Search

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.

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.

qRT-PCR of selected genes in the oxidoreductase pathway.

Knockdown of NRF2 blunts pevonedistat-mediated protection from cisplatin.

Genes altered by 2 fold change or more by pevonedistat treatment.

Pevonedistat does not alter OCT2 or CTR1 expression.

Pevonedistat stabilizes NRF2 expression in RPTECs.

Combination indices following treatment with pevonedistat and cisplatin.

Median survival time of FaDu tumor bearing mice.

Quantification of IHC staining.

Significantly upregulated oxidative stress response genes by pevonedistat.

ROC-325 combination data.

PDF file - 1755K, Supplemental Figure 1: Effects of targeted p65 knockdown on cisplatin sensitivity.

PDF file - 71K, Supplemental Table 1. Proteins Altered >2-fold by Treatment with MLN4924.

Cathepsin D knockdown studies.

Drug washout experiment.

Growth characteristics and HCQ sensitivity of ATG5 and ATG7 knockdown cells.

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

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.

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 vivo.Conclusions: 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.

Gene expression data

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.

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. We, and others, have reported that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8 activating enzyme (NAE) inhibitor pevonedistat. Here, we report that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augments 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 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. Citation Format: Trace M. Jones, Claudia M. Espitia, Aikseng Ooi, Julie E. Bauman, Jennifer S. Carew, Steffan T. Nawrocki. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2640.

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.

Despite the recent development of new agents for acute myeloid leukemia (AML) therapy, novel approaches are still needed for patients that do not benefit sufficiently from existing regimens. Reolysin (Pelareorep) is a proprietary clinical formulation of the naturally occurring oncolytic reovirus that is non-pathogenic and preferentially replicates in cancer cells, but not in normal tissue. Although Reolysin has been investigated in over 30 adult clinical trials and is very well tolerated when given alone and in combination with chemotherapy, it has never been evaluated for AML therapy. Our major goal was to determine the efficacy and mechanism of action of Reolysin alone and in combination with azacitidine in AML models and primary patient specimens. Reolysin exhibited dose-dependent effects against AML cells with respect to the reduction in leukemia cell viability and induction of apoptosis in all 8 cell lines evaluated. The combination of Reolysin with azacitidine yielded synergistic benefit across all cell lines that was dramatically superior to single agent treatments (p Disclosures No relevant conflicts of interest to declare.

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.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib and would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.

Head and neck cancer is a broad term for neoplasms of the epithelial lining of the anatomical sites of the upper aerodigestive tract. Very few novel therapeutics have been developed for the treatment of advanced head and neck cancer with cisplatin continuing to be a key standard of care agent. Innovative and more effective therapeutic strategies are urgently needed. The inappropriate loss of several NEDD8-cullin-RING ubiquitin ligase (CRL) targets has been linked to cancer progression and drug resistance. Our data show that overexpression of NEDD8 activating enzyme (NAE), a key proximal regulator of the NEDDylation cascade, is correlated with inferior survival in head and neck cancers suggesting that this pathway may be a promising new therapeutic target. We hypothesized that disrupting NEDDylation with the first-in-class NAE inhibitor pevonedistat (MLN4924, PEV) would be an effective new approach for the treatment of head and neck cancers. Initial testing demonstrated that PEV potently impaired the NEDDylation cascade as evidenced by decreased NEDDylated cullins and upregulation of the CRL target cell cycle inhibitors, p21 and p27. In addition, PEV induced a dramatic G2 cell cycle arrest, polyploidy, and activation of the DNA damage response. PEV treatment also potently decreased cell viability and induced apoptosis in a panel of head and neck cancer cell lines with diverse genetic features. Given the potent DNA damage-related aspects of PEV's mechanism of action, we hypothesized that it would be a rational combination partner for standard cisplatin therapy. Consistent with this hypothesis, co-treatment with PEV and cisplatin promoted synergistic levels of DNA damage as measured by comet assay, an accumulation of gamma-H2AX, and quantification of DNA fragmentation. The combination also cooperated to promote redox alterations that were necessary for apoptosis induction and linked to the DNA damaging effects of both drugs. Importantly, synergistic anticancer activity was observed in cell lines from multiple anatomical sites, suggesting that this combination has broad efficacy across many subtypes of head and neck cancer. To further evaluate the therapeutic benefit of the PEV and cisplatin combination, we determined its safety and efficacy in a head and neck cancer xenograft model. Consistent with our in vitro data, the combination significantly decreased tumor proliferation, induced apoptosis, and augmented DNA damage in vivo. While both PEV and cisplatin displayed significant activity as monotherapies, combination treatment resulted in a dramatic decrease in tumor burden and also led to long-term animal survival in every treated animal (10/10 mice remained disease-free at day 100). Importantly, the PEV and cisplatin combination was therapeutically selective and very well tolerated in mice. Our collective data provide a strong rationale for the clinical investigation of NAE inhibition as a novel strategy to augment cisplatin efficacy in patients with head and neck cancer. Citation Format: Trace Jones, Claudia Espitia, Jennifer Carew, Steffan T. Nawrocki. Targeting Neddylation synergistically augments the activity of cisplatin in head and neck cancer models to yield long-term survival by modulating key DNA damage and redox pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5211.

