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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. 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.

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.

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.

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.

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.

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