Genetic Silencing of AKT Induces Melanoma Cell Death via mTOR Suppression.

Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Melanoma central nervous system metastases: An update to approaches, challenges, and opportunities.

Brain metastases are the most common brain malignancy. This review discusses the studies presented at the third annual meeting of the Melanoma Research Foundation in the context of other recent reports on the biology and treatment of melanoma brain metastases (MBM). Although symptomatic MBM patients were historically excluded from immunotherapy trials, efforts from clinicians and patient advocates have resulted in more inclusive and even dedicated clinical trials for MBM patients. The results of checkpoint inhibitor trials were discussed in conversation with current standards of care for MBM patients, including steroids, radiotherapy, and targeted therapy. Advances in the basic scientific understanding of MBM, including the role of astrocytes and metabolic adaptations to the brain microenvironment, are exposing new vulnerabilities which could be exploited for therapeutic purposes. Technical advances including single-cell omics and multiplex imaging are expanding our understanding of the MBM ecosystem and its response to therapy. This unprecedented level of spatial and temporal resolution is expected to dramatically advance the field in the coming years and render novel treatment approaches that might improve MBM patient outcomes.

The role of PI3'-lipid signalling in melanoma initiation, progression and maintenance.

Phosphatidylinositol-3'-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3' hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5)P3 and PI(3,4)P2 regulate myriad biochemical and biological processes in both normal and cancer cells including responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3'-kinases (PI3Kα, ß, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K-lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3'-kinase signalling in cells. Indeed, in preclinical models, activation of the PI3K→AKT signalling axis cooperates with alterations such as expression of the BRAFV600E oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumour microenvironment, melanoma maintenance and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3K→AKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.

Prior Exposure to Coxsackievirus A21 Does Not Mitigate Oncolytic Therapeutic Efficacy.

Oncolytic viruses (OVs) are being developed as a type of immunotherapy and have demonstrated durable tumor responses and clinical efficacy. One such OV, Coxsackievirus A21 (CVA21), exhibited therapeutic efficacy in early phase clinical trials, demonstrating the ability to infect and kill cancer cells and stimulate anti-tumor immune responses. However, one of the major concerns in using this common cold virus as a therapeutic is the potential for innate and adaptive immune responses to mitigate the benefits of viral infection, particularly in individuals that have been exposed to coxsackievirus prior to treatment. In this study, we assess melanoma responses to CVA21 in the absence or presence of prior exposure to the virus. Melanomas were transplanted into naïve or CVA21-immunized C57BL6 mice and the mice were treated with intratumoral (IT) CVA21. We find that prior exposure to CVA21 does not dramatically affect tumor responses, nor does it alter overall survival. Our results suggest that prior exposure to coxsackievirus is not a critical determinant of patient selection for IT CVA21 interventions.

First line immunotherapy extends brain metastasis free survival, improves overall survival, and reduces the incidence of brain metastasis in patients with advanced melanoma.

BACKGROUND: Recent advances in targeted therapy and immunotherapy have improved the prognosis of melanoma patients but brain metastasis remains a major challenge. Currently, it is unclear how existing therapies can be best used to prevent or treat brain metastasis in melanoma patients. AIMS: We aimed to assess brain metastasis free survival (BMFS), overall survival (OS), incidence of brain metastases, and sequencing strategies of immunotherapy and targeted therapy in patients with BRAF-mutated advanced melanoma. METHODS AND RESULTS: We retrospectively analyzed 683 patients with BRAF-mutated advanced melanoma treated with first line (1L) immunotherapy (N = 266) or targeted therapy (N = 417). The primary outcome was BMFS. Secondary outcomes included OS of all patients and incidence of brain metastases in patients without documented brain metastases prior to 1L therapy. The median BMFS was 13.7 months [95% confidence interval (CI): 12.4-16.0] among all patients. The median BMFS for patients receiving 1L immunotherapy was 41.9 months [95% CI: 22.8-not reached (NR)] and targeted therapy was 11.0 months (95% CI: 8.8-12.5). Median OS results were qualitatively similar to BMFS results. The cumulative incidence of brain metastases for patients receiving 1L targeted therapy was higher than for patients receiving 1L immunotherapy (P < .001). Patients receiving 1L anti-CTLA4 plus anti-PD1 combination immunotherapy only or followed by second line (2L) targeted therapy had better BMFS (HR 0.40, 95% CI: 0.24-0.67, P = .001), improved OS (HR 0.49, 95% CI: 0.30-0.81, P = .005), and reduced incidence of brain metastases (HR 0.47, 95% CI: 0.24-0.67, P = .047) than patients receiving 1L combination BRAF and MEK targeted therapy followed by 2L immunotherapy. CONCLUSION: Patients with advanced BRAF mutant melanoma treated with 1L immunotherapy have significantly longer BMFS and OS, and reduced incidence of brain metastases, compared with those treated with 1L targeted therapy. Further studies evaluating the ability of immunotherapy and targeted therapy to improve OS and prevent brain metastases are warranted.

Melanoma models for the next generation of therapies.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Antitumor T-cell Immunity.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600, and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521.

