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There are 17 known ubiquitin-like proteins (UBLs) from nine phylogenetically distinct classes (NEDD8, SUMO, ISG15, FUB1, FAT10, Atg8, Atg12, Urm1, and UFM1) that have been identified to conjugate to substrates in a manner analogous to ubiquitin. NEDD8 is one of the most studied UBLs and shares the highest amino acid similarity to ubiquitin. Here, we review the current knowledge of the NEDD8 conjugation cascade derived from functional studies in genetic model organisms, structural insights from crystallographic studies, biochemical studies identifying a growing list of NEDD8 substrates with oncogenic implications, and attempts to pharmacologically target the NEDD8 pathway in cancer.

12093Background: dMAPKi with BRAFi and MEKi improves survival in BRAF V600E/K mt MM, but the effects of these inhibitors on nonV600 BRAF mt MM is poorly understood. We sought to characterize nonV60...

e15071Background: Therapeutics targeting inhibitory immune checkpoints have revolutionized the treatment of advanced and metastatic melanoma. Clinical trials of dual treatment with anti-CTLA4 and a...

Background: We identified a patient who presented with brain metastatic melanoma. Her tumor expressed a rare BRAF mutation (mt) (L597S). L597S is a non-V600 BRAF mt with intermediate BRAF kinase activity. Little is known about whether melanomas bearing these non-V600 mt are amenable to treatment with the same targeted therapies currently used for BRAF V600 mutant melanoma. Therefore, we employed a translational approach to characterize non-V600 mts in melanoma and to investigate their responsiveness to targeted therapies. Methods: We performed systemic review to quantify the incidence of non-V600 mts. Tumor fragments from patients BRAF WT, V600E and L597S melanoma brain metastases were used to generate 3 patient-derived xenograft and cell lines. We obtained several other cell lines bearing these mts, and mts within the p-loop of BRAF. Cells were treated with clinical inhibitors of BRAF (BRAFi; vemurafenib (V), dabrafenib (D), encorafenib (E)), and MEK (MEKi; cobimetinib (C), trametinib (T), binimetinib (B)). BRAFi, MEKi, or the combination thereof were tested in the following assays: immunoblots to analyze cell signalling, long-term in vitro growth assays, and subcutaneous and intracranial in vivo tumor growth experiments. Results: MEKi inhibited Erk in all cell lines whereas BRAFi induced paradoxical Erk activation in WT cells, but impaired Erk phosphorylation in V600 and L597 mutant cells. In long-term growth assays, all MEKi were capable of inhibiting growth in all 12 cell lines tested to varying degrees. BRAFi inhibited the growth of V600 and L597S mt cells but not BRAF WT or p-loop mutant cells. The addition of BRAFi consistently inhibited the growth of MEKi-treated L597S mt cells. E was more effective than either D or V at impairing the growth of MEKi-treated non-V600 cells. Remarkably, V potentiated the growth of MEKi-treated cells with BRAF p-loop mts, whereas E further inhibited their growth. In the L597S subcutaneous PDX model, all vehicle-treated tumors grew progressively, whereas T led to overall stability of tumor growth (25% ORR) and D + T caused 100% of tumors to shrink significantly. Moreover, D + T improved the survival of mice with intracranial L597S metastases, compared to vehicle or T treated mice. Conclusions: Non-V600 BRAF mts comprise 15% of all BRAF mts in melanoma. Like V600 mutant melanoma, L597S BRAF mutant melanoma are synergistically growth inhibited by the combination of BRAFi +MEKi in vitro and in vivo. MEKi-treated cells with BRAF p-loop mutations are further inhibited by E, whereas V potentiates growth, suggesting unique mechanisms of BRAFi between inhibitors. Taken together, our data provide a rationale for investigating the efficacy of dual MAPK inhibition with E or D + MEKi in patients with non-V600 mutant melanoma. Citation Format: April A.N. Rose, Matthew Dankner, Shivshankari Rajkumar, Ian R. Watson, Kevin Petrecca, Catalin Mihalcioiu, Peter M. Siegel. Non-V600 BRAF mutations in melanoma: actionable targets for rational drug combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B056.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

