Uveal melanoma: From diagnosis to treatment and the science in between.

Melanomas of the choroid, ciliary body, and iris of the eye are collectively known as uveal melanomas. These cancers represent 5% of all melanoma diagnoses in the United States, and their age-adjusted risk is 5 per 1 million population. These less frequent melanomas are dissimilar to their more common cutaneous melanoma relative, with differing risk factors, primary treatment, anatomic spread, molecular changes, and responses to systemic therapy. Once uveal melanoma becomes metastatic, therapy options are limited and are often extrapolated from cutaneous melanoma therapies despite the routine exclusion of patients with uveal melanoma from clinical trials. Clinical trials directed at uveal melanoma have been completed or are in progress, and data from these well designed investigations will help guide future directions in this orphan disease. Cancer 2016;122:2299-2312. © 2016 American Cancer Society.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

Genome-wide association study identifies novel loci predisposing to cutaneous melanoma.

We performed a multistage genome-wide association study of melanoma. In a discovery cohort of 1804 melanoma cases and 1026 controls, we identified loci at chromosomes 15q13.1 (HERC2/OCA2 region) and 16q24.3 (MC1R) regions that reached genome-wide significance within this study and also found strong evidence for genetic effects on susceptibility to melanoma from markers on chromosome 9p21.3 in the p16/ARF region and on chromosome 1q21.3 (ARNT/LASS2/ANXA9 region). The most significant single-nucleotide polymorphisms (SNPs) in the 15q13.1 locus (rs1129038 and rs12913832) lie within a genomic region that has profound effects on eye and skin color; notably, 50% of variability in eye color is associated with variation in the SNP rs12913832. Because eye and skin colors vary across European populations, we further evaluated the associations of the significant SNPs after carefully adjusting for European substructure. We also evaluated the top 10 most significant SNPs by using data from three other genome-wide scans. Additional in silico data provided replication of the findings from the most significant region on chromosome 1q21.3 rs7412746 (P = 6 × 10(-10)). Together, these data identified several candidate genes for additional studies to identify causal variants predisposing to increased risk for developing melanoma.

Slug expression during melanoma progression.

Slug (Snai2), a member of the Snail family of zinc finger transcription factors, plays a role in the epithelial-to-mesenchymal transformation (EMT) that occurs during melanocyte emigration from the neural crest. A role for Slug in the EMT-like loss of cell adhesion and increased cell motility exhibited during melanoma progression has also been proposed. Our immunohistochemical studies of melanoma arrays, however, revealed that Slug expression was actually higher in nevi than in primary or metastatic melanomas. Moreover, Slug expression in melanomas was not associated with decreased expression of E-cadherin, the canonical Slug target in EMT. Comparisons of endogenous Slug and E-cadherin expression in cultured normal human melanocytes and melanoma cell lines supported our immunohistochemical findings. Expression of exogenous Slug in melanocytes and melanoma cells in vitro, however, suppressed E-cadherin expression, enhanced N-cadherin expression, and stimulated cell migration and invasion. Interestingly, both in tumors and cultured cell lines, there was a clear relationship between expression of Slug and MITF, a transcription factor known to regulate Slug expression during development. Taken together, our findings suggest that Slug expression during melanomagenesis is highest early in the process and that persistent Slug expression is not required for melanoma progression. The precise role of Slug in melanomagenesis remains to be elucidated and may be related to its interactions with other drivers of EMT, such as Snail.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion. Significance: Collagen is a predominant component of the extracellular matrix that may influence the tumor immune microenvironment for cancer progression. We present here that mPGES-1 has specific roles in regulating tumor immunity, associated with several collagen-related genes and propose that pharmacologic inhibition of mPGES-1 may hold therapeutic promise for improving immune checkpoint-based therapies.

The role of melanoma tumor-derived nitric oxide in the tumor inflammatory microenvironment: its impact on the chemokine expression profile, including suppression of CXCL10.

