pevonedistat and Melanoma

Purpose The therapeutic index of proteasome inhibitors may be improved through selective inhibition of a sub-component of the ubiquitin-proteasome system, such as the NEDD8-conjugation pathway. This multicenter, phase I, dose-escalation study assessed safety and the maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and antitumor activity of pevonedistat, an investigational NEDD8-activating enzyme (NAE) inhibitor, in patients with metastatic melanoma. Methods Patients received intravenous pevonedistat on Days 1, 4, 8, 11 (schedule A) or 1, 8, 15 (schedule B) of 21-day cycles. Results 26 patients received pevonedistat 50-278 mg/m(2) on schedule A; 11 patients received pevonedistat 157 mg/m(2) on schedule B. The schedule A MTD was 209 mg/m(2): dose-limiting toxicities (DLTs) included grade 3 hypophosphatemia and grade 3 increased blood creatinine (associated with grade 3 hyperbilirubinemia). Two schedule A patients experienced acute organ failure toxicities, one of whom experienced grade 5 acute renal failure. Dose escalation did not occur in schedule B: DLTs included grade 3 myocarditis, grade 2 acute renal failure, and grade 2 hyperbilirubinemia in a single patient. Pevonedistat pharmacokinetics were approximately dose-proportional across the dose range studied, with a biphasic disposition profile characterized by a short elimination half-life (~10 h). Pharmacodynamic studies showed increases in NAE-regulated transcripts post-treatment; all post-dose biopsy samples were positive for pevonedistat-NEDD8 adduct. One schedule A patient achieved a partial response; 15 patients had stable disease (4 lasting ≥6.5 months). Conclusions Pevonedistat was generally well tolerated at the MTD. Anticipated pharmacodynamic effects of NAE inhibition were observed with single-agent pevonedistat in peripheral blood and tumor tissue.

Topics: Adult; Aged; Cyclopentanes; Female; Humans; Male; Maximum Tolerated Dose; Melanoma; Middle Aged; Pyrimidines; Treatment Outcome; Ubiquitin-Activating Enzymes

MLN4924 (pevonedistat) is a potent and selective NEDD8-activating enzyme (NAE) inhibitor. The NEDD8-regulated neddylation system is responsible for the regulated degradation of intracellular proteins with important cellular functions in cancer cell growth, apoptosis, angiogenesis and metastasis. In human melanoma, inhibition of NAE results in induction of DNA re-replication, S phase cell cycle arrest, DNA damage and apoptosis. The study aimed to assess the anti-cancer effect of MLN4924 on canine malignant melanoma cell lines and patient samples and to elucidate the underlying mechanisms. Canine melanoma cell lines and primary patient samples were evaluated for cell viability after incubation with varying concentrations of MLN4924 or dimethyl sulfoxide. Apoptosis, cell proliferation and senescence assays were performed to address underlying mechanisms of MLN4924-mediated anti-tumour effects. Gene expression of seven previously identified deregulated genes in human melanoma was compared in sensitive vs resistant samples. MLN4924 treatment significantly reduced the viability of canine melanoma cell lines and primary samples in a dose- and time-dependent manners. MLN4924 promoted cell apoptosis and inhibited cell growth through induction of DNA re-replication and cell senescence. While the majority of canine melanoma samples demonstrated sensitivity at nanomolar ranges, some samples were resistant to the treatment. Modulation of P21 levels correlated with canine melanoma cell sensitivity. These results provided justification for further exploration of MLN4924 as a treatment of canine melanoma.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclopentanes; DNA Replication; Dog Diseases; Dogs; Enzyme Inhibitors; Melanoma; Mouth Neoplasms; Pyrimidines

The NEDD8 pathway is a known activator of the ubiquitin-protease system, a complex that is partially responsible for the degradation of proteins involved in cell-cycle regulation and neoplastic growth. In this study, we evaluated the antitumor potential of MLN4924 (pevonedistat), a potent NEDD8 inhibitor. We hypothesized that MLN4924 treatment induces apoptosis in human melanoma cells. A375 and Mel39 BRAF V600E mutant melanoma cell lines were treated in vitro with MLN4924 alone or in combination with interferon-α (IFN-α) or vemurafenib - therapeutic agents utilized on melanoma patients. Annexin/propidium iodine flow cytometry analysis showed that treatment with MLN4924 for 72 h induced apoptosis in A375 and Mel39 melanoma cells with an IC50 of 1200 and 143 nmol/l, respectively. Combination therapy of A375 cells with 10 U/ml IFN-α and 1200 nmol/l MLN4924 led to a significant increase in cell death (78.2±3.7%) compared with single-agent treatment by IFN-α (17.5±2.5%) or MLN4924 (50.7±1.0%; P<0.005). Treatment of A375 cells with 1 μmol/l vemurafenib had a notable effect on cell viability. However, the addition of MLN4924 to vemurafenib had an inhibitory effect on apoptosis. Results from MTS proliferation assays indicate that MLN4924 has antiproliferative effects on melanoma cells in vitro, with the addition of IFN-α further inhibiting proliferation. Pretreatment with MLN4924 led to A375 cell sensitization to vemurafenib treatment and immunoblot analysis of MLN4924-treated cells revealed cleavage of caspase-3, caspase-7, caspase-9, and poly-ADP-ribose polymerase. These results show that MLN4924 does have an efficacy in treating melanoma in vitro alone or in combination with IFN-α, and thus it may have potential use in patients with advanced melanoma.

Topics: Cell Line, Tumor; Cyclopentanes; Enzyme Inhibitors; Humans; Melanoma; Pyrimidines; Skin Neoplasms; Survival Rate

Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclopentanes; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Pyrimidines; Ubiquitin-Activating Enzymes; Ubiquitination

The cullin-based CRL4-CDT2 ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle "rereplication" through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of melanoma cell lines through the induction of SET8- and p21-dependent DNA rereplication and senescence. Pevonedistat (MLN4924), a specific inhibitor of the NEDD8 activating enzyme (NAE), inhibits the activity of cullin E3 ligases, thereby stabilizing a vast number of cullin substrates and resulting in cancer cell inhibition in vitro and tumor suppression in nude mice. We demonstrate that pevonedistat is effective at inhibiting the proliferation of melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in melanoma cells demonstrated that the rereplication-mediated cytotoxicity of pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for melanoma suppression in nude mice. By contrast, pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit tumor growth in vivo. Pevonedistat additionally synergized with the BRAF kinase inhibitor PLX4720 to inhibit BRAF melanoma, and suppressed PLX4720-resistant melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by pevonedistat in melanoma and suggest that a broad patient population may benefit from pevonedistat therapy.. The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4(CDT2) by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4(CDT2) substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4(CDT2) therapeutically.

Topics: Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Cyclopentanes; Disease Models, Animal; Female; Gene Expression; Gene Silencing; Genes, ras; Histone-Lysine N-Methyltransferase; Humans; Kaplan-Meier Estimate; Melanoma; Mice; Mutation; Nuclear Proteins; Prognosis; Protein Stability; Proto-Oncogene Proteins B-raf; Pyrimidines; RNA Interference; Ubiquitin-Protein Ligases; Ubiquitination; Xenograft Model Antitumor Assays

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Topics: Antineoplastic Agents; Blotting, Western; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclopentanes; DNA Damage; Flow Cytometry; Humans; Melanoma; NEDD8 Protein; Polymerase Chain Reaction; Pyrimidines; Ubiquitins