agonists were shown to be highly synergistic, selective, and novel agents that enhance glucocorticoid activity in B-cell malignancies. Unexpectedly, A2A and β2AR agonists also synergize with melphalan, lenalidomide, bortezomib, and doxorubicin. An analysis of agonists, in combination with dexamethasone or melphalan in 83 cell lines, reveals substantial activity in multiple myeloma and diffuse large B-cell lymphoma cell lines. Combination effects are also observed with dexamethasone as well as bortezomib, using multiple myeloma patient samples and mouse multiple myeloma xenograft assays. Our results provide compelling evidence in support of development of A2A and β2AR agonists for use in multi-drug combination therapy for multiple myeloma. Furthermore, use of cHTS for the discovery and evaluation of new targets and combination therapies has the potential to improve cancer treatment paradigms and patient outcomes. Mol Cancer Ther; 11(7); 1432–42. ©2012 AACR.

Abstract Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.

Abstract Despite the promising antilymphoma activity of histone deacetylase (HDAC) inhibitors as a drug class, resistance is a significant clinical issue. Elucidating the molecular mechanisms driving HDAC inhibitor resistance and/or the specific targets that are altered in drug-resistant cells may facilitate the development of strategies that overcome drug resistance and are more effective for refractory patients. We generated novel T-cell lymphoma (TCL) cell line models of acquired resistance to the HDAC inhibitor belinostat to identify potential effective therapies. Belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including romidepsin, panobinostat, and vorinostat. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones. Drug-resistant cells featured significantly decreased expression of the key antiviral mediators IRF1 and STAT1. On the basis of these findings, we investigated the efficacy of the clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that HDAC inhibitor–resistant cells displayed enhanced vulnerability to reovirus replication and cell death in both in vitro and in vivo models compared with their parental counterparts. Importantly, Reolysin also significantly increased the antilymphoma activity of belinostat in HDAC inhibitor–resistant cells. Our data demonstrate that Reolysin alone or in combination with belinostat is a novel therapeutic strategy to treat TCL patients who develop resistance to HDAC inhibitors.

Abstract Purpose: Our preclinical studies showed that the oncolytic reovirus formulation pelareorep (PELA) has significant immunomodulatory anti-myeloma activity. We conducted an investigator-initiated clinical trial to evaluate PELA in combination with dexamethasone (Dex) and bortezomib (BZ) and define the tumor immune microenvironment (TiME) in patients with multiple myeloma treated with this regimen. Patients and Methods: Patients with relapsed/refractory multiple myeloma (n = 14) were enrolled in a phase Ib clinical trial (ClinicalTrials.gov: NCT02514382) of three escalating PELA doses administered on Days 1, 2, 8, 9, 15, and 16. Patients received 40 mg Dex and 1.5 mg/m2 BZ on Days 1, 8, and 15. Cycles were repeated every 28 days. Pre- and posttreatment bone marrow specimens (IHC, n = 9; imaging mass cytometry, n = 6) and peripheral blood samples were collected for analysis (flow cytometry, n = 5; T-cell receptor clonality, n = 7; cytokine assay, n = 7). Results: PELA/BZ/Dex was well-tolerated in all patients. Treatment-emergent toxicities were transient, and no dose-limiting toxicities occurred. Six (55%) of 11 response-evaluable patients showed decreased paraprotein. Treatment increased T and natural killer cell activation, inflammatory cytokine release, and programmed death-ligand 1 expression in bone marrow. Compared with nonresponders, responders had higher reovirus protein levels, increased cytotoxic T-cell infiltration posttreatment, cytotoxic T cells in significantly closer proximity to multiple myeloma cells, and larger populations of a novel immune-primed multiple myeloma phenotype (CD138+ IDO1+HLA-ABCHigh), indicating immunomodulation. Conclusions: PELA/BZ/Dex is well-tolerated and associated with anti–multiple myeloma activity in a subset of responding patients, characterized by immune reprogramming and TiME changes, warranting further investigation of PELA as an immunomodulator.