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 NAEβ 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 NAEβ. 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.

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.

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.

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.

Aberrant gene expression plays a pivotal role during tumorigenesis and cancer progression. The acetylation status of histones is an important determinant of gene expression and is controlled by two opposing classes of enzymes: histones acetyl transferases (HATs) and histone deacetylases (HDACs). The deacetylation of histones is associated with repression of key tumor suppressor genes and has been linked to HDAC overexpression in multiple forms of cancer including lymphomas. Several HDAC inhibitors have been FDA approved for T-cell lymphoma (TCL) therapy including belinostat, vorinostat, and romidepsin. Despite the promising anti-lymphoma activity of HDAC inhibitors as a drug class, resistance is a significant clinical issue. Identification of new strategies that are more effective in the drug resistant patient population is a high priority, but the mechanisms underlying HDAC inhibitor-induced cell death and the development of drug resistance are not completely understood. 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 effective for refractory patients. To pursue this goal, we generated novel TCL cell line models of acquired HDAC inhibitor resistance through repeated exposure to belinostat. The sensitivity of parental and resistant TCL cells to belinostat and other clinically relevant HDAC inhibitors was initially characterized using cell viability and flow cytometric assays. Notably, belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including vorinostat, romidepsin, panobinostat, and ricolinostat. This indicates that TCL patients that fail one HDAC inhibitor regimen may not benefit significantly from subsequent treatment with other drugs of this class. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones, tubulin acetylation (an HDAC6 target), and exhibited reduced upregulation of the CDK inhibitor p21 following belinostat treatment. In agreement with the absence of these hallmark characteristics of HDAC inhibition, belinostat also failed to cleave caspase-3 in the belinostat-resistant cells. Comprehensive transcriptome analysis was conducted to further characterize these new drug-resistant models and identify potential mechanisms of resistance and targets for second-line treatment. Drug resistant cells featured significantly increased basal levels of the reovirus receptor junctional adhesion molecule-A (JAM-A) as well as decreased JAK/STAT activity, a key antiviral response pathway. Based on these findings, we investigated the efficacy of the proprietary clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that belinostat-resistant cells displayed enhanced sensitivity to oncolytic reovirus-induced cell death compared to their parental counterparts. The increased benefit of Reolysin in resistant models was linked to elevated viral loads and more efficient endoplasmic reticular stress-mediated apoptosis. The heightened sensitivity of HDAC-resistant TCL cells to Reolysin was further validated in parental and belinostat-resistant T-cell lymphoma xenograft models. Collectively, these data demonstrate that oncolytic reovirus may be a novel therapeutic approach to treat T-cell lymphoma patients that are relapsed/refractory to HDAC inhibitors. We are currently planning an early phase clinical trial to further test the safety and benefit of this new approach. Disclosures Persky: Morphosys (IDMC): Consultancy; Merck: Research Funding; Spectrum: Research Funding; Genentech: Honoraria.