Brain Metastasis Cell Lines Panel: A Public Resource of Organotropic Cell Lines.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood-brain barrier, blood-tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell-Specific Gene Promoters.

BACKGROUND: Brain metastases (BrM) develop in 20-40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain because of the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems. METHODS: Green fluorescent protein (GFP)-transduced murine and nontransduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene-expression analysis and subsequent validation of a series of promoter-green fluorescent protein-reporter constructs in mice (n = 5). One of the promoters was used to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL vs control group). RESULTS: HSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (mean [SD] = 37.6% [7.2%] of all infiltrating cells for murine HSC progeny; 27.9% mean [SD] = 27.9% [4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3-53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (mean [SD] = 8.8% [7.8%]). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice. TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (mean [SD] = 19.0 [3.4] vs mean [SD] = 15.0 [2.0] days for TRAIL vs control group; two-sided P = .006), demonstrating therapeutic and translational potential of our approach. CONCLUSIONS: Our study establishes HSC gene therapy using a myeloid cell-specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions.

The Small GTPase ARF6 Activates PI3K in Melanoma to Induce a Prometastatic State.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.

AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis.

Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. IMPLICATIONS: This study suggests that AKT1E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.

Molecular Profiling Reveals Unique Immune and Metabolic Features of Melanoma Brain Metastases.

There is a critical need to improve our understanding of the pathogenesis of melanoma brain metastases (MBM). Thus, we performed RNA sequencing on 88 resected MBMs and 42 patient-matched extracranial metastases; tumors with sufficient tissue also underwent whole-exome sequencing, T-cell receptor sequencing, and IHC. MBMs demonstrated heterogeneity of immune infiltrates that correlated with prior radiation and post-craniotomy survival. Comparison with patient-matched extracranial metastases identified significant immunosuppression and enrichment of oxidative phosphorylation (OXPHOS) in MBMs. Gene-expression analysis of intracranial and subcutaneous xenografts, and a spontaneous MBM model, confirmed increased OXPHOS gene expression in MBMs, which was also detected by direct metabolite profiling and [U-13C]-glucose tracing in vivo. IACS-010759, an OXPHOS inhibitor currently in early-phase clinical trials, improved survival of mice bearing MAPK inhibitor-resistant intracranial melanoma xenografts and inhibited MBM formation in the spontaneous MBM model. The results provide new insights into the pathogenesis and therapeutic resistance of MBMs. SIGNIFICANCE: Improving our understanding of the pathogenesis of MBMs will facilitate the rational development and prioritization of new therapeutic strategies. This study reports the most comprehensive molecular profiling of patient-matched MBMs and extracranial metastases to date. The data provide new insights into MBM biology and therapeutic resistance.See related commentary by Egelston and Margolin, p. 581.This article is highlighted in the In This Issue feature, p. 565.

Melanoma central nervous system metastases: An update to approaches, challenges, and opportunities.

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.

Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo.

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anticancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mmol/L and 20 µmol/L, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD/SCID mice, salicylate was detected in plasma and skin at 4 hours and PGE2 levels were reduced up to 24 hours. Some human melanoma tumors xenografted into NOD/SCID mice were sensitive to chronic daily ASA administration, exhibiting reduced growth and proliferation. ASA-treated mice bearing sensitive and resistant tumors exhibited both decreased PGE2 in plasma and tumors and increased phosphorylated AMPK in tumors. We conclude that ASA inhibits colony formation, cell motility, and pigmentation through suppression of PGE2 and activation of AMPK and reduces growth of some melanoma tumors in vivo This preclinical model could be used for further tumor and biomarker studies to support future melanoma chemoprevention trials in humans. Cancer Prev Res; 11(10); 629-42. ©2018 AACR.

Mutant IDH1 Promotes Glioma Formation In Vivo.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.

A Nonsynonymous Variant in the GOLM1 Gene in Cutaneous Malignant Melanoma.

Background: Statistically significant linkage of melanoma to chromosome 9q21 was previously reported in a Danish pedigree resource and independently confirmed in Utah high-risk pedigrees, indicating strong evidence that this region contains a melanoma predisposition gene. Methods: Whole-exome sequencing of pairs of related melanoma case subjects from two pedigrees with evidence of 9q21 linkage was performed to identify the responsible predisposition gene. Candidate variants were tested for association with melanoma in an independent set of 454 unrelated familial melanoma case subjects and 396 unrelated cancer-free control subjects from Utah, and 1534 melanoma case subjects and 1146 noncancer control subjects from Texas (MD Anderson) via a two-sided Fisher exact test. Results: A rare nonsynonymous variant in Golgi Membrane Protein 1 (GOLM1), rs149739829, shared in two hypothesized predisposition carriers in one linked pedigree was observed. Segregation of this variant in additional affected relatives of the index carriers was confirmed. A statistically significant excess of carriers of the variant was observed among Utah case subjects and control subjects (odds ratio [OR] = 9.81, 95% confidence interval [CI] = 8.35 to 11.26, P < .001) and statistically significantly confirmed in Texas case subjects and control subjects (OR = 2.45, 95% CI = 1.65 to 3.25, P = .02). Conclusion: These findings support GOLM1 as a candidate melanoma predisposition gene.