Jennifer S. Carew

Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser(112), relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently induced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications.

Mitochondria play important roles in cellular energy metabolism, free radical generation, and apoptosis. Defects in mitochondrial function have long been suspected to contribute to the development and progression of cancer. In this review article, we aim to provide a brief summary of our current understanding of mitochondrial genetics and biology, review the mtDNA alterations reported in various types of cancer, and offer some perspective as to the emergence of mtDNA mutations, their functional consequences in cancer development, and therapeutic implications.

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide <mml:math><mml:mi>O</mml:mi><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mo>·</mml:mo><mml:mo>¯</mml:mo></mml:math> radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I. Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to an increase in <mml:math><mml:mi>O</mml:mi><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mo>·</mml:mo><mml:mo>¯</mml:mo></mml:math> generation and sensitization of the leukemia cells to anticancer agents whose action involve free radical generation. Using leukemia cells with genetic alterations in mitochondrial DNA and biochemical approaches, we further demonstrated that As₂O₃, a clinically active anti-leukemia agent, inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another <mml:math><mml:mrow><mml:mi>O</mml:mi><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mo>·</mml:mo><mml:mo>¯</mml:mo><mml:mo>-</mml:mo><mml:mi>generating</mml:mi></mml:mrow></mml:math> agent against cultured leukemia cells and primary leukemia cells isolated from patients. Our study shows that interfering mitochondrial respiration is a novel mechanism by which As₂O₃ increases generation of free radicals. This novel mechanism of action provides a biochemical basis for developing new drug combination strategies using As₂O₃ to enhance the activity of anticancer agents by promoting generation of free radicals.

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.

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (ρ-) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic 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.

The biology of acute myelogenous leukemia (AML) is characterized by a block in differentiation, increase in proliferation and inhibition of apoptosis, all of which when combined lead to an expansion of leukemic blasts.1 AML therapy cures ~20% of those affected,2 highlighting the need for a better understanding of leukemia biology in order to identify novel therapeutic targets. The Wilms’ tumor 1 (WT1) gene has an oncogenic role in leukemogenesis and its overexpression correlates with a poor prognosis.3 WTAP is a highly conserved protein that partners with WT14,5 to function as a switch gene, regulating the balance between quiescence and proliferation.6 WTAP-null mice exhibit embryonic lethality.7 WTAP has been recently described as an oncogenic factor in gliomas.8 We hypothesized that alterations in WTAP, whose role in leukemogenesis is unknown, might provide an alternate means of modulating the WT1 pathway in AML. We performed a series of experiments to test this possibility. First, we examined WTAP expression in several primary AML samples observing increased levels of WTAP in AML compared with normal peripheral blood mononuclear cells (Figure 1a) as well as in AML cell lines (data not shown). Next, the WTAP expression levels were determined in 511 newly diagnosed AML patients using the reverse-phase protein array (RPPA) technique. In comparison with normal bone marrow CD34+ cells, WTAP expression in bulk AML cells was above normal levels in 32% of patients (Figure 1b). Although WTAP levels were not associated with individual cytogenetic abnormalities, some specific molecular mutations such as NPM1 and FLT3-ITD were found to have significant correlation (P ≤ 0.05) with WTAP expression (Supplementary Figure 1A). In addition, RPPA analysis showed that WTAP levels were positively correlated (R > |0.2|) with levels of various cell proliferation-related proteins (cyclins and Hsp90), antiapoptotic proteins (Bcl-2 and Bax), oncoproteins (FLI1) and proteins important for stem cell functions such as Myc and Ash2L (Supplementary Figure 1B). To assess the functional significance of increased WTAP expression, its expression was silenced in K562 and HL-60 cells, leading to a significant reduction (P ≤ 0.05) in proliferation (Figure 1c), and clonogenic survival (P ≤ 0.01) (Figure 1d). Similar effects on proliferation were observed in the AML cell line OCI-AML3 and in primary AML cells (Supplementary Figure 1C), suggesting a pro-proliferative role for WTAP in AML. WTAP knockdown alone did not induce apoptosis but markedly increased (P ≤ 0.01) the extent of apoptosis following etoposide treatment (Figure 1e). These results provide evidence for an association between the increased expression of WTAP and chemoresistance in AML. Figure 1 Expression of WTAP in AML and effect of WTAP silencing on AML cell behavior. (a) Peripheral blood mononuclear cells from normal donors (NL) and AML patients (AML) were obtained by Ficoll–Paque density centrifugation, and protein extracts were ... To examine the role of WTAP in AML progression in vivo, we performed tumor xenograft experiments in nude mice. As shown in Figure 1f, the growth rates and masses of tumors derived from WTAP-knockdown cells were significantly reduced (P ≤ 0.01) compared with control. To complement this analysis, the in vitro transforming activity of WTAP was examined by investigating its effects on growth of the Ba/F3 cell line. This line depends on interleukin 3 (IL-3) for survival and proliferation, but this dependence can be released by the transgenic expression of suitable oncogenes.9 Whereas control Ba/F3 cells were not viable in the absence of IL-3 at 72 h, WTAP-expressing Ba/F3 cells were able to maintain growth factor-independent proliferation, as demonstrated by significantly higher (P ≤ 0.01) number of viable cells (Figure 1g), suggesting that WTAP harbors oncogenic activity. The aberrant cellular proliferation and terminal differentiation block of myeloid cells are two hallmarks of AML.10 Having shown that WTAP regulates growth and survival, we investigated whether WTAP has a role in myeloid cell differentiation. As shown in Figure 1h, knockdown of WTAP promoted phorbol 12-myristate 13-acetate (PMA)-induced myeloid differentiation, as revealed by an increase in the expression of myeloid differentiation markers CD11b and CD14 compared with control cells. These results suggest that increased expression of WTAP in AML not only supports cell proliferation but also induces the differentiation block. Our RPPA analysis suggested a link between WTAP and mammalian target of rapamycin (mTOR) expression; and given that the mTOR pathway is deregulated in a number of cancers including AML,11 we hypothesized a putative regulatory role of WTAP on mTOR activity in AML. As shown in Figure 1i, WTAP knockdown induced a decrease in the phosphorylation levels of mTOR and its downstream effector p70 ribosomal subunit 6 kinase (pS6K) compared with control shRNA. To further understand the participation of WTAP in leukemogenesis, we performed transcriptomic analysis with RNA-Seq on WTAP knockdown in K562 cells. Gene ontology analysis indicated that cell adhesion and regulation of cell proliferation are the most enriched functionalities (Supplementary Figure 1D and Supplementary Table 2). Among the most relevant genes affected by WTAP with recognized roles in leukemia are CD4, CD44, CEBPA, CSF1R, MPO, ABCG2, TCL1A, CYP1A1, CYP3A4, FGFR1, PTPRC (CD45), CD83, CD86, CD9 and CCR4. Consistent with its described role in RNA processing, we determined that WTAP knockdown affected exon usage of 93 genes, including important factors such as MLL and MSI2. Interestingly, we also observed that WTAP affects the processing of its own transcript, very likely via polyA site selection. Complete analysis can be accessed in GEO ({type:entrez-geo,attrs:{text:GSE46718,term_id:46718}}GSE46718—http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jbkjjswaqscegjsa Fisher’s exact test) in their mRNA levels as determined by RNA-Seq. Mutations of WTAP were not observed in the TCGA analysis of AML.12 Therefore, the etiology of increased WTAP expression in AML remains unexplained. We next sought to determine the potential mechanism that may contribute to an increase in WTAP expression in AML. The molecular chaperone Hsp90 maintains the stability of many tumor-promoting oncoproteins,13 including WT1.14 Keeping in mind the connection between WT1 and WTAP, we investigated the potential interaction between Hsp90 and WTAP. First, we determined that WTAP co-immunoprecipitates with Hsp90 (Figure 2a), whereas treatment with the Hsp90 inhibitor ganetespib significantly reduced the binding of Hsp90 to WTAP. Therefore, formation of the WTAP–Hsp90 complex is dependent on the chaperoning activity of Hsp90. Studies have shown that Hsp90 client proteins shift the primary chaperone association from Hsp90 to Hsp70 following inhibition of Hsp90 activity.15 Accordingly, our results showed that ganetespib treatment increased WTAP association with Hsp70 (Figure 2a). Furthermore, the GST pull-down assay showed a direct interaction between WTAP and Hsp90 (Figure 2b). To understand the functional significance of the association of Hsp90 with WTAP, we investigated the effects of Hsp90 inhibitors on WTAP protein stability. Ganetespib treatment promoted the degradation of WTAP in K562 (CML), MV4-11 (AML) and Kasumi-1 (AML) cell lines, previously shown to be highly sensitive to ganetespib (Figure 2c).16 Marked reduction in WTAP expression was similarly observed in blasts exposed to ganetespib (Figure 2d). Furthermore, ganetespib inhibited AML tumor growth in nude mice, as we reported previously,14 and WTAP expression in xenograft tumors (Figure 2e). Inhibition of Hsp90 induces polyubiquitination and proteasomal degradation of Hsp90 client proteins.17 We confirmed that pretreatment with proteasomal inhibitor bortezomib largely prevented ganetespib-induced degradation of WTAP (Figure 2f), and higher levels of ubiquitinated WTAP were observed in the presence of the combination of bortezomib and ganetespib compared with either agent alone (Figure 2g). Together, these data show that ganetespib-mediated degradation of WTAP is dependent on the ubiquitin-proteasome pathway, very similar to other bonafide Hsp90 client proteins. Figure 2 Hsp90 associates with WTAP and is necessary for its stability. (a) The K562 cells were treated with vehicle (−) or Hsp90 inhibitor, ganetespib (1 µm for 6 h) followed by immunoprecipitation (IP) with IgG and WTAP antibody. The immunoprecipitates ... In summary, our study shows that WTAP has an important role in abnormal proliferation and arrested differentiation of leukemia cells, and WTAP is a novel client protein of Hsp90. Therefore, WTAP may be a promising novel therapeutic target in AML. Further investigation aimed to elucidate the mechanism of action of WTAP is warranted.