Autophagy is an evolutionarily conserved degradation pathway that eliminates certain proteins, defective organelles, and protein aggregates in order to maintain protein homeostasis and prevent abnormal cells from replicating. However, the autophagy machinery may also be utilized to generate ATP from organelles and other cellular components to promote survival under metabolic/hypoxic stress conditions. Since nutrient and oxygen deprivation are hallmark characteristics of the tumor microenvironment, disrupting autophagic degradation may be a promising approach to selectively kill cancer cells. Chloroquine and its derivatives are the only clinically relevant autophagic inhibitors that are currently being evaluated for their potential applications in cancer therapy. However, these drugs are known to cause ocular toxicity, especially irreversible retinopathy, and this underscores the need for additional inhibitors of autophagy with more favorable therapeutic indices. Lucanthone is an anti-schistome agent that is being investigated for cancer therapy as a sensitizer to chemotherapy and radiation. Here we report a novel mechanism of action for lucanthone that is characterized by the disruption of lysosomal function, inhibition of autophagy, and induction of apoptosis. Exposure to lucanthone resulted in the processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but induced lysosomal membrane permeabilization and impaired autophagic degradation as revealed by electron microscopy and the accumulation of p62/SQSTM1. Importantly, lucanthone displayed more potent activity against a panel of breast cancer cell lines compared to chloroquine. Cathepsin D is a lysosomal protease that functions to promote apoptosis following its release into the cytosol upon lyososomal disruption. In order to elucidate the mechanism by which lucanthone induces apoptosis in cancer cells, we investigated the potential effects of lucanthone on cathepsin D expression and subcellular localization. As expected based on its effects on the integrity of lysosomes, lucanthone treatment stimulated a highly significant increase in cytosolic cathepsin D expression, which was associated with apoptosis induction. Accordingly, cathepsin D-targeted siRNA blocked the ability of lucanthone to stimulate its expression and significantly reduced lucanthone-mediated apoptosis. Subsequent experiments revealed that the anticancer activity of lucanthone occurred through a mechanism independent of the functional status of the p53 tumor suppressor. Considering that p53 is functionally inactivated in the majority of cancers, the ability of lucanthone to retain anticancer activity under p53 deficient conditions could have important clinical implications. Collectively, this study establishes a novel mechanism of action for lucanthone as an autophagy inhibitor and provides a rationale for its use in combination with anticancer agents that induce this important survival pathway. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-08.

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. Mol Cancer Ther; 14(6); 1404–13. ©2015 AACR.

PURPOSE: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major problem that limits overall survival and novel therapeutic strategies are urgently needed. The inappropriate loss of several NEDD8-cullin-RING ubiquitin ligase (CRL) targets has been linked to cancer progression and drug resistance, suggesting that this pathway may be a promising new therapeutic target. MLN4924 is a first-in-class NEDD8 activating enzyme (NAE) inhibitor currently under investigation in multiple clinical studies. We investigated its anticancer activity and pharmacodynamic effects in cisplatin-sensitive and cisplatin-resistant ovarian cancer models. EXPERIMENTAL PROCEDURES: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, senescence, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference approaches were used to identify and validate regulators of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamic endpoints were assessed in cisplatin-sensitive and -resistant xenograft models. RESULTS: Intrinsic resistance to cisplatin did not significantly impact the efficacy of MLN4924 against ovarian cancer cells, indicating that this agent may be useful for patients with platinum-resistant disease. Detailed pharmacodynamic analyses showed that treatment with MLN4924 induced the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, cisplatin-resistant cells displayed significantly higher basal levels of NEDDylated cullins and NEDDylation activity than cisplatin-sensitive cells. Further investigation demonstrated that MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells. This suggests that aberrant NEDDylation may contribute to drug resistance and that targeting this pathway may be a promising strategy to restore chemosensitivity. 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 pro-apoptotic effects of the MLN4924/cisplatin combination. In addition, administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and cisplatin-resistant tumors. CONCLUSIONS: Our collective data provide a rationale for the clinical investigation of NAE inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies where platinum-based chemotherapy is utilized. Citation Format: Steffan T. Nawrocki, Kevin R. Kelly, Claudia M. Espitia, and Jennifer S. Carew. TARGETING NEDDYLATION TO OVERCOME CISPLATIN RESISTANCE IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-102.