Melanoma appears to be heterogeneous in terms of its molecular biology, etiology and epidemiology. We previously reported that the expression of inducible nitric-oxide synthase (iNOS) in melanoma tumor cells is strongly correlated with poor patient survival. Therefore, we hypothesized that nitric oxide (NO) produced by iNOS promotes the melanoma inflammatory tumor microenvironment associated with poor outcome. To understand the role of NO and iNOS in the melanoma inflammatory tumor microenvironment, polymerase chain reaction arrays of inflammatory and autoimmunity genes were performed on a series of stage III melanoma lymph node metastasis samples to compare the gene expression profiles of iNOS-expressing and nonexpressing tumor samples. The results indicate that expression of CXC chemokine ligand 10 (CXCL10) was inversely correlated with iNOS expression, and the high CXCL10-expressing cases had more favorable prognoses than the low CXCL10-expressing cases. Functional studies revealed that treating iNOS-negative/CXCL10-positive melanoma cell lines with a NO donor suppressed the expression of CXCL10. Furthermore, scavenging NO from iNOS-expressing cell lines significantly affected the chemokine expression profile. Culture supernatants from NO scavenger-treated melanoma cells promoted the migration of plasmacytoid dendritic cells, which was diminished when the cells were treated with a CXCL10-neutralizing antibody. CXCL10 has been reported to be an antitumorigenic chemokine. Our study suggests that the production of NO by iNOS inhibits the expression of CXCL10 in melanoma cells and leads to a protumorigenic tumor microenvironment. Inhibiting NO induces an antitumorigenic environment, and thus, iNOS should be considered to be an important therapeutic target in melanoma.

Association of activated c-Met with NRAS-mutated human melanomas.

Cutaneous melanomas can be divided into three mutually exclusive genetic subsets: tumors with mutated BRAF, tumors with mutated NRAS and tumors wild type at both loci (wt/wt). Targeted therapy for melanoma has been advancing with agents directed to mutated BRAF, accounting for 50% of melanoma patients. The c-Met pathway is known to play a role in melanoma tumorigenesis and preliminary data from our laboratory suggested that this pathway is preferentially activated in NRAS-mutated tumors. The objective of this study was to test the hypothesis that melanomas carrying the mutated NRAS genotype are uniquely sensitively to c-Met inhibition, thus providing rationale for therapeutic targeting of c-Met in this patient cohort. Using primary human melanomas with known BRAF/NRAS genotypes, we observed greater immunostaining for phosphorylated (activated) c-Met in NRAS-mutated and wt/wt tumors, compared to BRAF-mutated tumors. NRAS-mutated and wt/wt cell lines also demonstrated more robust c-Met activation in response to hepatocyte growth factor (HGF). Knock-down of mutated N-Ras, but not wild type N-Ras, by RNA interference resulted in decreased c-Met phosphorylation. Compared to BRAF mutants, NRAS-mutated melanoma cells were more sensitive to pharmacologic c-Met inhibition in terms of c-Met activation, Akt phosphorylation, tumor cell proliferation, migration and apoptosis. This enhanced sensitivity was observed in wt/wt cells as well, but was a less consistent finding. On the basis of these experimental results, we propose that c-Met inhibition may be a useful therapeutic strategy for melanomas with NRAS mutations, as well as some tumors with a wt/wt genotype.

Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma.

Soluble forms of receptors play distinctive roles in modulating signal-transduction pathways. Soluble CD74 (sCD74) has been identified in sera of inflammatory diseases and implicated in their pathophysiology; however, few relevant data are available in the context of cancer. Here we assessed the composition and production mechanisms, as well as the clinical significance and biological properties, of sCD74 in melanoma. Serum sCD74 levels were significantly elevated in advanced melanoma patients compared with normal healthy donors, and the high ratio of sCD74 to macrophage-migration inhibitory factor (MIF) conferred significant predictive value for prolonged survival in these patients (p = 0.0035). Secretion of sCD74 was observed primarily in melanoma cell lines as well as a THP-1 line of macrophages from monocytes and primary macrophages, especially in response to interferon-γ (IFN-γ). A predominant form that showed clinical relevance was the 25-KDa sCD74, which originated from the 33-KDa isoform of CD74. The release of this sCD74 was regulated by either a disintegrin and metalloproteinase-mediated cell-surface cleavage or cysteine-protease-mediated lysosomal cleavage, depending on cell types. Both recombinant and THP-1 macrophage-released endogenous sCD74 suppressed melanoma cell growth and induced apoptosis under IFN-γ stimulatory conditions via inhibiting the MIF/CD74/AKT-survival pathway. Our findings demonstrate that the interplay between sCD74 and MIF regulates tumor progression and determines patient outcomes in advanced melanoma.

Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth.

Uveal melanoma originating in the eye and metastasizing to the liver is associated with poor prognosis and has only one approved therapeutic option. We hypothesized that liver-borne growth factors may contribute to UM growth. Therefore, we investigated the role of IGF-1/IGF-1R signaling in UM. Here, we found that IRS-1, the insulin receptor substrate, is overexpressed in both UM cells and tumors. Since we previously observed that IGF-1R antibody therapy was not clinically effective in UM, we investigated the potential of NT157, a small molecule inhibitor of IRS-1/2, in blocking this pathway in UM. NT157 treatment of multiple UM cell lines resulted in reduced cell growth and migration and increased apoptosis. This treatment also significantly inhibited UM tumor growth in vivo, in the chicken egg chorioallantoic membrane (CAM) and subcutaneous mouse models, validating the in vitro effect. Mechanistically, through reverse phase protein array (RPPA), we identified significant proteomic changes in the PI3K/AKT pathway, a downstream mediator of IGF-1 signaling, with NT157 treatment. Together, these results suggest that NT157 inhibits cell growth, survival, and migration in vitro, and tumor growth in vivo via inhibiting IGF-1 signaling in UM.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

Zyflamend mediates therapeutic induction of autophagy to apoptosis in melanoma cells.

Melanoma is the most aggressive form of skin cancer. The rising incidence of melanoma and its poor prognosis in advanced stages are compelling reasons to identify novel therapeutic agents. Though isolated dietary components such as lycopene, resveratrol, and isothiocyanate compounds have been shown to provide limited protection against cancer development, the use of whole herbs and herbal extracts for the treatment of cancer remains of great interest. As suggested by earlier studies, the antiinflammatory activity of many plants available as intact products or as extracts has long been considered for supplemental therapeutics for cancer. Zyflamend, a unique multiherbal extract preparation, is a promising antiinflammatory agent that has also been suggested to regulate multiple pathways in cancer progression. As Zyflamend contains ingredients that can suppress tumor cell proliferation, invasion, angiogenesis, and metastasis through regulation of inflammatory pathway products, we hypothesized that this preparation might inhibit melanoma proliferation. To test this hypothesis, we studied the effect of Zyflamend on melanoma proliferation. Here, we present that Zyflamend inhibits melanoma growth by regulating the autophagy-apoptosis switch. Based on the responsible molecular mechanisms of Zyflamend, our study highlights the importance of the use of herbal preparations for the prevention and treatment of cancer.

iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation.

We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO-driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

IL-24 is expressed during wound repair and inhibits TGFalpha-induced migration and proliferation of keratinocytes.

Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.

The Expression of CD74-Regulated Inflammatory Markers in Stage IV Melanoma: Risk of CNS Metastasis and Patient Survival.