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.

Mitochondrial DNA (mtDNA) mutations and deletions are frequently observed in cancer, and contribute to altered energy metabolism, increased reactive oxygen species (ROS), and attenuated apoptotic response to anticancer agents. The mechanisms by which cells maintain mitochondrial genomic integrity and the reason why cancer cells exhibit more frequent mtDNA mutations remain unclear. Here, we report that the tumor suppressor molecule p53 has a novel role in maintaining mitochondrial genetic stability through its ability to translocate to mitochondria and interact with mtDNA polymerase gamma (pol gamma) in response to mtDNA damage induced by exogenous and endogenous insults including ROS. The p53 protein physically interacts with mtDNA and pol gamma, and enhances the DNA replication function of pol gamma. Loss of p53 results in a significant increase in mtDNA vulnerability to damage, leading to increased frequency of in vivo mtDNA mutations, which are reversed by stable transfection of wild-type p53. This study provides a mechanistic explanation for the accelerating genetic instability and increased ROS stress in cancer cells associated with loss of p53.

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.

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.

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.

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

Mutation of Bcr-Abl is an important mechanism by which chronic myelogenous leukemia (CML) cells become resistant to Gleevec. The T315I mutation is clinically significant since CML cells harboring this mutation are insensitive to Gleevec and other Bcr-Abl-targeted drugs. Identification of new agents capable of effectively killing CML cells with T315I mutation would have important therapeutic implications in Gleevec-resistant CML. Here, we showed that β-phenylethyl isothiocyanate (PEITC), a natural compound found in vegetables, is effective in killing CML cells expressing T315I BCR-ABL. Treatment of leukemia cell lines harboring wild-type or mutant Bcr-Abl with 10 μM PEITC resulted in an elevated ROS stress and a redox-mediated degradation of the BCR-ABL protein, leading to massive death of the leukemia cells. Antioxidant NAC attenuated the PEITC-induced oxidative stress in CML cells and prevented the degradation of BCR-ABL, caspase-3 activation and cell death. We further showed that the ROS-induced degradation of BCR-ABL was mediated partially by caspase-3 and the proteasome pathway. The ability of PEITC to effectively kill T315I-positive CML cells was further confirmed using primary leukemia cells isolated from CML patients. Our results suggest that PEITC is a promising compound capable of killing Gleevec-resistant CML cells through a ROS-mediated mechanism and warrants further investigations.

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.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due, in part, to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) have a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that perhexiline, an antiangina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity, leading to rapid depolarization and massive CLL cell death. The therapeutic activity of perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only four drug injections. Our study suggests that targeting CPT using an antiangina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.

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.

The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.

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.

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]

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.

Abstract Purpose: The prognosis of patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor, and novel therapies are needed. The proteasome pathway represents a potential therapeutic target. A phase I trial of the second-generation proteasome inhibitor ixazomib in combination with MEC (mitoxantrone, etoposide, and cytarabine) was conducted in patients with R/R AML. Patients and Methods: Dose escalation of ixazomib was performed using a standard 3 × 3 design. Gene-expression profiling was performed on pretreatment and posttreatment bone marrow or blood samples. Results: The maximum tolerated dose of ixazomib in combination with MEC was 1.0 mg. The dose limiting toxicity was thrombocytopenia. Despite a poor risk population, the response rate [complete remission (CR)/CR with incomplete count recovery (CRi)] was encouraging at 53%. Gene-expression analysis identified two genes, IFI30 (γ-interferon inducible lysosomal thiol reductase) and RORα (retinoic orphan receptor A), which were significantly differentially expressed between responding and resistant patients and could classify CR. Conclusions: These results are encouraging, but a randomized trial is needed to address whether the addition of ixazomib to MEC improves outcome. Gene-expression profiling also helped us identify predictors of response and potentially novel therapeutic targets.

Medical Journal of AustraliaVolume 2, Issue 20 p. 1016-1017 Reports of Cases SINDBIS VIRUS INFECTION IN AUSTRALIA K. L. Doherty M.B., B.S., M.P.H., M.R.A.C.P., M.C.P.A., K. L. Doherty M.B., B.S., M.P.H., M.R.A.C.P., M.C.P.A. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria Director, Queensland Institute of Medical ResearchSearch for more papers by this authorA. S. Bodey M.B., B.S., B.SC., M.C.P.A., M.C.PATH., A. S. Bodey M.B., B.S., B.SC., M.C.P.A., M.C.PATH. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria Regional Pathologist, Mildura Base HospitalSearch for more papers by this authorJ. S. Carew M.B., B.S., J. S. Carew M.B., B.S. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria General practitioner, The Medical Clinic, MilduraSearch for more papers by this author K. L. Doherty M.B., B.S., M.P.H., M.R.A.C.P., M.C.P.A., K. L. Doherty M.B., B.S., M.P.H., M.R.A.C.P., M.C.P.A. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria Director, Queensland Institute of Medical ResearchSearch for more papers by this authorA. S. Bodey M.B., B.S., B.SC., M.C.P.A., M.C.PATH., A. S. Bodey M.B., B.S., B.SC., M.C.P.A., M.C.PATH. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria Regional Pathologist, Mildura Base HospitalSearch for more papers by this authorJ. S. Carew M.B., B.S., J. S. Carew M.B., B.S. Queensland Institute of Medical Research, Brisbane Mildura Base Hospital, Mildura, Victoria General practitioner, The Medical Clinic, MilduraSearch for more papers by this author First published: 01 November 1969 https://doi.org/10.5694/j.1326-5377.1969.tb107563.xCitations: 27 Address for reprints: Dr R. L. Doherty, Director, Queensland Institute of Medical Research, Herston Road, Herston, Queensland. 4006 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract A child with a febrile illness with vesicular rash showed serological evidence of infection with Sindbis virus. Sindbis virus has been shown to cause similar syndromes in Africa, and further assessment of its importance in human disease in Australia is suggested. Citing Literature Volume2, Issue20November 1969Pages 1016-1017 RelatedInformation

While the bone marrow in most patients with myelodysplastic syndromes (MDS) has normal or increased cellularity (hyper-MDS), approximately 10%–15% of MDS patients will present with a hypocellular bone marrow (hypo-MDS).1 Since the diagnosis of MDS relies mainly on the presence of dyplastic morphology of myeloid precursors and/or the presence of recurrent chromosomal abnormalities, low cellularity aspirates in patients with hypo-MDS may compromise morphologic evaluation and karyotypic analyses, and ultimately contribute to difficulties in distinguishing hypo-MDS from other bone marrow failure syndromes. Further, approximately 50% of MDS patients, including those with hypo-MDS, have normal karyotype by metaphase cytogenetics (MC), which makes the distinction even more difficult.2 We previously reported that single nucleotide polymorphism (SNP) array karyotyping can improve the detection of chromosomal lesions that can distinguish hypo-MDS patients from those with aplastic anemia (AA).3 This finding suggests that clonal abnormalities can be detected in hypo-MDS and AA by using higher sensitivity methods other than MC.