Multiple myeloma (MM) is the second most common adult hematologic malignancy in the United States and is characterized by a unique form of progressive bone destruction. Despite the development of new treatments such as proteasome inhibitors and immunomodulatory agents, patients with high-risk disease have a median survival of only 2-3 years, highlighting the demand for more effective therapeutic strategies. Oncolytic viral formulations represent a promising new class of anticancer agents that may have important applications in precision medicine. Recent studies have demonstrated that reoviruses specifically replicate only in cancer cells and this led to the development of a reovirus-based oncolytic viral therapy called Reolysin. We have demonstrated that reoviruses selectively replicate in MM cells and Reolysin 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(programmed death 1 ligand, 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. Adaptive resistance mediated by inhibitory ligands such as PD-L1 has emerged as an important mechanism of malignant cell survival and has led to the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. These agents have exhibited dramatic efficacy in certain forms of cancer including melanoma and lung cancer. Analysis of specimens from patients treated on clinical trials with these agents indicates that high basal expression of PD-L1 on tumor cells may be necessary to elicit significant clinical benefit. Notably, most 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 benefiting from immune checkpoint therapy. However, novel immune priming strategies that stimulate transient upregulation of PD-L1 on malignant cells could potentially render agents that target the PD-L1/PD-1 axis significantly more effective for a broader range of malignancies including MM. We hypothesized that Reolysin could be used as a precision immune priming agent to potentiate the anti-MM efficacy of PD-L1 targeted therapy by rendering MM cells vulnerable to PD-L1 inhibition through the transient upregulation of target expression. 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: Novartis: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment.

Multiple myeloma (MM) is an incurable plasma cell malignancy and represents the second most common adult hematologic malignancy in the United States. MM is relatively asymptomatic during its early stages and as a result, the majority of patients have advanced disease at diagnosis. Innovations in the treatment of MM, including the development of proteasome inhibitors such as bortezomib (Velcade) have improved clinical outcomes. However, many patients fail to respond to these agents or relapse after initial response highlighting the need for novel therapeutic strategies. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression. Aberrant MYC transcriptional activity can increase the levels of a number of factors that are associated with disease progression and drug resistance making it an appealing therapeutic target. Recent studies have demonstrated that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BET antagonist JQ1 decreased the expression of c-MYC, BCL-xL, and BCL-2, induced p21 levels, diminished cell viability, and triggered apoptosis in MM cell lines. Comprehensive gene expression profiling of the pharmacodynamic effects of JQ1 revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with this agent. Several earlier studies demonstrated that aberrant HDAC6 expression/activity contributes to malignant progression and resistance to a number of classes of anticancer agents including proteasome inhibitors. Based on the roles of HDAC6 in malignant pathogenesis, we hypothesized that its induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized both genetic and pharmacological approaches to impair HDAC6 function [shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat)] and evaluated the consequential impact on the anti-MM effects of JQ1. Notably, antagonzing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with further reductions in c-MYC, BCL-2, and BCL-xL along with significant increases in apoptosis induction as evidenced by enhanced caspase-3 cleavage and DNA fragmentation. Importantly, administration of ACY-1215 was very well tolerated (less than 5% mean transient reduction in body weight) and significantly augmented the in vivo anti-myeloma activity of JQ1 in the RPMI-8226 MM xenograft model as disease burden in combination treated animals was substantially lower than those that received either monotherapy. Immunohistochemical analyses demonstrated that the combination of JQ1 and ACY-1215 led to significantly lower MM cell proliferation (PCNA), increased apoptosis (active caspase-3), and diminished expression of c-MYC and BCL-2. These data suggest for the first time that induction of HDAC6 may represent a key mechanism that promotes drug resistance and limits the efficacy of bromodomain inhibitor therapy. Taken together, our findings demonstrate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel approach to augment the efficacy of bromodomain inhibitors in MM that warrants further investigation. Disclosures Carew: Boehringer Ingelheim: Research Funding.