Abstract Background: While novel agents have improved the outcome for multiple myeloma (MM), the disease remains incurable. Our preclinical work has shown that MM cells from relapsed/refractory patients are very sensitive to the combination of Reolysin (a proprietary formulation of an oncolytic reovirus) and bortezomib (BZ), resulting in synergistic levels of endoplasmic reticulum (ER) stress. A pilot phase 1 study showed that Reolysin was well tolerated in relapsed/refractory MM patients and was associated with prolonged stable disease. Methods: Relapsed/refractory MM patients including patients refractory to BZ were included. This is a phase 1b study of 3 escalating doses of Reolysin (cohort 1; 3 x1010 TCID50, cohort 2; 4.5 x1010 TCID50, and cohort 3; 9 x1010 TCID50). Reolysin is given on days 1, 2, 8, 9, 15, and 16. Patients receive 40 mg dexamethasone and 1.5 mg/m2bortezomib on days 1, 8, and 15. Cycles are repeated every 28 days in the absence of disease progression or unacceptable toxicity. Results: Eight patients have been enrolled, seven were male and the median age was 55 (range, 33 - 66). The median number of prior therapies was 4 (range 1 to 6). All patients were previously exposed to BZ, and 6 patients were previously exposed to both an immunomodulatory agent and carfilzomib. Most patients had ISS stage I disease at study entry (n=5), 2 had stage II and 1 had stage III. The combination was well tolerated and most treatment-related toxicities were transient and easily managed with supportive care. The most common treatment-related toxicities were grade 1 diarrhea (n=4), grade 1 fatigue (n=4), grade 1 flu-like symptoms (n=5) and grade 1 headache (n=4). No dose limiting toxicities occurred in cohort 1 or 2. Three patients completed 1 cycle of treatment only, 2 completed 3 cycles, and 1 patient completed 4 cycles. One patient remains on treatment. Reasons for treatment discontinuation included disease progression (n=4), clinical deterioration (n=1) and patient withdrawal (n=2). Six patients were evaluable for response, 3 patients had stable disease lasting at least 3 cycles whereas 3 patients had progressive disease at the end of cycle 1. Ex vivo treatment of primary MM cells and MM cell lines (U266 and RPMI-8226) with Reolysin revealed a dramatic induction of PD-L1 expression as measured by qRT-PCR and flow cytometry. Furthermore, ex vivo treatment of MM patient mononuclear cells with Reolysin resulted in NK and T cell activation. These results suggest that the addition of an anti-PD-1 or anti-PD-L1 agent may augment the anti-MM activity of Reolysin. Conclusions: The combination of Reolysin, BZ and dexamethasone is well tolerated in a heavily pretreated MM patient population. Cohort 3 is currently enrolling at the 9 x1010 TCID50 dose level of Reolysin. The potential anti-MM effects of immune activation following Reolysin infusion may be mitigated by MM expression of PD-L1. Additional cohorts exploring the tolerability, efficacy and pharmacodynamics of the combination of Reolysin, BZ or carfilzomib, dexamethasone and an immune checkpoint inhibitor will be added following completion of cohort 3. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment. Gill:Oncolytics Biotech: Employment.

