Tumour necrosis is a valuable histopathological prognostic parameter in melanomas of the vulva and vagina.

Vulvar and vaginal melanomas (VVMs) are rare and aggressive malignancies with limited prognostic models available and there is no standard reporting protocol. VVMs were selected from six tertiary Canadian hospitals from 2000-2021, resected from patients aged ≥18 years, with 6 months or longer follow-up data, and confirmation of melanocytic differentiation by at least two immunohistochemical markers. Cases were reviewed by pathologists to identify histological biomarkers. Survival outcomes were tested with Kaplan-Meier log-rank, univariate Cox, and multivariate Cox regression. There were 79 VVMs with median follow-up at 26 months. Univariate analysis revealed that tumour necrosis, tumour ulceration, positive lymph nodes, and metastasis at diagnosis were significantly associated with disease-specific mortality, progression, and metastasis. Multivariate analysis identified tumour necrosis as an independent prognostic factor for disease-specific mortality (HR 4.803, 95% CI 1.954-11.803, p<0.001), progression (HR 2.676, 95% CI 1.403-5.102, p=0.003), and time-to-metastasis for non-metastatic patients at diagnosis (HR 3.761, 95%CI 1.678-8.431, p=0.001). Kaplan-Meier survival analyses demonstrated that tumour necrosis was a poor prognostic factor for disease-specific, progression-free, and metastasis-free survival (p<0.001 for all comparisons). Vaginal melanomas displayed decreased survival compared to vulvar or clitoral melanomas. This study identifies tumour necrosis as an independent prognostic factor for VVMs. Vaginal melanomas specifically showed worse survival outcomes compared to vulvar or clitoral melanomas, consistent with previously reported findings in the literature, emphasising the importance of differentiating between these primary tumour epicentres for prognostication and treatment planning in the care of genital melanoma patients.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

Setdb1 -loss induces type-I interferons and immune clearance of melanoma.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

The Crossroads of Cancer Epigenetics and Immune Checkpoint Therapy.

Immune checkpoint inhibitors (ICI) have significantly improved treatment outcomes for several types of cancer over the past decade, but significant challenges that limit wider effectiveness of current immunotherapies remain to be addressed. Certain "cold" tumor types, such as pancreatic cancer, exhibit very low response rates to ICI due to intrinsically low immunogenicity. In addition, many patients who initially respond to ICI lack a sustained response due to T-cell exhaustion. Several recent studies show that epigenetic modifiers, such as SETDB1 and LSD1, can play critical roles in regulating both tumor cell-intrinsic immunity and T-cell exhaustion. Here, we review the evidence showing that multiple epigenetic regulators silence the expression of endogenous antigens, and their loss induces viral mimicry responses bolstering the response of "cold" tumors to ICI in preclinical models. Similarly, a previously unappreciated role for epigenetic enzymes is emerging in the establishment and maintenance of stem-like T-cell populations that are critical mediators of response to ICI. Targeting the crossroads of epigenetics and immune checkpoint therapy has tremendous potential to improve antitumor immune responses and herald the next generation of sustained responses in immuno-oncology.

Leukoderma Induced by Rotigotine Patch, a Transdermal Dopamine Agonist.

Leukoderma, or hypomelanosis of the skin, can occur in response to various chemical and pharmacologic substances ranging from topical medications to optic preparations and systemic medications. In this case report, we present a 78-year-old man with a history of restless leg syndrome (RLS) who had been using rotigotine transdermal patches once daily for 1 year and developed leukoderma on the bilateral anterior shoulders in the area of patch application. Histopathologic examination showed an absence of melanocytes at the dermal-epidermal junction confirmed by Melan A stain. While the patient was not bothered by the depigmentation and elected to continue the rotigotine patch for his RLS, this case highlights leukoderma as a potential side effect of dopamine transdermal patches and offers insight into the potential mechanism of hypopigmentation in response to dopamine agonism.

KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

Structure-Guided Identification of DNMT3B Inhibitors.

Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC50 values of 13-72 µM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 µM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC50 of 8.0 µM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC50 of 4.8 µM. Structure-activity data are rationalized based on computed structures for DNMT3B complexes.

RAP80 and BRCA1 PARsylation protect chromosome integrity by preventing retention of BRCA1-B/C complexes in DNA repair foci.

BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.

PD-L1 methylation regulates PD-L1 expression and is associated with melanoma survival.

The aim of this study is to determine the significance of programmed death ligand 1 (PD-L1 or CD274) methylation in relation to PD-L1 expression and survival in melanoma. Despite the clinical importance of therapies targeting the PD-1/PD-L1 immune checkpoint in melanoma, factors regulating PD-L1 expression, including epigenetic mechanisms, are not completely understood. In this study, we examined PD-L1 promoter methylation in relation to PD-L1 expression and overall survival in melanoma patients. Our results suggest that DNA methylation regulates PD-L1 expression in melanoma, and we identify the key methylated CpG loci in the PD-L1 promoter, establish PD-L1 methylation as an independent survival prognostic factor, provide proof of concept for altering PD-L1 expression by hypomethylating agents, and uncover that PD-L1 methylation is associated with an interferon signaling transcriptional phenotype. Based on our findings, measuring and altering PD-L1 promoter DNA methylation may have potential prognostic and therapeutic applications in melanoma.