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

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N1,N11-diethylnorspermine (DENSPM) or α-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N1-acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma. The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N1,N11-diethylnorspermine (DENSPM) or α-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N1-acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma. The fluoropyrimidine 5-fluorouracil (5-FU) 1The abbreviations used are: 5-FU, 5-fluorouracil; CI, combination index; CM-H2DCFDA, 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; DENSPM, N1,N11-diethylnorspermine; DFMO, α-difluoromethylornithine; IC25, 25% of inhibitory concentration; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Rho123, rhodamine 123; SSAT, spermidine/spermine N1-acetyltransferase; wt-p53, wild-type p53; PBS, phosphate-buffered saline. 1The abbreviations used are: 5-FU, 5-fluorouracil; CI, combination index; CM-H2DCFDA, 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; DENSPM, N1,N11-diethylnorspermine; DFMO, α-difluoromethylornithine; IC25, 25% of inhibitory concentration; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Rho123, rhodamine 123; SSAT, spermidine/spermine N1-acetyltransferase; wt-p53, wild-type p53; PBS, phosphate-buffered saline. has been widely used a chemotherapy agent in the treatment of many types of solid tumors (1Ansfield F. Klotz J. Nealon T. Ramirez G. Minton J. Hill G. Wilson W. Davis Jr., H. Cornell G. Cancer. 1997; 39: 34-40Crossref Scopus (147) Google Scholar, 2Moertel C.G. Schutt A.J. Reitemeier R.J. Hahn R.G. Cancer Res. 1972; 12: 2717-2719Google Scholar, 3Fine S. Erlichman C. Kaizer L. Warr D. Gadalla T. Breast Cancer Res. Treat. 1994; 30: 205-209Crossref PubMed Scopus (8) Google Scholar). To improve the efficacy of 5-FU, several other drugs such as leucovorin, irinotecan, or oxaliplatin have been used recently in combination with 5-FU in preclinical and clinical trials that have produced promising results (4Calvo E. Cortes J. Rodriguez J. Fernandez-Hidalgo O. Rebollo J. Martin-Algarra S. Garcia-Foncillas J. Martinez-Monge R. de Irala J. Brugarolas A. Clin. Colorectal Cancer. 2002; 2: 104-110Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 5Gil-Delgado M.A. Guinet F. Castaing D. Adam R. Coeffic D. Durrani A.K. Bismuth H. Khayat D. Am. J. Clin. 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Our recent functional genomic studies have rediscovered that 5-FU also affects polyamine pathways to reduce the levels of polyamines in treated colon cancer cells (10Zhang W. Ramdas L. Shen W. Song S.W. Hu L. Hamilton S.R. Cancer Biol. Ther. 2003; 2: 572-578Crossref PubMed Scopus (33) Google Scholar), an observation that was originally made by Russell et al. in 1974 in a liver cancer xenograft mouse model (11Russell D.H. Looney W.B. Kovacs C.J. Hopkins H.A. Marton L.J. LeGendre S.M. Morris H.P. Cancer Res. 1974; 34: 2382-2385PubMed Google Scholar). Thus, it appears that 5-FU targets two major cellular processes, i.e. polyamine metabolism as well as processing and synthesis of DNA and RNA. These two processes may have intimate inter-relationships because polyamines bind DNA and RNA and affect chromosomal structure, RNA stability, and translation (12Snyder R.D. Biochem. J. 1989; 260: 697-704Crossref PubMed Scopus (88) Google Scholar). Spermidine, spermine, and their diamine precursor putrescine are natural polyamines in living organisms and play critical roles in proliferation, differentiation, and homeostasis in both normal and cancer cells. The balance between polyamine biosynthesis and catabolism is tightly regulated to maintain cellular polyamine levels. Mice with disrupted genes important for polyamine biosynthesis, such as ornithine decarboxylase, die early in embryonic development (13Pendeville H. Carpino N. Marine J.C. Takahashi Y. Muller M. Martial J.A. Cleveland J.L. Mol. Cell. Biol. 2001; 21: 6549-6558Crossref PubMed Scopus (189) Google Scholar). Insufficient levels of polyamines result in retarded cell growth and sometimes cell death. Therefore, cellular polyamine depletion through inhibition of the polyamine biosynthetic pathway has been used for chemotherapeutic intervention (14Elmore E. Stringer D.E. Steele V.E. Gerner E.W. Redpath J.L. Anticancer Res. 2001; 21: 1163-1165PubMed Google Scholar, 15Subhi A.L. Diegelman P. Porter C.W. Tang B. Lu Z.J. Markham G.D. Kruger W.D. J. Biol. Chem. 2003; 278: 49868-49873Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Belting M. Borsig L. Fuster M.M. Brown J.R. Persson L. Fransson L.A. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 371-376Crossref PubMed Scopus (104) Google Scholar). DFMO, which is a suicide inhibitor of ornithine decarboxylase, the enzyme that decarboxylates ornithine to produce putrescine, is a well studied inhibitor of the biosynthetic pathway. However, clinical trials using DFMO yielded disappointing results (17Fabian C.J. Kimler B.F. Brady D.A. Mayo M.S. Chang C.H. Ferraro J.A. Zalles C.M. Stanton A.L. Masood S. Grizzle W.E. Boyd N.F. Arneson D.W. Johnson K. Clin. Cancer Res. 2002; 8: 3105-3117PubMed Google Scholar, 18Einspahr J.G. Nelson M.A. Saboda K. Warneke J. Bowden G.T. Alberts D.S. Clin. Cancer Res. 2002; 8: 149-155PubMed Google Scholar, 19Levin V.A. Uhmk J.H. Jaeckle K.A. Choucair A. Flynn P.J. Yung W.K.A. Prados M.D. Bruner J.M. Chang S.M. Kyritsis A.P. Gleason M.J. Hess K.R. Clin. Cancer Res. 2000; 6: 3878-3884PubMed Google Scholar), perhaps because polyamines are often present at high levels in human diet and intestinal lumen, thus bypassing the suppression of polyamine biosynthesis (20Sarhan S. Knodgen B. Seiler N. Anticancer Res. 1992; 12: 457-466PubMed Google Scholar). Nevertheless, because of the low toxicity of DFMO, it has been tested as a chemopreventive agent for colon carcinoma (21Meyskens Jr., F.L. Gerner E.W. Clin. Cancer Res. 1999; 5: 945-951PubMed Google Scholar). Interest in polyamine metabolism has shifted to its catabolism, especially because catabolism has been associated with the anti-tumor effects of several polyamine analogues. DENSPM is an analogue of spermine that has been shown to deplete polyamines by inducing expression of the polyamine catabolism enzyme SSAT through Nrf-2 and polyamine-modulated factor-1 interaction on the SSAT promoter (22Wang Y. Xiao L. Thiagalingam A. Nelkin B.D. Casero Jr., R.A. J. Biol. Chem. 1998; 273: 34623-34630Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 23Wang Y. Devereux W. Stewart T.M. Casero Jr., R.A. J. Biol. Chem. 1999; 274: 22095-22101Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). SSAT acetylates spermine and spermidine, which in turn are substrates for a flavin-dependent polyamine oxidase to produce hydrogen peroxide that is believed to account for the cytotoxicity of DENSPM (24Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 25Chen Y. Kramer D.L. Diegelman P. Vujcic S. Porter C.W. Cancer Res. 2001; 61: 6437-6444PubMed Google Scholar). However, the cytotoxic effect of DENSPM is not observed in all cell types and is often observed only after long exposure, suggesting that the cytotoxicity of DENSPM alone is not very potent. In this study, we investigated whether combinations of 5-FU with drugs that modulate polyamine metabolism may have synergistic effects in inducing colon cancer cell apoptosis. In addition, to evaluate the role of p53 in the response to combination therapy, we used two isogenic HCT116 colon cancer cell lines that were either wild-type for p53 (wt-p53) or had the p53 gene deleted (p53-null) (26Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar, 27Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2527) Google Scholar). Reagents—5-FU, DFMO, and antioxidant GSH were purchased from Sigma, and DENSPM was purchased from Tocris (Ellisville, MI). 