Abstract Autophagy contributes to therapeutic resistance and malignant progression by generating alternative metabolic fuel to maintain cell survival under stress conditions including those imposed by hypoxia, radiation, chemotherapy, and targeted agents. The FDA approved anti-malarial drug hydroxychloroquine (HCQ) inhibits autophagy through the disruption of lysosomal function. Robust efforts to repurpose HCQ for cancer therapy based on these properties stimulated numerous clinical trials where HCQ was combined with an array of other anticancer regimens and ultimately produced modest clinical activity. However, it was unclear if the maximum tolerated dose of HCQ was sufficient to completely inhibit autophagy in tumors. New autophagy inhibitors with increased potency and more favorable therapeutic indices are clearly needed to rigorously investigate the potential benefit of this approach. Polyvalent molecules can yield nonlinear, multifold potency against their respective targets compared to their corresponding monomers. We generated a series of novel dimeric compounds containing modified core elements of HCQ, CQ and the anti-schistosomal drug lucanthone with the goal of developing new autophagy inhibitors with superior potency, tolerability, and anticancer efficacy. Initial screens that tested for drug-induced increased expression of p62, a protein that is specifically turned over by autophagy, and anticancer activity identified ROC-325 as a lead agent. The structure of ROC-325 was confirmed by NMR and MS analyses. Direct comparison of the in vitro activity of ROC-325 and HCQ in 13 different genetically and histologically diverse human cancer cell lines demonstrated that ROC-325 was approximately 10-fold more potent than HCQ based on IC50 analyses. Acute myeloid leukemia (AML) was selected as a primary malignancy for intensive investigation based on the high sensitivity of FLT3-ITD+ MV4-11 cells to this agent in preliminary studies. Transmission electron microscopy analyses demonstrated that treatment with ROC-325 triggered all of the hallmark features of autophagy inhibition including the accumulation of autophagosomes with undegraded cargo, an increase in lysosomal membrane permeability, deacidification of lysosomes, and elevated LC3B, p62, and cathepsin D expression. Bafilomycin A1 clamp experiments showed that ROC-325 potently inhibited autophagic flux. Genetic impairment of two different genes that are essential for functional autophagy, ATG5 and ATG7, using lentiviral shRNA approaches significantly diminished the anticancer effects of ROC-325, thus indicating that autophagy inhibition is a key component of its anticancer mechanism of action.RNA sequencing and gene level analyses demonstrated that treatment with ROC-325 (1 µM) in MV4-11 cells altered the levels of autophagy-dependent degradation pathways while preserving protein synthesis through the upregulation of post-translational ribosomal, methylation, and splicing components. In vitro treatment of a panel of human AML cell lines and normal human bone marrow progenitors demonstrated that ROC-325 diminished AML cell viability (IC50 range 0.7-2.2 µM), antagonized clonogenic survival, and induced apoptosis in a manner that was therapeutically selective. Analysis of primary blasts from patients with AML showed that its activity was not significantly affected by adverse cytogenetics or multi-drug resistance due to relapsed/refractory clinical status. Oral administration of 50 mg/kg ROC-325 (QDx5) to mice bearing disseminated MV4-11 human AML xenografts significantly increased lifespan (P&lt;0.05) and enhanced the efficacy of azacitidine (5 mg/kg IV, 2X per week). ROC-325 was well tolerated and no notable toxicities were observed other than a modest, non-significant reversible reduction in mean body weight. Immunohistochemical analysis of specimens collected from animals treated with ROC-325 demonstrated significant increases in the autophagic markers LC3B and p62 and apoptotic blasts. Our data demonstrate that ROC-325 is a novel orally available autophagy inhibitor that is well tolerated, significantly more potent than HCQ, and has promising anti-AML activity. These findings support further investigation of the safety and efficacy of ROC-325 as a novel agent for the treatment of AML and other disorders where lysosomal activity contributes to disease pathogenesis. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau.

Autophagy is a mechanism of lysosomal proteolysis that is utilized to degrade damaged organelles, proteins, and other cellular components. Although key studies demonstrate that autophagy functions as a mechanism of tumor suppression via the degradation of defective pre-malignant cells, autophagy can also be used as a mechanism to break down cellular components under stress conditions to generate the required metabolic materials for cell survival. Autophagy has emerged as an important mediator of resistance to radiation, chemotherapy, and targeted agents. This series of articles highlight the role of autophagy in cancer progression and drug resistance and underscores the need for new and more effective agents that target this process.