SMARCB1 (INI1/BAF47/SNF5) encodes a part of a multiprotein complex that regulates gene expression through chromatin remodeling. SMARCB1 expression is lost or downregulated in multiple human tumors, including epithelioid sarcoma, meningioma and rhabdoid tumors of the brain, soft tissue and kidney.A 46-gene or 50-gene next-generation sequencing AmpliSeq Cancer Panel (Life Technologies; San Francisco, CA, USA) was applied to ∼1400 primary or metastatic melanoma tissues.We identified eight cases of melanoma harboring mutations in SMARCB1. Immunohistochemistry demonstrated preservation of SMARCB1 protein expression in all cases. SMARCB1 mutations occurred together with TP53 mutations in five of the eight cases, suggesting a functional relationship between these tumor suppressors in melanoma.Because single-base substitutions in SMARCB1 occur in a small subset of melanomas and do not affect SMARCB1 protein expression, such mutations would only be discovered by sequencing approaches. Our findings highlight the potential for next-generation sequencing platforms to identify mutations unexpected for melanoma that may contribute to its oncogenic potential. Though rare, the identification of SMARCB1 mutations adds to the growing literature regarding the role of epigenetic control mechanisms in melanoma progression and therapeutic resistance and provide a rationale for strategies targeting such alterations (via chromatin remodeling agents) in clinical trials.

Mutations in p73 are rare in cancer. Emerging evidence suggests that the relative expression of various p73 isoforms may contribute to tumorigenesis. Alternative promoters and N-terminal splicing result in the transcription and processing of either full-length (TA) or N-terminally truncated (deltaN) p73 isoforms. TAp73 possesses pro-apoptotic functions, while deltaNp73 has anti-apoptotic properties via functional inhibition of TAp73 and p53. Here, we report that TAp73, but not deltaNp73, is covalently modified by NEDD8 under physiologic conditions in an Mdm2-dependent manner. Co-expression of NEDP1, a cysteine protease that specifically cleaves NEDD8 conjugates, was shown to deneddylate TAp73. In addition, blockage of the endogenous NEDD8 pathway increased TAp73-mediated transactivation of p53- and p73-responsive promoter-driven reporter activity, and in conjunction, neddylated TAp73 species were found preferentially in the cytoplasm. These results suggest that Mdm2 attenuates TAp73 transactivation function, at least in part, by promoting NEDD8-dependent TAp73 cytoplasmic localization and provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73.

Hypoxia-inducible factor (HIF) is a central regulator of cellular responses to hypoxia, and under normal oxygen tension the catalytic α subunit of HIF is targeted for ubiquitin-mediated destruction via the VHL-containing E3 ubiquitin ligase complex. Principally known for its association with oncogenesis, HIF has been documented to have a role in the antibacterial response. Interferons, cytokines with antiviral functions, have been shown to upregulate the expression of HIF-1α, but the significance of HIF in the antiviral response has not been established. Here, using renal carcinoma cells devoid of VHL or reconstituted with functional wild-type VHL or VHL mutants with various abilities to negatively regulate HIF as an ideal model system of HIF activity, we show that elevated HIF activity confers dramatically enhanced resistance to vesicular stomatitis virus (VSV)-mediated cytotoxicity. Inhibition of HIF activity using a small-molecule inhibitor, chetomin, enhanced cellular sensitivity to VSV, while treatment with hypoxia mimetic CoCl2promoted resistance. Similarly, targeting HIF-2α by RNA interference also enhanced susceptibility to VSV. Expression profiling studies show that upon VSV infection, the induction of genes with known antiviral activity, such as that encoding beta interferon (IFN-β), is significantly enhanced by HIF. These results reveal a previously unrecognized role of HIF in the antiviral response by promoting the expression of the IFN-β gene and other genes with antiviral activity upon viral infection.

The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.