5-FU was dissolved in Me2SO, and DFMO and DENSPM were dissolved in water according to the manufacturer's instructions. CM-H2DCFDA and Rho123 were purchased from Molecular Probes (Eugene, OR) and dissolved in Me2SO. Cell Culture—The isogenic HCT116 cell lines with wt-p53 or p53-null background were provided by Dr. Bert Vogelstein (Johns Hopkins University). The cell lines were cultured in Dulbecco's modified Eagle's medium with 10% Nu-serum (Collaborative Research Products, Bedford, MA) at 37 °C in a 5% CO2 incubator. Because fetal bovine serum has abundant thymine and polyamines, we used dialyzed serum to remove these moieties that would interfere with the experimental results. Later experiments showed that fetal bovine serum did not affect the pattern of cellular responses but did shift the dose of drugs required to achieve the same results (data not shown). MTS Cell Viability Assay—CellTiter 96 Aqueous One Solution Reagent (Promega, G358B, Madison, WI) was used to detect viable cells in cytotoxicity experiments. After 20 μl of MTS solution were added into each well, the cells were incubated at 37 °C with 5% CO2 for 2 h before absorbance at 490 nm was measured with a Micro-plate reader (MRX, Danatech Laboratory, Houston, TX). All the MTS assays included 3 to 5 replicates for each treatment condition, and the experiments were repeated at least twice. Drug Treatment Experiments—A total of 3000 cells per well in a volume of 100 μl were plated in each 96-well plate. On the second day, various concentrations of 5-FU were added to the wells, and MTS assays were performed to estimate IC25 values (2.5 μm for HCT116 wt-p53 and 15 μm for HCT116 p53-null). Twenty-four hours after the initial cell seeding, the IC25 dose of 5-FU alone or combinations with various concentrations of DENSPM (1.25, 2.5, 5, and 10 μm) were added. Three different dosing schedules were evaluated. The post-5-FU schedule was 5-FU for 48 h followed by DENSPM for 48 h; the pre-5-FU schedule was DENSPM for 48 h followed by 5-FU for 48 h; and the simultaneous schedule was 5-FU and DENSPM together for 48 h followed by medium alone for 48 h. After drug exposure, the MTS assay was used to measure cell viability. Measurement of Cellular Levels of Polyamines—Treated cells were collected and lysed. The polyamine concentrations were determined by high-pressure liquid chromatography as described previously (28Tome M.E. Fiser S.M. Payne C.M. Gerner E.W. Biochem. J. 1997; 328: 847-854Crossref PubMed Scopus (85) Google Scholar). Cell Cycle Analysis by Flow Cytometry—Cells were seeded in a 10-cm2 dish (one million cells/dish) in 10 ml of medium supplemented with 10% dialyzed fetal bovine serum. After 24 h at 37 °C, drugs were added for 72 h. After being washed with phosphate-buffered saline, cells were harvested by trypsinization and centrifugation, fixed with 70% of ethanol, and then stained with propidium iodide with RNase, followed by analysis on a FACS-Calibur instrument (BD Biosciences, Franklin Lakes, NJ) with CELL Quest software. Real-time Reverse Transcriptase-PCR Methods for SSAT Expression—SSAT and 18 S rRNA were quantitated using the Applied Biosystem TaqMan method in conjunction with Assays-On-Demand (ABI PRISM 7700 Sequence Detection System, Applied Biosystems, Foster City, CA). The PCR mixture consisted of Taqman Master Mix (without uracil-N-glycosylase), MultiScribe reverse transcriptase, RNase inhibitors, and 10 ng of total RNA in a final volume of 50 μl. Thermal cycling was initiated with reverse transcription at 48 °C for 30 min and the Taq Gold activation step at 95 °C for 10 min. The thermal profile for the PCR was 95 °C for 15 s and 60 °C for 1 min. Data were obtained during 40 cycles of amplification. PCR were performed in triplicate for SSAT with 18 S as an internal standard. The levels of SSAT in drug-treated samples relative to untreated control were determined. Microarray Methods for Transcriptome Analysis—Total RNA was isolated, labeled, and hybridized as described (29Hu L. Wang J. Baggerly K. Wang H. Fuller G.N. Hamilton S.R. Coombes K.R. Zhang W. BMC Genomics. 2002; 3: 16-23Crossref PubMed Scopus (61) Google Scholar). After being washed, the slides were scanned with a GeneTAC UC 4 laser scanner (Genomic Solutions, Ann Arbor, MI), and the signal intensities were quantified with ArrayVision™ (Imaging Research Inc., St. Catherine's, Ontario, Canada). The data were analyzed using the in-house program for microarray analysis (30Baggerly K.A. Coombes K.R. Hess K.R. Stivers D.N. Abruzzo L.V. Zhang W. J. Comput. Biol. 2001; 8: 639-659Crossref PubMed Scopus (85) Google Scholar). Each differentially expressed gene was determined from a cutoff value of its T value. Generally, a cutoff value of 3 was considered to be statistically significant based on the statistical variations (30Baggerly K.A. Coombes K.R. Hess K.R. Stivers D.N. Abruzzo L.V. Zhang W. J. Comput. Biol. 2001; 8: 639-659Crossref PubMed Scopus (85) Google Scholar). The pathway array includes 2,016 functionally well characterized genes involved in cell death, cell growth, metabolism, cell invasion, cell communication, immune response, DNA repair, and transcription (generated by the M. D. Anderson Cancer Center Genomics Core Laboratory, www3.mdanderson.org/~genomics). GoMiner was used (discover.nci.nih.gov/gominer) for the analysis of functional gene groups. Western Blotting—Total cellular protein extract was isolated from harvested cells, protein concentration was determined, and Western blotting was carried out as described previously (31Fuller G.N. Rhee C.H. Hess K.R. Caskey L.S. Wang R. Bruner J.M. Yung W.K. Zhang W. Cancer Res. 1999; 59: 4228-4232PubMed Google Scholar). The antibodies used were anti-p53 monoclonal antibody (Ab-6, Oncogene Science, Manhasset, NY), anti-p21Waf1 mouse hybridoma (a gift from Dr. Wade Harper at Baylor College of Medicine, Houston, TX), anti-caspase 3 (R & D Systems, Minneapolis, MN), anti-mdm2, anti-cyclin A2, anti-cyclin B1, anti-Fas, anti-β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and antibody for cleaved caspase 9 (Cell Signaling, Beverly, MA). Cytochrome c Release Assay—Drug-treated cells were harvested for separation of mitochondria and cytosol by use of ApoAlert™ cell fractionation kit (BD Biosciences) according to the instructions from the manufacturer. Antibody for cytochrome c included in the kit was used in Western blotting analysis. Cellular Hydrogen Peroxide (H2O2) Staining Assay—Cells were seeded in 6-well plates and treated with 5-FU and DENSPM for 48 h. CM-H2DCFDA was applied to the cells at 1 μm final concentration and incubated for 1 h at 37 °C. The cells were harvested with trypsin and washed twice with ice-cold phosphate-buffered saline. Ten thousand cells were analyzed on the FACS-Calibur instrument (BD Biosciences) with CELL Quest Pro software. Mitochondrial Transmembrane Potential (Δψm) Assay—Cells were treated with Rho123 at 100 nm final concentration to detect the disruption of mitochondrial membrane potential. Statistical Analysis—The effect of drug combinations was analyzed using median effect algorithms developed by Chou and Talalay (32Chou T.C. Talalay P. Adv. Enzyme Regul. 1984; 22: 27-55Crossref PubMed Scopus (5891) Google Scholar). Dose-response interactions (antagonism, additivity, synergism) between 5-FU and DENSPM were expressed as a combination index (CI) for every fraction affected. A CI value that is smaller than 1 indicates synergism, a CI value higher than 1 indicates antagonism, and a CI value of 1 indicates additivity. Differences in means were evaluated by two-tailed t test assuming unequal variances. Synergism by Combination of 5-FU with DENSPM—As shown in Fig. 1, treatment of both wt-p53 and p53-null HCT116 cells with combined 5-FU and DENSPM markedly reduced tumor cell viability. For wt-p53 cells, 5-FU and DENSPM had a strong synergistic effect, with a Chou and Talalay CI value in the range of 0.3–0.6. Moderate synergistic effect (0.7 < CI < 0.85) was seen with a post-5-FU schedule of initial 5-FU followed by DENSPM. Interestingly, an antagonistic effect was observed when the wt-p53 cells were pre-treated with DENSPM followed by 5-FU. In p53-null cells, simultaneous drug treatment also had the most synergistic effect, whereas pretreatment with DENSPM before 5-FU had an antagonistic effect. Flow cytometric analysis (Fig. 2) indicated that simultaneous treatment in both isogenic lines resulted in a marked increase of the apoptotic sub-G1 population, indicating cytotoxic effect and apoptosis. Absence of p53 alleles in the null cells appeared to reduce apoptosis in response to either DENSPM alone or the combination treatment. In contrast to the results for 5-FU with DENSPM, the combination of 5-FU with DFMO did not produce synergistic cell killing (Fig. 3).Fig. 3Effects of combined 5-FU and ornithine decarboxylase inhibitor DFMO on colon carcinoma cells. HCT116 colon cancer cells were treated with 5-FU at 2.5 μm (IC25 for wt-p53 line) or 15 μm (IC25 for p53-null line) in combination with increasing doses of DFMO (simultaneous or one drug following the other). Cell viability at the 96-h end point was measured with MTS assay. A, dose-response curves. The straight line at around 70% viability shows the effect by 5-FU alone. The four curves represent different regimens: •, DFMO alone; ▴, 5-FU + DFMO (simultaneous treatment for 48 h and drug-free medium for 48 h); ▪, DFMO (48 h) then 5-FU (48 h); ○, 5-FU (48 h) then DFMO (48 h). No combination (simultaneous treatment, DFMO post-treatment, or DFMO pre-treatment) produced synergistic cytotoxicity. Some pre-treatment and post-treatment regiments of DFMO are antagonistic to 5-FU depending on the p53 status. DFMO post-treatment and DFMO pre-treatment had the strongest antagonistic effects in wt-p53 and p53-null lines, respectively. B, analysis of the combination effect of 5-FU and DFMO using the Chou and Talalay CI. Dose-response interactions between 5-FU and DFMO were expressed as a CI value (CI < 1: synergism, CI = 1: additivity, CI > 1: antagonism). Simultaneous treatment with 5-FU and DFMO only had additive effects in most regimens. 