Head and neck cancer is diagnosed in nearly 900,000 new patients worldwide each year. Despite this alarming number, patient outcomes, particularly for those diagnosed with late-stage and human papillomavirus (HPV)-negative disease, have only marginally improved in the last three decades. New therapeutics that target novel pathways are desperately needed. NEDDylation is a key cellular process by which NEDD8 proteins are conjugated to substrate proteins in order to modulate their function. NEDDylation is closely tied to appropriate protein degradation, particularly proteins involved in cell cycle regulation, DNA damage repair, and cellular stress response. Components of the NEDDylation pathway are frequently overexpressed or hyperactivated in many cancer types including head and neck cancer, which contribute to disease progression and drug resistance. Therefore, targeting NEDDylation could have a major impact for malignancies with alterations in the pathway, and this has already been demonstrated in preclinical studies and clinical trials. Here, we will survey the mechanisms by which aberrant NEDDylation contributes to disease pathogenesis and discuss the potential clinical implications of inhibiting NEDDylation as a novel approach for the treatment of head and neck cancer.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 1-year survival rate of approximately 18% for all stages of the disease. Currently, FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone, often limiting their use to patients with good performance status (PS). REOLYSIN® (proprietary isolate of reovirus Type 3 Dearing) has shown promising antitumor activity in preclinical models, attributed to the ability of reovirus to preferentially replicate in cells with activated RAS pathway. In addition to direct cytotoxic effects on cancer cells, REOLYSIN can also trigger an antitumor immune response. Due to the high frequency of RAS mutations in PDAC, we hypothesized REOLYSIN would promote selective reovirus replication in pancreatic tumors and enhance anticancer activity of gemcitabine. Methods: Patients with chemotherapy-naïve, advanced or metastatic PDAC were eligible for this phase 2 study. The primary objective was to assess Clinical Benefit Rate (CBR) defined as Complete Response (CR) + Partial Response (PR) + Stable Disease (SD) ≥12 weeks. Secondary objectives included determination of progression-free survival (PFS), overall survival (OS), toxicity profile, and pharmacodynamic analysis of viral replication in patients consenting to on-treatment biopsy. Patients were treated every 3 weeks with 800mg/m2 gemcitabine on days 1 + 8 and REOLYSIN 1x1010 TCID50 REOLYSIN IV on days 1,2,8 + 9. Tumor assessment was performed every 6 weeks. A Simon-two stage design was used where at least 3/17 patients must achieve CBR in order to proceed to stage 2. Results: 34 patients were enrolled. Mean age was 66 years (range 48-85) with 53% of patients above 65 years. M/F: 53/47%, 71% Caucasian, and 94% with PS 0-1; 94% of patients had metastatic disease (62% with liver metastases, 18% with peritoneal involvement). Median number of cycles was 4 with 29 patients evaluable for response. One confirmed PR, 23 SD, and 5 had progressive disease as best response, with 70% of patients having a CA19.9 ≤20% from baseline. The treatment was well tolerated with manageable non-hematological toxicities, including grade 3-4 asthenia (38%), fever (12%), chills (3%), flu-like syndrome (3%), nausea/vomiting (3%), diarrhea (9%), neutropenia (18%). Median PFS was 4 months; 53% of patients received post-PD chemotherapy, including 12% nab-paclitaxel. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24% respectively, with a median duration of follow-up of 2 years. One patient with KRAS G12D mutation displayed positive staining for reoviral protein and activated caspase 3 by immunohistochemistry (IHC) in tumor biopsies. Fluorescent in situ hybridization demonstrated co-expression of reoviral protein and caspase-3 consistent with a productive lytic infection. Further, we also reveal upregulation of immune markers including PD-L1 on IHC following Reolysin therapy. Conclusion: REOLYSIN in combination with gemcitabine is tolerable and an effective alternative to single agent gemcitabine with a promising survival advantage that requires further validation in randomized trials. Pharmacodynamic analysis reveals reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1, suggest future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors.

Here we investigate single codon SF3B1 mutations (K700E, K666, H662) to identify whether discrete amino acid substitutions may influence clinical phenotypes, survival and response to treatments in ...