Background: The duration of survival benefit from MAPKi in B-raf mutant melanoma (MEL) is limited by the near universal development of resistance (RES). Recently, MiTF, a transcription factor and master regulator of melanocyte differentiation - was identified as a hub of MAPKi RES. Given its dual role as both a tumor promoter and inducer of MEL senescence, MiTF is a problematic therapeutic target. Pigmentation is a MiTF-dependent feature of MELs correlates with poor survival. We have interrogated the MAPKi/MiTF-dependent regulation of a panel of MITF-regulated melanosomal differentiation antigens (MDAs) for their potential as therapeutic targets to overcome MAPKi RES. Methods: The TCGA MEL gene expression data set was interrogated. B-raf mutant MEL cells were exposed to dabrafenib (D), or trametinib (T). qRT-PCR, Immunoblot or FACS were used to assess MiTF and MDA expression. Serial biopsy tissue and serum samples from MEL patients taken before treatment, during treatment, and at progression with MAPKi therapy were obtained, and analyzed by IHC and qRT-PCR, RNAseq, and ELISA. Transient siRNA-mediated knockdown was used to assess the requirement for MiTF, in MAPKi-mediated induction of MDAs, including GPNMB. To assess the interaction between MAPKi and CDX011 (C), a GPNMB- targeted antibody drug conjugate, mice bearing A375 and WM2664 xenografts were treated with T or DT + C in combination. Results: MiTF is strongly correlated with MDAs, including GPNMB in gene-expression datasets derived from human MEL tumors. A MiTF driven MDA-gene-signature correlates with poor survival among 291 melanoma patients. MAPKi treatment increased MiTF and MDAs in MEL cells, and induced pigmentation in vivo. This phenotype was reversible as MEL acquired RES to MAPKi. Knockdown of MiTF leads to abrogation of MAPKi-mediated GPNMB induction. GPNMB promotes MEL invasion in vitro. MiTF and MDA were elevated in tumors and serum from patients receiving MAPKi treatment and returned to baseline in tumors that acquired RES. The addition of C to either T or DT led to, decreased pigmentation, prolonged tumor regression and inhibited the development RES to T or DT treatment in vivo. Tumors that acquired RES to DT displayed increased c-Met expression and MAPK activation; tumors treated with DT+C failed to develop this phenotype. Conclusions: MAPKi induce MDA in a MiTF-dependent manner in MEL. This process leads to melanocytic differentiation and increased pigmentation, which is subsequently reversed as a tumor acquires RES to MAPKi. This demonstrates the plasticity of MEL in response to MAPKi. The pigmented phenotype is less proliferative but more invasive which is in part mediated by GPNMB. This transient phenotype may allow tumors to acquire characteristics (ie. c-Met upregulation and MAPK re-activation) which contribute to MAPKi RES. Targeting a pro-invasive MDA, GPNMB with CDX-011 subverts this plasticity and impairs the development of MAPKi RES. Citation Format: April A.N. Rose, Matthew G. Annis, Dennie T. Frederick, Zhifeng Dong, Lawrence Kwong, Tibor Keller, Thomas Hawthorne, Ian R. Watson, Keith T. Flaherty, Peter M. Siegel. Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 296.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5–55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)—a PTEN-interacting protein and negative regulator of PTEN in breast cancer—as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma., National Human Genome Research Institute (U.S.)

p53 mutations are rarely detected in clear cell renal cell carcinoma (CCRCC), but, paradoxically, these tumors remain highly resistant to chemotherapy and death receptor–induced death. Here, we show that the accumulation of hypoxia-inducible factor 2α (HIF2α), a critical oncogenic event in CCRCC following the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser166), which is concomitant with low p53 expression. The abrogation of Hdm2-p53 interaction using the small-molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2α via HIF2α-specific short hairpin RNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2α and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC. [Cancer Res 2009;69(23):9056–64]

We describe here the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 resected primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification based on the pattern of the most prevalent significantly mutated genes into one of four subtypes: mutant BRAF, RAS, NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of mutations in KIT, focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We find no significant outcome correlation with genomic classification, but samples assigned to a transcriptomic subclass enriched for immune gene expression are significantly associated with improved patient survival. That favorable prognosis of the ‘immune’ transcriptomic subclass is associated with high LCK protein expression, a T-cell marker, and presence of lymphocyte infiltrate on pathology review. This clinicopathological and multi-dimensional analysis suggests prognosis of melanoma patients with nodal metastases is influenced by immunobiology of the tumor stroma, offering insights to further personalize therapeutic decision-making. Citation Format: Ian R. Watson, Chang-Jiun Wu, Lihua Zou, Jeffrey E. Gershenwald, Lynda Chin, on behalf of the Melanoma Analysis Working Group and The Cancer Genome Analysis Research Network. Genomic classification of cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2015-2972