Multiple myeloma (MM) is the second most common adult hematologic malignancy in the United States and is characterized by a unique form of progressive bone destruction. Although new treatments such as bortezomib (BZ), carfilzomib and IMIDs have provided benefit to many patients with MM, drug resistance remains a very significant problem. Patients diagnosed with high-risk MM in particular are in urgent need of more effective therapeutic strategies in order to improve their survivorship. The sirtuin deacetylases (SIRTs, SIRT1-7) are critical epigenetic regulators that control longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in primary MM cells from patients with high-risk disease than those with favorable risk MM or normal CD138+ controls. We investigated the role of SIRT1 as a potential regulator of MM pathogenesis and evaluated the benefit of SIRT inhibition as a novel strategy for MM therapy. A series of experiments established a mechanistic relationship between MYC and SIRT1 in that SIRT1 increased MYC protein stability and transcriptional activity. Disruption of SIRT1 function through targeted knockdown or with the small molecule inhibitor tenovin-6 (T6) directly led to the reduction in the expression of key MYC transcriptional targets that play important roles in MM pathogenesis including cyclin D and MAF. Subsequent analyses revealed that U266 and RPMI-8226 cells with acquired BZ resistance exhibited significantly higher basal SIRT1 expression levels (p Disclosures Carew: Boehringer Ingelheim: Research Funding.

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

Multiple myeloma (MM) is a plasma cell disease that represents the second most common adult hematologic malignancy in the United States. Advances in the treatment of MM, including the development of proteasome inhibitors have improved clinical outcomes. However, many patients fail to respond to these newer agents or relapse after initial response. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression making it an ideal therapeutic target. Recent studies suggest that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors such as BCL-2 and displays significant activity against MM. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BRD4 antagonist JQ1 decreased the expression of c-MYC, BCL-2, and BCL-XL, diminished cell viability, disrupted clonogenic survival, and triggered apoptosis in a panel of MM cell lines. Subsequent assays conducted in MM cell lines confirmed a strong reduction in MYC levels and activity following treatment with JQ1 along with consistent increases in the expression of genes regulated by BRD4 including CDKN1A and HEXIM1. Interestingly, gene expression profiling assays revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with JQ1. Based on its ability to alter cellular survival capacity, we hypothesized that HDAC6 induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat) and evaluated the impact of each on the anti-MM effects of JQ1. Abrogation of HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with apoptosis induction as evidenced by enhanced caspase-3 cleavage and further reductions in c-MYC expression. These data suggest that HDAC6 induction may represent a key mechanism that promotes drug resistance and/or limits the efficacy of bromodomain inhibitor therapy. Our collective findings indicate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel and promising approach to augment the efficacy of bromodomain inhibitors for MM that warrants further investigation. Disclosures No relevant conflicts of interest to declare.