5-FU followed by DFMO had a strong antagonistic effect in the wt-p53 cells and DFMO followed by 5-FU had a strong antagonistic effect in the p53-null cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Depletion of Polyamines and Accumulation of Acetylated Spermidine by Combined 5-FU and DENSPM Treatment— 5-FU at the IC25 dose did not markedly reduce polyamines. DENSPM decreased the levels of spermidine and spermine as expected, but increased the putrescine level (Fig. 4). However, the combination treatment with 5-FU and DENSPM produced a marked reduction in spermidine and spermine in tumor cells. The levels of acetylated spermidine in the wt-p53 line were markedly increased compared with controls. However, the levels of acetylated spermidine increased only minimally in the p53-null line despite a marked decrease in spermidine in the treated cells. The cellular polyamine levels in cells treated with DFMO showed a reduction of putrescine and spermidine, which confirmed the effect of DFMO as a biosynthetic inhibitor (Fig. 4). Combination of 5-FU and DFMO also markedly reduced putrescine by half but did not lead to an increase in acetylated polyamines. Induction of SSAT Gene Expression by 5-FU and DENSPM— To test whether the 5-FU and DENSPM combination had a stronger effect on SSAT expression than each individual agent, a real-time PCR analysis was performed to quantify SSAT transcript levels in the tumor cells. As shown in Fig. 5, 5-FU alone led to only a minor increase in SSAT expression, but DENSPM produced a major increase. The combination of the two drugs, however, resulted in a marked induction of SSAT expression (24 times higher in wt-p53 cells and 10 times higher in p53-null cells). The extent of SSAT mRNA induction appeared to match the levels of acetylated spermidine in wt-p53 cells but not in p53-null cells. Although we did not measure SSAT enzyme activity levels, increased expression of the gene and the marked reduction of spermidine and spermine levels (see above) indicated that an increase in SSAT enzymatic activity occurred. H2O2 Production and Antioxidant Protective Effect—Because SSAT catabolizes polyamines to produce acetylated polyamines, which serve as substrates for polyamine oxidase that generates hydrogen peroxide (24Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar), we investigated whether combined 5-FU and DENSPM treatment resulted in an increased level of H2O2 that would play a role in apoptotic responses as a toxic reactive oxygen species. Measurements of H2O2 using CM-H2DCFDA showed that incubation of cells with simultaneous 5-FU and DENSPM led to increased H2O2 production (Fig. 6A). Interestingly, 5-FU alone also caused a marked accumulation of H2O2, especially in p53-null cells. To determine whether the observed apoptosis involved reactive oxygen species, the cells were incubated with 5-FU and DENSPM in the presence of the antioxidant GSH, and attenuated cytotoxicity was observed, as expected (Fig. 6B). Transcriptome Analysis—From the MTS assay, the combination of 5-FU and DENSPM showed synergistic growth inhibition and cell-killing effects. The gene expression profiles of drug-treated cells at 48 h were investigated to elucidate the molecular pathways of the synergism. Both the wt-p53 and p53-null cell lines showed that a larger number of genes were significantly altered after combination treatment with 5-FU and DENSPM as compared with the single-agent treatment groups (Tables I and II). In the wt-p53 cell line, the numbers of differentially expressed genes among 2,016 cancer pathway-related genes examined in cells treated with 5-FU alone, DENSPM alone, or their combination were 61, 30, and 76, respectively. In the p53-null cell line, 41, 54, and 132 genes were altered. Consistent with the real-time PCR results, marked induction of SSAT mRNA was observed with combined 5-FU and DENSPM treatment in both cell lines (data not shown).Table IAltered expression of redox genes encoded by nuclear and mitochondria DNAGene descriptionAccession numberSourceHCT116 wt-p53HCT116 p53-null5-FUDENSPM5-FU + DENSPM5-FUDENSPM5-FU + DENSPMMTCO1NM_173704MT1.481.250.780.900.750.27MTCO2NM_173705MT1.560.890.661.160.580.14MTATP6NM_173702MT1.490.810.660.950.640.23MTATP8NM_173703MT1.490.760.640.890.540.21MTND3NM_173710MT0.870.620.620.680.290.19MTND4NM_173711MT1.440.820.630.630.470.15MTND6NM_173714MT0.750.460.650.550.760.54MTND4LNM_173712MT1.340.740.630.620.360.14WEE1XM_049184Nuclear0.490.680.310.680.840.90SDHBNM_003000Nuclear1.070.751.521.071.241.62UQCRHNM_006004Nuclear0.951.161.151.151.331.67COX8NM_004074Nuclear1.251.291.571.121.121.69SFNNM_006142Nuclear1.661.893.031.170.851.20FDXRXM_056248Nuclear2.002.602.361.181.061.20DUTU31930Nuclear0.890.760.500.941.481.38 Open table in a new tab Table IIAltered expression of DNA genes and p53 regulated genesGene descriptionAccession numberHCT116 wt-p53HCT116 p53-null5-FUDENSPM5-FU + DENSPM5-FUDENSPM5-FU + DENSPMDNA replication and chromosome cycle TREX1NM_0336280.660.440.500.730.470.41 TOP2AJ040880.740.910.350.791.061.13 TOP3U434311.090.480.520.901.120.73 MSH2U039110.730.620.380.750.840.65 WEE1XM_0491840.490.680.310.680.840.90p53 regulated genes MDM2M924241.250.994.630.681.010.75 CDKN1AL256103.500.925.703.241.981.25 BBC3NM_0144172.117.842.281.801.661.53 FDXRXM_0562482.002.612.361.181.061.20 MIC-1AF0197701.993.973.950.710.710.50 DDIT1L244981.330.912.530.631.051.26 SFNNM_0061421.661.893.031.170.851.20 Open table in a new tab In the wt-p53 HCT116 cell line, the expression of p53 downstream genes (p21WAF1, mdm2, bbc3, mic-1, ferredoxin reductase, 14-3-3-σ, etc.) was induced, demonstrating that the p53 pathway was activated. These genes were not induced in the p53-null cells. Consistent with the flow cytometry results, the expression of cell cycle arrest genes (p21WAF1, cyclin D2, cyclin A, 14-3-3-σ) were also altered. Genes involved in mitochondrial functions were statistically significantly down-regulated (Table I, p = 0.04). In the p53-null HCT116 cell line, genes in the categories of oxidative phosphorylation (p = 0.0007) and electron transport chain (p = 0.0001) were altered. Most of the genes in respiratory chain complex I were suppressed (Table I). Some of the gene expression results were also confirmed at the protein level by Western blot analysis (see Fig. 8). Loss of Mitochondria Potential and Cytochrome c Release— To test the hypothesis that mitochondria are the potential target for the drug treatment, we measured the disruption of mitochondrial membrane potential (Δψm) using Rho123. Combined 5-FU and DENSPM treatment resulted in a significant decrease in Δψm, as shown by a shift in the cell population from high to low fluorescence (Fig. 7). Consistent with the loss of mitochondrial membrane potential, cytochrome c was released from mitochondria into the cytoplasm after the combination drug treatment in both cell lines (Fig. 8). Caspase 9 Dependence of 5-FU/DENSPM-induced Cell Death—On Western blot using antibodies that specifically recognize activated caspase 3 and cleaved caspase 9, caspase 3 was not activated in 5-FU and DENSPM-treated cells, but activated caspase 9 was detected (Fig. 8). Protein extracts from HL-60 cells with and without Ara-C treatment were used as positive and negative controls for the cleaved and activated forms of caspase 3. This result showed that 5-FU/DENSPM-induced cell death is caspase 9-dependent and caspase 3-independent, which is one of the four types of apoptosis involving different combinations of caspase dependence (33Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. de la Elia A. Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar). Western blotting analyses also showed that cyclin A and cyclin B1 were decreased after the combination treatment, confirming our microarray results. Consistent with our previous report (10Zhang W. Ramdas L. Shen W. Song S.W. Hu L. Hamilton S.R. Cancer Biol. Ther. 2003; 2: 572-578Crossref PubMed Scopus (33) Google Scholar) that Fas expression was increased in cells treated with 5-FU, 5-FU as well as combined 5-FU and DENSPM also caused Fas up-regulation. Consistent with the results from our transcriptome analysis, 5-FU and DENSPM activated p53 and protein expression of genes of p53 downstream (phosphorylated p53, serine 15, p21Waf1, and mdm2) in the HCT116 wt-p53 cell line but not in the p53-null cells (Fig. 8). We reported previously that high-dose 5-FU modulates the polyamine pathway in colon carcinoma cells (10Zhang W. Ramdas L. Shen W. Song S.W. Hu L. Hamilton S.R. Cancer Biol. Ther. 2003; 2: 572-578Crossref PubMed Scopus (33) Google Scholar). This observation led us to speculate that 5-FU in combination with polyamine-targeting drugs may have synergistic effects in cytotoxicity. This present study showed that indeed the combination of 5-FU with the polyamine catabolism-enhancing drug DENSPM synergistically killed colon carcinoma cells independent of their p53 status. Interestingly, antagonistic effects depending on the drug treatment sequence were observed in p53 wild-type and null lines. Most notably, DENSPM pretreatment followed by 5-FU produced a antagonistic effect in both cell lines. This finding indicates that the synergistic effect of simultaneous 5-FU and DENSPM is not because of “priming” of one drug for the other. Rather, the effects of the two drugs may need to augment each other, although we do not know the exact mechanism involved. One possibility is that both 5-FU and DENSPM damage DNA but through different mechanisms (34Basu H.S. Schwietert H.C. Feuerstein B.G. Marton L.J. Biochem. J. 1990; 269: 329-334Crossref PubMed Scopus (134) Google Scholar, 35Edwards M.L. Snyder R.D. Stemerick D.M. Med. Chem. 1991; 34: 2414-2420Crossref Scopus (58) Google Scholar), which may make DNA repair less effective. In contrast to 5-FU and DENSPM, combination of 5-FU with the polyamine synthesis inhibitor DFMO did not have synergistic cell killing effects. Furthermore, 5-FU followed by DFMO in the wt-p53 line and DFMO pretreatment followed by 5-FU in the p53-null line had marked antagonistic effects. Although we do not yet understand the mechanisms that account for these different effects, the results underscore the impact of the genetic background of tumor cells on the cellular response to drug treatment and therefore the importance of understanding these relationships. Modulation of polyamine levels and their cellular responses is a complex process. The synergistic effect of combined DENSPM and 5-FU must be specific at molecular levels. One of the molecular effectors appears to be that SSAT is synergistically induced by the combination of 5-FU and DENSPM but not by 5-FU and DFMO. Previous studies have shown that DENSPM-induced SSAT expression, which is responsible for the depletion of spermine and spermidine via acetylation (36Fogel-Petrovic M. Kramer D.L. Vujcic S. Miller J. McManis J.S. Bergeron R.J. Porter C.W. Mol. Pharmacol. 1997; 52: 69-74Crossref PubMed Scopus (43) Google Scholar). Consistent with this finding, cellular polyamine levels were markedly reduced by the combination of 5-FU and DENSPM. Acetylated spermidine accumulated in wt-p53 cells but not in p53-null cells. Surprisingly, the levels of putrescine were increased by combined 5-FU and DENSPM in wt-p53 cells but decreased in p53-null cells, indicating that the p53 status has an impact on how polyamines are depleted. p53 could be suppressing the diamine exporter, resulting in enhanced retention of polyamine catabolic products in wt-p53 cells (37Xie X. Gillies R.J. Gerner E.W. J. Biol. Chem. 1997; 272: 20484-20489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Increased putrescine in the absence of spermidine may have another cellular effect because increased putrescine was shown to inhibit hypusine formation and transfer of the butylamine moiety of spermidine to a lysine residue in eukaryotic initiation factor 5A (eIF-5A), resulting in induction of apoptosis (28Tome M.E. Fiser S.M. Payne C.M. Gerner E.W. Biochem. J. 1997; 328: 847-854Crossref PubMed Scopus (85) Google Scholar). This mechanism may involve the ability of deoxyhypusine synthetase to produce homospermidine, as recently shown by Park et al. (38Park J.H. Wolff E.C. Folk J.E. Park M.H. J. Biol. Chem. 2003; 278: 32683-32691Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the polyamine catabolic pathway, acetylated spermine or spermidine is oxidized by polyamine oxidase. This reaction produces hydrogen peroxide, which at high levels damages mitochondrial membranes resulting in cytochrome c release. Release of cytochrome c activates members of caspase families and triggers the cascade to apoptosis (25Chen Y. Kramer D.L. Diegelman P. Vujcic S. Porter C.W. Cancer Res. 2001; 61: 6437-6444PubMed Google Scholar). The anti-mitochondrial effects of acetylated polyamines were reported in previous studies (39Snyder R.D. Beach D.C. Loudy D.E. Anticancer Res. 1994; 14: 347-356PubMed Google Scholar, 40Marverti G. Piccinini G. Ghiaroni S. Barbieri D. Quaglino D. Moruzzi M.S. Int. J. Cancer. 1998; 78: 33-40Crossref PubMed Scopus (22) Google Scholar). In addition, it was shown that acetylated polyamines cause the loss of mitochondrial DNA (40Marverti G. Piccinini G. Ghiaroni S. Barbieri D. Quaglino D. Moruzzi M.S. Int. J. Cancer. 1998; 78: 33-40Crossref PubMed Scopus (22) Google Scholar). Results from our study showed that the combination of 5-FU with DENSPM indeed elevated cellular hydrogen peroxide and damaged mitochondrial membranes. Consistent with mitochondria being an important target for combined 5-FU and DENSPM, our transcriptome analyses showed that, regardless of the p53 status, genes involved in mitochondrial functions were preferentially affected. However, mitochondrial alterations are more apparent in p53-null cells where 11 of 136 altered genes are involved in mitochondrial function. Among the 13 genes essential for electron transport chain subunits in the inner mitochondrial membrane (41Chinnery P.F. Howell N. Andrews R.M. Turnbull D.M. J. Med. Genet. 1999; 36: 425-436Crossref PubMed Scopus (77) Google Scholar), 8 genes were suppressed in the p53-null line. Among the mitochondrial function-related genes that were markedly attenuated in p53-null cells were cytochrome c oxidase I and II (MTCO1 and MTCO2), which are subunits of cytochrome c oxidase. MTCO2 transfers an electron from cytochrome c to the binuclear center of cytochrome a3-CuB (42Hill B.C. J. Bioenerg. Biomembr. 1993; 25: 115-120Crossref PubMed Scopus (45) Google Scholar) and translocates protons, which are associated with the mitochondrial transmembrane potential (43Babcock G.T. Wikstrom M. Nature. 1992; 356: 301-309Crossref PubMed Scopus (1081) Google Scholar). NADH dehydrogenase resides in the first complex in the electron transfer chain and catalyzes the transfer of electrons from NADH to coenzyme Q (44Gerhard M. Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons Inc., New York1998Google Scholar). Thus, suppression of MTCO1, MTCO2, and NADH dehydrogenases by the combined drug treatment would likely lead to decreased efficiency of the mitochondrial electron transport chain and subsequently increase the production of superoxides. In wt-p53 cells, in addition to mitochondria-related genes, many genes involved in the p53 pathway were affected, indicating that the p53-mediated apoptosis pathway most likely played a dominating role in the observed cell death, as expected. One other interesting gene was dUTPase, which hydrolyzes dUTP to dUMP and pyrophosphate. Remarkably, increased dUTPase levels in colon cancer cell lines have a major role in their resistance to 5-FU and fluorodeoxyuridine (45Canman C.E. Radany E.H. Parsels L.A. Davis M.A. Lawrence T.S. Maybaum J. Cancer Res. 1994; 54: 2296-2298PubMed Google Scholar, 46Pugacheva E.N. Ivanov A.V. Kravchenko J.E. Kopnin B.P. Levine A.J. Chumakov P.M. Oncogene. 2002; 21: 4595-4600Crossref PubMed Scopus (86) Google Scholar). Our results showed that the expression of dUTPase was suppressed when 5-FU and DENSPM were applied simultaneously in the wt-p53 cell line. This reduction may represent another mechanism that is responsible for the synergistic effects of combined 5-FU and DENSPM in the wt-p53 cell line. Thus, the presence of wt-p53 appears to sensitize cells to 5-FU and DENSPM through multiple processes. In summary, our study shows that the combination of 5-FU and DENSPM provides more effective cytotoxicity in colon cancer in a cell culture system through effects on the polyamine pathway. The potential of 5-FU and DENSPM as a therapeutic combination for treating colon cancer patients awaits results from preclinical animal model experiments and future clinical trials. We thank David Stringer for assistance with the polyamine measurements.