Innate inflammatory features have been found in melanoma tumors from patients at all stages, and molecular analysis has identified definitive inflammatory proteins expressed by tumors cells in patients who presents the worst prognosis. We have previously observed weakened outcomes in patients with constitutive expression of inducible nitric oxide synthase (iNOS), macrophage migration inhibitory factor (MIF) and improved outcomes with CD74 expression in stage III melanoma. In our current study, we tested our hypothesis on CD74-regulated inflammatory markers' expression in stage IV melanoma tumors whether the signature is associated with survival outcome and/or risk of developing CNS metastasis. We retrospectively identified 315 patients with stage IV melanoma. In a tissue microarray (TMA), we examined the expression of cells with CD74, its receptor MIF, and downstream inflammatory markers iNOS, nitrotyrosine (NT), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES1). We analyzed the association of those inflammatory markers with overall survival time (OS) and time to CNS metastasis using Kaplan-Meier survival analyses. Our data validates CD74 as a useful prognostic tumor cell protein marker associated with favorable OS as in stage III melanomas, while the tumor NT expression strongly predicts an increased risk of developing CNS metastasis (p = 0.0008) in those patients.

Genetic deficiency of complement isoforms C4A or C4B predicts improved survival of metastatic renal cell carcinoma.

PURPOSE: Autoimmune phenomena during immunotherapy are associated with favorable outcomes in patients with metastatic renal cell carcinoma. We have reported improved survival in patients with stage IV renal cell carcinoma who carry autoimmunity associated HLA class II haplotypes. We propose that the clinical benefit is mediated by products of other autoimmunity associated genes linked to these haplotypes. A candidate gene is complement C4, which replicates as part of the RCCX module, can be present in multiple copies and exists as C4A and C4B isoforms. Deficiencies of either isoform are associated with autoimmunity. In the current study we tested the hypothesis that C4A or C4B deficiency predicts improved survival of patients with RCC. MATERIALS AND METHODS: The total C4 copy number was determined by simultaneous amplification of RP1 and TNXA/RP2 to quantitate RCCX modules. C4A and C4B alleles were distinguished by PshAI restriction fragment length polymorphism. RESULTS: Genetic complotypes were determined in 61 patients. Individuals with a solitary copy of either C4 isoform experienced longer survival. Median survival from the diagnosis of metastatic disease in patients with a solitary copy of C4A or C4B was 7.75 years vs 1.25 in the comparison group (p = 0.001). This was independent of the benefit derived from autoimmune class II genotypes. CONCLUSIONS: Improved survival is seen in patients with C4A or C4B deficiency and renal cell carcinoma treated with cytokine therapy with or without surgery. These data support our hypothesis that patients with renal cell carcinoma who have autoimmune genotypes have favorable outcomes resulting from autoimmune mechanisms directed to the tumor.

A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells.

PURPOSE: Activation of signal transducers and activators of transcription 3 (STAT3) has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. EXPERIMENTAL DESIGN: The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. RESULTS: WP1066 achieved an IC(50) of 1.6, 2.3, and 1.5 mumol/L against melanoma cell line A375, B16, and B16EGFRvIII, respectively. WP1066 suppressed the phosphorylation of Janus-activated kinase 2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunologic memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (transforming growth factor-beta, RANTES, MCP-1, vascular endothelial growth factor), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of T cells. CONCLUSIONS: The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain.

Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells.

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide, a promising new derivative of dacarbazine, is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as temozolomide correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Interleukin-24 (IL-24; mda-7) is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple preclinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome temozolomide resistance in human melanoma cells. We have shown that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of temozolomide-induced cell killing in temozolomide-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced down-regulation of MGMT. Using either MGMT small interfering RNA, p53 small interfering RNA, or a p53 dominant-negative mutant to block MGMT protein expression resulted in increased sensitization to temozolomide. However, MGMT blockade in combination with IL-24 + temozolomide resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of temozolomide resistance in melanoma cells. This study shows that IL-24 can play a significant role in overcoming temozolomide resistance and that the clinical efficacy of temozolomide may be improved by using a biochemotherapy combination with IL-24.

Elevated Endogenous SDHA Drives Pathological Metabolism in Highly Metastatic Uveal Melanoma.