Renal clear-cell carcinoma (RCC) is the predominant form of kidney cancer and is highly refractory to conventional anti-cancer therapies. The status of p53 tumor suppressor gene has been correlated with the efficacy of radio- and chemotherapies, where presence of mutant p53 is associated with reduced responsiveness to treatment. However, p53 itself is rarely mutated in RCC, rather suggesting that the p53 pathway might be compromized in RCC cells. In support of this notion, the transactivation property of normal p53 was shown to be repressed in various transformed kidney epithelial cells via an unknown dominant-negative mechanism. Mutation of the von Hippel-Lindau ( VHL ) gene causes familial VHL disease, which includes predisposition to RCC. Moreover, biallelic inactivation of VHL has been observed in the vast majority of sporadic RCC. Recently, the expression of pVHL in RCC cells was demonstrated to elevate the expression of p53 by inducing the binding of RNA-stabilizing protein HuR to the 3′untranslated region of p53 mRNA. Contrary to this finding, we report here that the reconstitution of a variety of VHL (−/−) RCC lines including 786-O, RCC4, and A498 or non-RCC cells with wild-type pVHL does not influence the expression of p53 and fails to induce p53-responsive gene p21 CIP1/WAF1 or p53-responsive reporters. These results suggest that the expression of p53 in RCC cells is independent of pVHL.

Following recurrent mutations in BRAF and NRAS, the RAC1 c.85C > T single nucleotide variant encoding P29S amino acid change represents the next most frequently observed hotspot mutation in melanoma. However, the biological and clinical relevance of the RAC1 P29S somatic mutation in approximately 4-9% of patients is not entirely clear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (dabrafenib and vemurafenib). Enforced expression of RAC1 P29S in sensitive BRAF mutant melanoma cell lines resulted in increased viability and in vivo tumor growth upon treatment with RAF inhibitors compared to controls. Conversely, knockdown of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to dabrafenib and vemurafenib. Our results suggest that RAC1 P29S status in melanoma may be an important predictor of RAF inhibitor resistance in patients, and consequently should be evaluated as a predictive biomarker to such therapies in the clinic. Citation Format: Ian R. Watson, Liren Li, Peter K. Cabeceiras, Mozhdeh Mahdavi, Zhuangna Fang, Katherine Stemke-Hale, Gordon B. Mills, Lynda Chin. The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2014-LB-214

Melanoma has emerged as the latest success story for both immuno- and genomics-guided targeted therapy following the advent of both monoclonal antibodies directed toward the inhibitory immune receptors CTLA-4, PD-1, and PD-L1, as well as BRAF inhibitors targeting mutant BRAF found in over 50% of melanoma patients. To characterize the genomic alterations in cutaneous melanoma, The Cancer Genome Atlas Project (TCGA) analyzed exome sequence, DNA structural variations (copy number and translocations), promoter methylation, mRNA, miRNA, and protein expression in primary and metastatic melanoma samples originating from non-glabrous skin in up to 336 samples. Interestingly, clustering analysis of RNA-seq data demonstrated 3 expression module subgroups of melanomas, categorized by elevated lymphocytic, keratin, and melanocyte differentiation markers. Consistent with previous findings, the high lymphocytic expression subgroup was found to possess a better overall survival. Mutation analysis from exome sequencing identified a broad spectrum of samples possessing a signature of UV mutagenesis dominated by C > T transitions at dipyrimidines. We observed a mean somatic mutation rate of 17 mutations per megabase (Mb), almost doubling the highest mutation rate reported previously for a cancer type analyzed by TCGA. The InVEx algorithm identified 13 significantly mutated genes (P. Bonferroni Citation Format: Ian R. Watson, Chang-Jiun Wu, Jeffrey E. Gershenwald, Lynda Chin, On Behalf of the Melanoma Analysis Working Group and The Cancer Genome Analysis Research Network. Comprehensive genomic characterization of cutaneous melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-315. doi:10.1158/1538-7445.AM2014-LB-315

Background: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. Results: To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. Conclusion: This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNA-imediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability via NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death. Citation Information: Cancer Res 2009;69(23 Suppl):B1.