Abstract Introduction: Paraneoplastic leukemoid reaction (PLR) comprises 10% of leukemoid reactions among patients with solid tumors, especially those with pulmonary malignancy and metastatic disease. Defined as a white blood cell (WBC) count of &gt;50 x 109/L with mature, non-clonally derived neutrophils, and without tumor involvement in the bone marrow, PLR is associated with a poor prognosis. Systemic inflammation promotes tumor growth and metastasis; however, the mechanisms underlying PLR are not well elucidated. We performed a comprehensive clinico-pathological-molecular analysis of cytokines and gene expression in bone marrow, metastatic tumor, and serum from a 76-year-old man with metastatic poorly differentiated non-small cell lung cancer (NSCLC), hyperleukocytosis (WBC 146.5 x 109/L) and extreme neutrophilia (96% neutrophils; absolute neutrophil count 140.6 x 109/L). Methods: After informed consent, we conducted an extensive clinical evaluation of the patient's neutrophilic hyperleukocytosis. We examined the patient's peripheral blood, serum and bone marrow via histologic examination, flow cytometry, cytogenetics, and fluorescent in situ hybridization (FISH) following established protocols. We evaluated levels of 12 cytokines (G-CSF, GM-CSF, IFN-gamma, IL-1a, -2, -4, -6, -8, -10, -12, and -17a, and TNF-a) in serum by enzyme-linked immunosorbent assay (ELISA; Quantikine, R&amp;D Systems). We compared gene expression of 30 cytokines and their receptors (CSF2/R, CSF3/R, IFN-gamma/R1, IL-1A/B/R1/RN, IL-2/RA/RB/RG, IL-4/R, IL-6/R, CXCL8/CXCR1, IL-10/RA, IL-12A/RB2, IL17/RA, and TNF-a/RSF1A/1B) on paraffin-fixed samples of the patient's NSCLC and on an age- and gender-matched sample of NSCLC from a patient without PLR (Geneticist Inc. Biorepository), using real-time polymerase chain reaction (PCR; RT2 Profiler, QIAGEN). Results: We confirmed the diagnosis of PLR after an extensive evaluation did not show any infectious or clonal myeloproliferative process. The peripheral blood smear showed marked leukocytosis, composed mainly of mature neutrophils and mild absolute monocytosis without circulating blasts or atypical cells; there was also a normochromic, normocytic anemia (hemoglobin 11.9 g/dL, hematocrit 35.7%, and MCV 94 fl) and mild thrombocytopenia (platelets 148 x 109/L). Flow cytometric analysis of peripheral blood showed granulocyte predominance (98.8% of the events) and no blast population. Bone marrow showed 40-50% cellularity, trilineage hematopoiesis, and myeloid: erythroid ratio 14.3, without dysplasia, increased blasts and metastatic cancer. FISH analysis with extended acute leukemia panel probe showed no malignancy. Cytogenetics showed 46, XY, and PCR studies were negative for mutations of JAK2 V617F and CSF3R. Serum levels of IFN-G, IL-2, IL-4, IL-10, IL-12, and IL-17a were modestly elevated relative to normal values (3.1- to 6.3-fold increase), while level of IL-1a was decreased (0.7 normal level). In contrast, the serum levels of GM-CSF (40.06 pg/mL), G-CSF (1880.63 pg/mL), and IL-6 (361.91 pg/mL) were all markedly elevated above normal by 48.2-fold, 40.1-fold, and 72.4-fold, respectively. When compared with control non-PLR NSCLC tissue, the patient's tumor showed 3-fold overexpression of the G-CSF receptor, 13.3-fold overexpression of the GM-CSF receptor, and 1.7-fold overexpression of the IL-6 receptor. However, neither PLR nor control NSCLC samples showed increased expression of genes for those cytokines (Table 1). Conclusion: In this comprehensive mechanistic analysis of PLR, we have shown that the metastatic NSCLC tumor overexpressed genes for receptors for G-CSF, GM-CSF, and IL-6, but did not overexpress the genes for those cytokines. Significantly elevated serum levels of G-CSF, GM-CSF and IL-6, synthesized from non-tumor tissues, caused hyperleukocytosis. We hypothesize that an autocrine positive feedback loop, in which these cytokines led to autostimulation of their respective aberrantly expressed receptors on tumor cells, resulted in tumor proliferation as well as off-target stimulation of granulocytopoiesis and corresponding PLR. Disclosures No relevant conflicts of interest to declare.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects at least 10 million people worldwide and is associated with the development of T-cell lymphoma (TCL). The treatment of TCL remains challenging and new treatment options are urgently needed. With the goal of developing a novel therapeutic approach for TCL, we investigated the activity of the clinical formulation of oncolytic reovirus (Reolysin, Pelareorep) in TCL models. Our studies revealed that HTLV-1-negative TCL cells were highly sensitive to Reolysin-induced cell death, but HTLV-1-positive TCL cells were resistant. Consistent with these data, reovirus displayed significant viral accumulation in HTLV-1-negative cells, but failed to efficiently replicate in HTLV-1-positive cells. Transcriptome analyses of HTLV-1-positive vs. negative cells revealed a significant increase in genes associated with retroviral infection including interleukin-13 and signal transducer and activator of transcription 5 (STAT5). To investigate the relationship between HTLV-1 status and sensitivity to Reolysin, we infected HTLV-1-negative cells with HTLV-1. The presence of HTLV-1 resulted in significantly decreased sensitivity to Reolysin. Treatment with the JAK inhibitor ruxolitinib suppressed STAT5 phosphorylation and expression of the key anti-viral response protein MX1 and enhanced the anti-TCL activity of Reolysin in both HTLV-1-positive and negative cells. Our data demonstrate that the inhibition of the JAK/STAT pathway can be used as a novel approach to antagonize the resistance of HTLV-1-positive cells to oncolytic virus therapy.