Purpose: Metastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy. Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy. Methods: We assessed two sets of oxidative phosphorylation (OxPhos) genes within 9858 tumors across 31 cancer types. An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM. Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities. Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non-M3 UM. We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition. Results: We identified UM to have among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile. M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the tricarboxylic acid cycle with OxPhos by oxidizing (lowering) succinate. We showed that SDHA-high M3 UM have elevated expression of key OxPhos molecules, exhibit abundant mitochondrial reserve respiratory capacity, and are resistant to OxPhos antagonism, which can be reversed by SDHA knockdown. Conclusions: Our study has identified a critical metabolic program within poor prognostic M3 UM. In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediate resistance to therapy that is reversible with targeted treatment.

The COX2 Effector Microsomal PGE2 Synthase 1 is a Regulator of Immunosuppression in Cutaneous Melanoma.

PURPOSE: Microsomal prostaglandin E2 synthase 1 (mPGES1) was evaluated as an important downstream effector of the COX2 pathway responsible for tumor-mediated immunosuppression in melanoma. EXPERIMENTAL DESIGN: The analysis of a stage III melanoma tissue microarray (n = 91) was performed to assess the association between mPGES1, COX2, CD8, and patient survival. Pharmacologic inhibitors and syngeneic mouse models using PTGES-knockout (KO) mouse melanoma cell lines were used to evaluate the mPGES1-mediated immunosuppressive function. RESULTS: We observed correlations in expression and colocalization of COX2 and mPGES1, which are associated with increased expression of immunosuppressive markers in human melanoma. In a syngeneic melanoma mouse model, PTGES KO increased melanoma expression of PD-L1, increased infiltration of CD8a+ T cells, and CD8a+ dendritic cells into tumors and suppressed tumor growth. Durable tumor regression was observed in mice bearing PTGES KO tumors that were given anti-PD-1 therapy. Analysis of a stage III melanoma tissue microarray revealed significant associations between high mPGES1 expression and low CD8+ infiltration, which correlated with a shorter patient survival. CONCLUSIONS: Our results are the first to illustrate a potential role for mPGES1 inhibition in melanoma immune evasion and selective targeting in supporting the durability of response to PD-1 checkpoint immunotherapy. More research effort in this drug development space is needed to validate the use of mPGES1 inhibitors as safe treatment options.

Genetic variants and haplotypes of the caspase-8 and caspase-10 genes contribute to susceptibility to cutaneous melanoma.

Caspase-8 (CASP8) and caspase-10 (CASP10) play key roles in regulating apoptosis, and their functional polymorphisms may alter apoptosis and cancer risk. However, no reported studies have investigated the association between such polymorphisms and the risk of cutaneous melanoma (CM). In a hospital-based study of 805 non-Hispanic white patients with CM and 835 cancer-free age-, sex-, and ethnicity-matched controls, we genotyped three reported putatively functional polymorphisms of CASP8 and CASP10-CASP8 D302 H (rs1045485:G>C), CASP8 -652 6N del (rs3834129:-/CTTACT), and CASP10 I522L (rs13006529:A>T)-and assessed their associations with risk of CM and interactions with known risk factors for CM. We also calculated the false-positive report probability (FPRP) for significant findings. CASP8 302 H variant genotypes (DH: adjusted odds ratio [OR], 0.70; 95% confidence interval [CI], 0.50-0.98; DH+HH: unadjusted OR, 0.78; 95% CI, 0.62-0.98; FPRP, 0.79) and CASP8 -652 6N del variant genotypes (ins/del: OR, 0.74; 95% CI, 0.57-0.97; ins/del+del/del: OR, 0.76; 95% CI, 0.61-0.95; FPRP, 0.61) were associated with significantly lower CM risk than were the DD and ins/ins genotypes, respectively. However, the CASP10 522L variant genotypes were not associated with significantly altered CM risk. Also, the D-del-I haplotype was associated with a significantly lower CM risk (OR, 0.52; 95% CI, 0.37-0.74; FPRP, 0.04) than was the most common haplotype, D-ins-I. Furthermore, multivariate logistic regression analysis revealed that CASP8 D302 H, CASP8 -652 6N del, and CASP10 I522L were independent risk factors for CM. Therefore, these CASP8 and CASP10 polymorphisms may be biomarkers for susceptibility to CM.