Inhibition of isoprenylation synergizes with MAPK blockade to prevent growth in treatment-resistant melanoma, colorectal, and lung cancer.

This study evaluates the use of HMG-CoA reductase inhibitors, or statins, as an adjunctive to BRAF and MEK inhibition as a treatment in melanomas and other tumors with driver mutations in the MAPK pathway. Experiments used simvastatin in conjunction with vemurafenib and selumetinib in vitro and simvastatin with vemurafenib in vivo to demonstrate additional growth abrogation beyond MAPK blockade alone. Additional studies demonstrated that statin anti-tumor effects appeared to depend on inhibition of isoprenoid synthesis given rescue with add-back of downstream metabolites. Ultimately, we concluded that statins represent a possible useful adjunctive therapy in MAPK-driven tumors when given with current approved targeted therapy.

p90RSK Blockade Inhibits Dual BRAF and MEK Inhibitor-Resistant Melanoma by Targeting Protein Synthesis.

Despite improvements in survival in metastatic melanoma with combined BRAF and mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor treatment, the overwhelming majority of patients eventually acquire resistance to both agents. Consequently, new targets for therapy in resistant tumors are currently being evaluated. Previous studies have identified p90 subfamily of ribosomal S6 kinase (p90RSK) family kinases as key factors for growth and proliferation, as well as protein synthesis via assembly of the 7-methyl-guanosine triphosphate cap-dependent translation complex. We sought to evaluate inhibitors of p90RSK family members: BI-D1870 and BRD7389, for their ability to inhibit both proliferation and protein synthesis in patient-derived melanoma cell lines with acquired resistance to combined treatment with the BRAF inhibitor vemurafenib and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor selumetinib. We found that the RSK inhibitors blocked cell proliferation and protein synthesis in multiple dual-resistant melanoma lines. In addition, single agent RSK inhibitor treatment was effective in drug-naïve lines, two of which are innately vemurafenib resistant. We also used Reverse Phase Protein Array screening to identify differential protein expression that correlates with BI-D1870 sensitivity, and identified prognostic biomarkers for survival in human melanoma patients. These findings establish p90RSK inhibition as a therapeutic strategy in treatment-resistant melanoma and provide insight into the mechanism of action.

UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1-/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.

Aberrant DNA methylation in melanoma: biomarker and therapeutic opportunities.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, known to play important roles in melanoma formation and progression. Recent advances in genome-wide methylation methods have provided the means to identify differentially methylated genes, methylation signatures, and potential biomarkers. However, despite considerable effort and advances in cataloging methylation changes in melanoma, many questions remain unanswered. The aim of this review is to summarize recent developments, emerging trends, and important unresolved questions in the field of aberrant DNA methylation in melanoma. In addition to reviewing recent developments, we carefully synthesize the findings in an effort to provide a framework for understanding the current state and direction of the field. To facilitate clarity, we divided the review into DNA methylation changes in melanoma, biomarker opportunities, and therapeutic developments. We hope this review contributes to accelerating the utilization of the diagnostic, prognostic, and therapeutic potential of DNA methylation for the benefit of melanoma patients.

Attenuation of genome-wide 5-methylcytosine level is an epigenetic feature of cutaneous malignant melanomas.

Epigenetic modification of DNA, namely covalent changes of cytosine residues, plays a key role in the maintenance of inactive chromatin regions, both in health and in disease. In the vast majority of malignant melanomas, the most notable known epigenetic abnormality is the attenuation of 5-hydroxymethylcytosine (5-hmC) residues. However, it remains unknown whether a decrease in 5-hmC represents a primary defect of melanoma cancer epigenome or whether it is secondary to the loss of 5-methylcytosine (5-mC), a chemical substrate for 5-hmC. Here, we evaluated 5-mC levels in a spectrum of melanocytic proliferations. To study the epigenetic features of melanocytic nuclei, we began by measuring 5-mC levels in histologic specimens semiquantitatively by immunohistochemistry. We next treated established melanoma cell lines with S-adenosyl methionine (SAM), a universal methyl group donor, in an effort to cause changes in 5-mC levels. We detected a marked reduction in 5-mC levels in both primary and metastatic melanomas compared with 5-mC levels in benign melanocytic nevi. We also empirically induced changes in 5-mC in melanoma cell lines by incubation with SAM. To our surprise, we observed a significant cytoreductive effect of SAM on all melanoma cell lines examined. At subcytotoxic levels, SAM treatment is accompanied by a genome-wide increase in 5-mC. Moreover, we recorded a dose-dependent increase in genome-wide 5-mC levels in melanoma cell lines following SAM treatment. Taken together, we report that genome-wide attenuation of 5-mC is a hallmark of malignant melanomas. We propose that genome-wide attenuation of 5-mC is not merely an epiphenomenon as it is required for melanoma cell growth, albeit by an as of yet undetermined mechanism. Given its potential benefit in slowing down the growth of melanoma cells, SAM should be studied further to determine its role in epigenome modulation.