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.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.

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.

Abstract Abstract 578 MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures: Smith: Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

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.

SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production.MDS-RS are characterized by significant anemia. Patients with MDS-RS die from a shortage of red blood cells and the side effects of iron overload due to their constant need for transfusions. Our study has implications for the development of therapies to achieve long-lasting hematologic responses. This article is highlighted in the In This Issue feature, p. 476.

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.

Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.

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.

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 ...

Adults with acute myeloid leukemia (AML) have a high rate of remission; however, more than 50% relapse. C-kit is expressed in approximately 60% of patients with de novo AML and represents a potential therapeutic target.Patients with newly diagnosed AML received 12 months of imatinib mesylate as maintenance therapy after the completion of post-remission therapy. The primary objective was to determine whether this approach improved progression-free survival (defined as no relapse and no death) compared with historical controls.The median progression-free survival of patients < 60 years of age was 52.1 months (historical control, 13 months) and for patients ≥ 60 years of age was 10.7 months (historical control, 8 months). The median level of AF1q expression was high (9.59), and 84% of patients had moderate or high levels of drug-resistance factors.Imatinib maintenance therapy may improve the outcome of newly diagnosed patients with AML who are < 60 years of age.

Abstract MEC (mitoxantrone, etoposide, cytarabine) is a standard regimen for relapsed/ refractory (R/R) acute myeloid leukemia (AML), but outcomes remain poor. The overexpression of proteasomes and constitutive activation of NF-KB in AML cells suggest that proteasome inhibitors (PI) such as bortezomib (Bz) may be effective anti-leukemia therapy. PI or a decoy NF-KB oligonucleotide increase chemosensitivity to both anthracyclines and cytarabine. To test the hypothesis that PI may improve the efficacy of MEC, we conducted a phase 1 trial of Bz in combination with MEC. Here, we present final results of this trial: response rate, toxicity, and correlation of outcomes with mutation analysis. As CD74 expression may identify a subset NF-KB-dependent AML with predicted increased sensitivity to PI (Clin Can Res 2008; 14: 1446-54), we also explored this correlation. Methods: All pts were treated at the Cleveland Clinic from Aug 2010-Mar 2014. This protocol was approved by the institution’s review board. Eligibility included: age 18-70 yrs, R/R AML, cardiac ejection fraction ≥ 45%. CD74 was assessed by flow cytometry using CD45 PE (BD Biosciences San Jose, CA) and CD74-Alexa 488 (AbD Serotec Raleigh, NC). A myeloid panel mutational analysis was performed on extracted DNA in pts with banked samples (n=26). All pts received 1 cycle of MEC: mitoxantrone (6 mg/m2/d), etoposide (80 mg/ m2), and cytarabine (1000 mg/ m2) Days 1-6. Bz was administered IV on Days 1, 4, 8, and 11. Dose was escalated using a standard 3 x 3 design. Dose levels (DL) were: -1 (0.40 mg/ m2), 1 (0.70 mg/ m2), 2 (1.0 mg/ m2), and 3 (1.3 mg/m2). Response was defined by IWG criteria (Cheson, 2006). The maximum tolerated dose (MTD) of Bz with MEC was 1.0 mg/m2 (Advani et al, ASH 2012, Abstract 3595). Results: Of 35 pts enrolled, the median age was 55 yrs (range 33-69), 13 (38%) were male, and median baseline WBC was 4.0 K/ µL (range 0.82-84.7). The median time from initial diagnosis of AML to enrollment was 8.4 months (range 1.1-88.2) and 6 pts (17%) had an antecedent hematologic disorder. Salvage status (S) at enrollment: S1 (24 pts, 69%), S2 (7 pts, 20%), S4 (4 pts, 11%). Nine pts (26%) were refractory to all prior therapies, and 3 pts (9%) had received prior allogeneic hematopoietic cell transplant (AHCT). Adverse cytogenetics per CALGB/ Alliance 8461 criteria occurred in 19% of pts at study entry and 15 of 26 pts (58%) had poor-risk molecular mutations (RUNX1, ASXL1, TET2, p53, IDH1, MECOM, FLT3 ITD). Ten pts were enrolled on DL1, 13 pts on DL2, 11 pts on DL3, and 1 pt died prior to treatment. Overall, 3 pts (9%) died during induction. In addition to febrile neutropenia and Gr 4 hematologic toxicity, the most commonly reported adverse events (AEs) were metabolic, constitutional, gastrointestinal (GI), and dermatologic, with the majority of these being Gr 1 or 2. GI toxicity was the only reported AE attributable to Bz: 12 pts had constipation or ileus (10: Gr 1 or 2; 2: Gr 3 or 4). Seventeen of the 33 evaluable pts (52%) have achieved a complete remission (CR) or complete remission with incomplete count recovery (CRi); with 1 pt inevaluable due to donor lymphocyte infusion. The estimated median overall survival was 7.2 months; median duration of response was 10.3 months. DL did not correlate with response. Eleven pts (32%) went on to receive AHCT. Among pts with poor-risk molecular mutations, 64% achieved CR/ CRi. Inhibition of NF-KB signaling in leukemia cells with mutated RUNX1 efficiently blocks growth and development of leukemia (Blood 2011; 118: 6626-37). Of the 5 pts with RUNX1 mutations, 3 (60%) achieved CR/ CRi, suggesting that Bz may have promising clinical benefit in this difficult subset of pts. Among the 17 pts with CD74 expression testing who were evaluable for response, the mean CD74 expression trended higher in non-responding pts (32.6%) than in responders (11.1%) (p=0.14). Conclusions: The combination of MEC/Bz was well-tolerated and resulted in high response rates, even within a molecularly-defined poor risk population of pts with R/R AML. Our data do not confirm the expectation that higher CD74 expression would correlate with response in this R/ R AML cohort, but larger pt numbers are needed. These results, especially in pts with poor-risk mutations, support development of a randomized study to address the benefit of adding Bz to MEC in the treatment of R/R AML. Disclosures Advani: Takeda: Research Funding. Carew:Takeda: Research Funding. Sekeres:Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