Drug resistance is a major cause of treatment failure for patients with acute myeloid leukemia (AML) and novel approaches that circumvent or overcome resistance mechanisms are critically needed. Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays an important role in regulating cell cycle progression in M phase. It is overexpressed in a broad range of solid and hematological malignancies. Volasertib (BI 6727) is a novel PLK inhibitor that is currently under investigation in a Phase III clinical trial for patients with AML. Despite its reported clinical anti-AML activity, the efficacy and pharmacodynamics (PD) of volasertib have not been rigorously investigated in AML models in the preclinical setting. Here, we studied the activity and PD effects of volasertib in a panel of human AML cell lines and primary patient specimens. Low nanomolar concentrations of volasertib potently disrupted the viability and clonogenic survival of AML cells. Notably, the specific cytogenetic background of individual cell lines did not significantly impact cellular sensitivity to volasertib. Similarly, primary AML blasts from patients demonstrated ex vivo sensitivity to volasertib regardless of their cytogenetic and molecular features or prior treatment history. This in vitro data set is in line with recent Phase II clinical data in AML patients showing efficacy of volasertib across all cytogenetic risk categories. Volasertib triggered caspase-dependent apoptosis in AML cells in a manner that was preceded by disruption of the spindle assembly checkpoint. Notably, analysis of the activity of volasertib in three different paired cytarabine-sensitive and cytarabine-resistant AML cell line models revealed that volasertib retained its ability to diminish viability and stimulate apoptosis in cells with three log-fold increased cytarabine resistance. These findings suggest that volasertib may be effective for patients that have relapsed or are refractory to cytarabine-based regimens. Combination studies showed that volasertib and cytarabine yielded additive effects in cytarabine-sensitive cells. Gene expression profiling studies demonstrated that the chemokine CXCL10 was the most significantly upregulated gene following short-term treatment with low concentrations of volasertib. Subsequent qRT-PCR and ELISA experiments confirmed drug-induced CXCL10 expression and elevated CXCL10 secretion by AML cells. To our knowledge, this is the first report linking PLK1 inhibition to alterations in CXCL10 expression and secretion. We are currently investigating the specific contribution of CXCL10 to the anti-leukemic activity of volasertib and are elucidating the molecular mechanisms that mediate this effect. Collectively, our findings demonstrate that inhibition of PLK1 with volasertib is a promising new strategy for the treatment of AML that is able to overcome cytarabine resistance. Disclosures No relevant conflicts of interest to declare.

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.

Mutations in the RAS oncogene are associated with transformation and proliferation and are prevalent in many tumor types. Since RAS is frequently hyperactivated in pancreatic cancer and plays an essential role in tumor progression, it is an ideal therapeutic target. Reoviruses are naturally occurring viruses that are non-pathogenic and have been reported to specifically replicate in cancer cells with an activated RAS pathway, but not in normal tissue. To take advantage of this observation therapeutically, the reovirus-based anticancer agent Reolysin was recently developed. Reolysin has already progressed into clinical trials, but its mechanisms of action have not been fully elucidated. We hypothesized that Reolysin exposure would result in the production of viral products, stimulate endoplasmic reticular (ER) stress, and induce apoptosis. Reolysin treatment triggered the selective replication of reoviruses as visualized by immunocytochemistry and electron microscopy in KRAS-transformed immortalized human pancreatic duct epithelial cells and in a panel of pancreatic cancer cell lines. Reovirus accumulation was associated with an increase in the expression of ER stress-related genes as assessed by microarray and quantitative RT-PCR analyses, cleavage of caspase-4, and induction of apoptosis. In support of our hypothesis that Reolysin induces ER stress-mediated apoptosis, co-treatment with tunicamycin or brefeldin A further augmented Reolysin-induced cell death. Previous studies have demonstrated that the proteasome inhibitor bortezomib causes ER stress-mediated apoptosis. Here we show that co-treatment with bortezomib and Reolysin stimulates the simultaneous accumulation of ubiquitinated and viral proteins resulting in enhanced levels of ER stress-related gene expression, caspase-4 cleavage, and apoptosis. The role of caspase-4 as a regulator of therapeutic sensitivity to these agents was investigated by targeting caspase-4 expression with siRNA. Knockdown of caspase-4 blunted apoptosis stimulated by Reolysin, bortezomib, and the combination. Importantly, bortezomib also dramatically augmented the anticancer activity of Reolysin in xenograft models of pancreatic cancer in vivo. Analysis of tumor specimens from mice treated with Reolysin and bortezomib displayed reovirus accumulation, enhanced expression of GRP78/Bip, a marker of ER stress and increased levels of apoptosis. Collectively, our results demonstrate that the abnormal protein accumulation induced by Reolysin and bortezomib promotes heightened ER stress and apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2717. doi:1538-7445.AM2012-2717