Abstract Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202). We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off &gt;20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1K700E compared to SF3B1WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1K700E patients showed increased autophagosomes compared to SF3B1WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08). In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.

&lt;div&gt;Abstract&lt;p&gt;The use of combination drug regimens has dramatically improved the clinical outcome for patients with multiple myeloma. However, to date, combination treatments have been limited to approved drugs and a small number of emerging agents. Using a systematic approach to identify synergistic drug combinations, combination high-throughput screening (cHTS) technology, adenosine A2A and β-2 adrenergic receptor (β2AR) agonists were shown to be highly synergistic, selective, and novel agents that enhance glucocorticoid activity in B-cell malignancies. Unexpectedly, A2A and β2AR agonists also synergize with melphalan, lenalidomide, bortezomib, and doxorubicin. An analysis of agonists, in combination with dexamethasone or melphalan in 83 cell lines, reveals substantial activity in multiple myeloma and diffuse large B-cell lymphoma cell lines. Combination effects are also observed with dexamethasone as well as bortezomib, using multiple myeloma patient samples and mouse multiple myeloma xenograft assays. Our results provide compelling evidence in support of development of A2A and β2AR agonists for use in multi-drug combination therapy for multiple myeloma. Furthermore, use of cHTS for the discovery and evaluation of new targets and combination therapies has the potential to improve cancer treatment paradigms and patient outcomes. &lt;i&gt;Mol Cancer Ther; 11(7); 1432–42. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Although cisplatin remains a backbone of standard-of-care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30%–40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models. We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin-interacting protein (TXNIP)-mediated mechanism. Cotreatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism. Significance: Cisplatin therapy is associated with significant nephrotoxicity, which limits its clinical use. Here we demonstrate that NEDDylation inhibition with pevonedistat is a novel approach to selectively prevent cisplatin-induced oxidative damage to the kidneys while simultaneously enhancing its anticancer efficacy. Clinical evaluation of the combination of pevonedistat and cisplatin is warranted.