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 Background Achieving a complete remission (CR) in patients with newly diagnosed acute myeloid leukemia (AML) after induction chemotherapy with cytarabine and an anthracycline (7+3) remains an important treatment goal associated with better overall survival (OS). Approximately 25-30% of younger, and up to 50% of older patients (pts) fail to achieve CR. AML pts with residual leukemia at day 14 receive a second cycle of the same regimen; whether these pts have worse survival than pts not requiring re-induction is unclear. Information on pts with primary refractory AML and the best treatment strategy in this setting are limited. Methods Pts with newly diagnosed AML treated at our institution between 1/2000 and 1/2015 were included. Pts received standard induction chemotherapy with cytarabine for 7 days and an anthracycline for 3 days (7+3). Bone marrow biopsies were obtained at day 14 and a second cycle of the same regimen (7+3 for younger adults, 5+2 for older adults) was given to pts with residual leukemia (blasts &gt; 5%). All responses were assessed at day 30 +/- 5 days post induction. Response was defined as CR and CR with incomplete hematologic recovery (CRi) or platelet recovery (CRp) per International Working Group (IWG) 2003 response criteria. Cytogenetic risk stratifications were based on CALGB/Alliance criteria. OS was calculated from the time of diagnosis to time of death or last follow up. A panel of 62 gene mutations that have been described as recurrent mutations in myeloid malignancies was used to evaluate whether genomic data can be used to predict response. Results: Among 227 pts with AML, 123 received 7+3 and had clinical and mutational data available. Median age was 60 years (range, 23-82). Median baseline WBC was 8.2 X 109/L (range, 0.3-227), hemoglobin 8.9 g/L (range, 4.7-13.8), platelets 47 X 109/L (range, 9-326), and BM blasts 46% (range, 20-95). Cytogenetic risk groups were: favorable in 12 (10%), intermediate in 68 (56%) [normal karyotype in 44 (36%)], and unfavorable in 42 (34%). A total of 93 pts (76%) responded, 69 (74%) received 1 cycle of induction and 24 (26%) required re-induction at day 14 due to residual leukemia. A total of 39 pts (32%) received allogeneic stem cell transplant (ASCT): 18 (46%) from a matched sibling donor, 16 (41%) from a matched unrelated donor and 5 (13%) had an umbilical cord transplant. With a median follow up of 13.5 months, the median OS for the entire group was 13 months (m, range, 0.1-120). The median OS for pts who failed 1-2 cycles of 7+3 was significantly worse than pts who responded (median 2.6 vs 16.9 m, p = 0.002). When pts undergoing ASCT were censored, the median OS was 2.3 vs 9.9 m, p= 0.003, respectively. Overall, 33 pts (27%) had residual leukemia at day 14 and received re-induction, 24 (72%) achieved a response at day 30+/- 5 days. The median OS for pts who received re-induction was inferior compared to pts who did not (10.1 vs. 16.1 months, p= 0.02). When pts who received ASCT were censored, the OS was similar (8.5 vs. 7.4 months, p = 0.49, respectively). Among the 30 pts with persistent disease following induction therapy at day 30, 11 (37%) died from induction complications, 6 (20%) received salvage therapy with mitoxantrone/etoposide/cytarabine, 3 (10%) received high dose cytarabine, 2 (7%) received azacitidine, and 8 (27%) received best supportive care. Among pts who received salvage chemotherapy 56% achieved CR and proceeded with ASCT. Two pts had ASCT with residual leukemia and relapsed within 3 m of ASCT. Pts who received ASCT after induction failure had a significantly better OS compared to non-transplant pts (median OS 22.0 vs. 1.4 months, p &lt; 0.001, respectively); however, this benefit was only seen in pts who had ASCT in CR. We then investigated if genomic mutations can predict response or resistance to chemotherapy. Out of the 62 genes tested, only a TP53 mutation was associated with resistance, p = 0.02. Further, pts with TP53 mutations had significantly inferior OS compared to TP53 wild type regardless of ASCT status (1.4 vs 14.8 m, p&lt; 0.001) Conclusion: Pts with newly diagnosed AML who fail induction chemotherapy with a 7+3 regimen have a poor outcome. Re-induction with the same regimen at day 14 for residual leukemia converted most non-responders to responders, but was associated with worse OS. ASCT improves outcome only in pts who achieve CR with salvage therapy. TP53 mutations predicted resistance to chemotherapy with 7+3. Disclosures Carew: Boehringer Ingelheim: Research Funding. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

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.

During many years, very limited data had been available on specific gene mutations in MDS in particular due to the fact that balanced chromosomal translocations (which have allowed to discover many “leukemia” genes) are very rare in MDS, while chromosomal deletions are generally very large, making it difficult to identify genes of interest. Recently, the advent of next generation sequencing (NGS) techniques has helped identify somatic gene mutations in 75–80% of MDS, that cluster mainly in four functional groups, i.e. cytokine signaling (RAS genes), DNA methylation, (TET2, IDH1/2, DNMT3a genes) histone modifications (ASXL1 and EZH2 genes), and spliceosome (SF3B1 and SRSF2 genes) along with mutations of RUNX1 and TP 53 genes. Most of those mutations, except SF3B1 and TET2 mutations, are associated with an overall poorer prognosis, while some gene mutations (mainly TET2 mutation), may be associated to better response to hypomethylating agents.The frequent mutations of epigenetic modulators in MDS appear to largely contribute to the importance of epigenetic deregulation (in particular gene hypermethylation and histone deacetylation) in MDS progression, and may account at least partially for the efficacy of hypomethylating agents in the treatment of MDS.

Correction to: Leukemia (2014) 28, 1171–1174; doi:10.1038/leu.2014.16 Since the publication of this article, the authors have identified an error concerning one of the author names. D Proia should be listed as DA Proia. The corrected list is shown above. The Authors would like to apologise for any inconvenience this may have caused.

&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;