You have accessJournal of UrologyCME1 Apr 2023MP28-15 A NEW STRATEGY TO ENHANCE CISPLATIN EFFICACY WHILE REDUCING NEPHROTOXICITY THROUGH TARGETED NEDDYLATION INHIBITION Kyle Garcia, Trace Jones, Claudia Espitia, Juan Chipollini, Benjamin Lee, Jason Wertheim, Jennifer Carew, and Steffan Nawrocki Kyle GarciaKyle Garcia More articles by this author , Trace JonesTrace Jones More articles by this author , Claudia EspitiaClaudia Espitia More articles by this author , Juan ChipolliniJuan Chipollini More articles by this author , Benjamin LeeBenjamin Lee More articles by this author , Jason WertheimJason Wertheim More articles by this author , Jennifer CarewJennifer Carew More articles by this author , and Steffan NawrockiSteffan Nawrocki More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003256.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although cisplatin remains a backbone of standard of care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30-40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the anticancer efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models METHODS: The effects of pevonedistat and cisplatin on normal renal proximal epithelial cells were evaluated using in vitro and in vivo models. Molecular and biological experiments were performed to investigate the cytoprotective effects of pevonedistat from cisplatin-mediated nephrotoxicity. RESULTS: We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin interacting protein (TXNIP)-mediated mechanism. Co-treatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. CONCLUSIONS: Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism. A clinical trial further investigating the safety and efficacy of this approach is being planned. Source of Funding: Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award numbers T32CA009213, R01CA268383, R01CA190789 and P30CA023074 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e377 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyle Garcia More articles by this author Trace Jones More articles by this author Claudia Espitia More articles by this author Juan Chipollini More articles by this author Benjamin Lee More articles by this author Jason Wertheim More articles by this author Jennifer Carew More articles by this author Steffan Nawrocki More articles by this author Expand All Advertisement PDF downloadLoading ...

&lt;div&gt;Abstract&lt;p&gt;Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH&lt;sub&gt;2&lt;/sub&gt;-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy. [Cancer Res 2008;68(12):4783–90]&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21&lt;sup&gt;+/+&lt;/sup&gt; cells but not in the HCT116 p21&lt;sup&gt;−/−&lt;/sup&gt; cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA–mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab. [Cancer Res 2007;67(14):6987–94]&lt;/p&gt;&lt;/div&gt;

Supplementary Methods and Figure 1 from The Novel Polyamine Analogue CGC-11093 Enhances the Antimyeloma Activity of Bortezomib

&lt;div&gt;Abstract&lt;p&gt;Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH&lt;sub&gt;2&lt;/sub&gt;-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy. [Cancer Res 2008;68(12):4783–90]&lt;/p&gt;&lt;/div&gt;

Supplementary Figures 1-2 from Histone Deacetylase Inhibitors Enhance Lexatumumab-Induced Apoptosis via a p21&lt;sup&gt;Cip1&lt;/sup&gt;-Dependent Decrease in Survivin Levels

Supplementary Figures 1-2 from Histone Deacetylase Inhibitors Enhance Lexatumumab-Induced Apoptosis via a p21&lt;sup&gt;Cip1&lt;/sup&gt;-Dependent Decrease in Survivin Levels

&lt;div&gt;Abstract&lt;p&gt;Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21&lt;sup&gt;+/+&lt;/sup&gt; cells but not in the HCT116 p21&lt;sup&gt;−/−&lt;/sup&gt; cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA–mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab. [Cancer Res 2007;67(14):6987–94]&lt;/p&gt;&lt;/div&gt;

Supplementary Methods and Figure 1 from The Novel Polyamine Analogue CGC-11093 Enhances the Antimyeloma Activity of Bortezomib

&lt;p&gt;PDF file - 71K, Supplemental Table 1. Proteins Altered &gt;2-fold by Treatment with MLN4924.&lt;/p&gt;

&lt;p&gt;PDF file - 3554K, Supplemental Figure 2: Effects of caspase inhibition and antioxidant treatment on MLN4924/cisplatin-induced DNA damage.&lt;/p&gt;

&lt;p&gt;PDF file - 1755K, Supplemental Figure 1: Effects of targeted p65 knockdown on cisplatin sensitivity.&lt;/p&gt;

&lt;p&gt;Supplemental Table 1. Clinical characteristics of AML patients who provided specimens for this study.&lt;/p&gt;

&lt;p&gt;Supplemental Figure 1 Part 3. Quantification of metabolomic data continued.&lt;/p&gt;