BET inhibition sensitizes innate checkpoint inhibitor resistant melanoma to anti-CTLA-4 treatment.

Approximately 50% of melanoma patients fail to respond to immune checkpoint blockade (ICB), and acquired resistance hampers long-term survival in about half of initially responding patients. Whether targeting BET reader proteins, implicated in epigenetic dysregulation, can enhance ICB response rates and durability, remains to be determined. Here we show elevated BET proteins correlate with poor survival and ICB responses in melanoma patients. The BET inhibitor IBET151, combined with anti-CTLA-4, overcame innate ICB resistance however, sequential BET inhibition failed against acquired resistance in mouse models. Combination treatment response in the innate resistance model induced changes in tumor-infiltrating immune cells, reducing myeloid-derived suppressor cells (MDSCs). CD4+ and CD8+ T cells showed decreased expression of inhibitory receptors, with reduced TIM3, LAG3, and BTLA checkpoint expression. In human PBMCs in vitro, BET inhibition reduced expression of immune checkpoints in CD4+ and CD8+ T cells, restoring effector cytokines and downregulating the transcriptional driver TOX. BET proteins in melanoma may play an oncogenic role by inducing immune suppression and driving T cell dysfunction. The study demonstrates an effective combination for innately unresponsive melanoma patients to checkpoint inhibitor immunotherapy, yet highlights BET inhibitors' limitations in an acquired resistance context.

Repurposing Melanoma Chemotherapy to Activate Inflammasomes in the Treatment of BRAF/MAPK Inhibitor Resistant Melanoma.

The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy.

Melanoma with osseous or chondroid differentiation: a report of eight cases including SATB2 expression and mutation analysis.

Melanoma can present with osteocartilaginous differentiation, however few reports exist on this rare subtype. We present eight cases of melanoma with osteocartilaginous differentiation to highlight its clinical, pathological and molecular features. The cases showed no association with gender (5 males and 3 females) or age (range 23-84 years). Cases included both primary melanomas and distant metastases (6 and 2, respectively), with the majority arising from cutaneous sites (7/8) and the remaining case from a mucosal site. Tumour-infiltrating lymphocyte (TIL) score ranged from 0 to 3 (median 1), and 2/8 lesions had evidence of inflammatory changes or antecedent trauma. No recurrent mutations were found in the tumours by next generation sequencing, and the mutations observed were typical of melanoma rather than osteosarcomatous lesions. The majority of tumours stained positive for melanoma markers including S100, HMB45, Melan-A, SOX10 and MITF. Staining of the osteoblastic marker SATB2 varied from negative to widespread positive. We demonstrate that melanomas with osteocartilaginous differentiation are heterogeneous in presentation and are not typified by a recurrent mutation in cancer associated genes. Where uncertainty exists in diagnosing an osteocartilaginous lesion, a diagnosis of melanoma can be supported by the presence of genomic mutations typical of melanoma such as BRAF, NRAS and NF1, and IHC staining positive for S100, HMB45, Melan-A, SOX10 and MITF. SATB2 may be positive in these lesions and thus should not be used to rule out melanoma.

A Combination of Epigenetic BET and CDK9 Inhibitors for Treatment of Human Melanoma.

Dysregulation of epigenetic modifiers is a frequent event in melanoma and underlies many aspects of melanoma biology, including resistance to targeted therapy and immunotherapies. Here, we report that dual targeting of BET and CDK9 proteins have synergistic effects against melanoma cells in vitro and in vivo. The BET inhibitor (IBET151) and CDK9 inhibitor (CDKI73) synergistically killed melanoma cells in vitro independent of their BRAF or NRAS mutation status. The combination of drugs markedly inhibited the growth of human melanoma C002M cells in vitro in three-dimensional spheroids and in vivo in NOD-SCID gamma mice compared with vehicle control and the individual drugs (P < 0.05). Cell death was associated with mitochondrial depolarization, caspase-dependent apoptosis with cleavage of PARP1, and downregulation of anti-apoptotic proteins BCL2, BCLXL, and MCL1. Gene set enrichment analysis revealed downregulation of hallmark gene sets associated with E2F, G2M checkpoint, and c-MYC. Survival analysis showed worse prognosis with high G2M, E2F, or c-MYC gene signatures, suggesting biomarkers of response of BET and CDK9 inhibitors in melanoma. This combination of epigenetic inhibitors targets multiple downstream genes, leading to cell death of melanoma cells in vitro and in vivo, and warrants further investigation for treatment of melanoma in patients not responding to current therapies.

Sex bias of females in survival from cancer and infections. Is X the answer?

Major differences in survival of men and women from infectious diseases and cancers have been highlighted by death rates from COVID-19 infections. In cancer, attention has been focussed on differences in gene expression from X chromosomes in men and women with a preponderance of genes involved in immune responses being expressed in women. Important findings have been that some of the genes are important epigenetic regulators that play fundamental roles in immune responses.

Study of the Female Sex Survival Advantage in Melanoma-A Focus on X-Linked Epigenetic Regulators and Immune Responses in Two Cohorts.

BACKGROUND: Survival from melanoma is strongly related to patient sex, with females having a survival rate almost twice that of males. Many explanations have been proposed but have not withstood critical scrutiny. Prior analysis of different cancers with a sex bias has identified six X-linked genes that escape X chromosome inactivation in females and are, therefore, potentially involved in sex differences in survival. Four of the genes are well-known epigenetic regulators that are known to influence the expression of hundreds of other genes and signaling pathways in cancer. METHODS: Survival and interaction analysis were performed on the skin cutaneous melanoma (SKCM) cohort in The Cancer Genome Atlas (TCGA), comparing high vs. low expression of KDM6A, ATRX, KDM5C, and DDX3X. The Leeds melanoma cohort (LMC) on 678 patients with primary melanoma was used as a validation cohort. RESULTS: Analysis of TCGA data revealed that two of these genes-KDM6A and ATRX-were associated with improved survival from melanoma. Tumoral KDM6A was expressed at higher levels in females and was associated with inferred lymphoid infiltration into melanoma. Gene set analysis of high KDM6A showed strong associations with immune responses and downregulation of genes associated with Myc and other oncogenic pathways. The LMC analysis confirmed the prognostic significance of KDM6A and its interaction with EZH2 but also revealed the expression of KDM5C and DDX3X to be prognostically significant. The analysis also confirmed a partial correlation of KDM6A with immune tumor infiltrates. CONCLUSION: When considered together, the results from these two series are consistent with the involvement of X-linked epigenetic regulators in the improved survival of females from melanoma. The identification of gene signatures associated with their expression presents insights into the development of new treatment initiatives but provides a basis for exploration in future studies.

EZH2 Cooperates with DNA Methylation to Downregulate Key Tumor Suppressors and IFN Gene Signatures in Melanoma.

The histone methylase EZH2 is frequently dysregulated in melanoma and is associated with DNA methylation and silencing of genes involved in tumor suppression. In this study, we used chromatin immunoprecipitation and sequencing to identify key suppressor genes that are silenced by histone methylation in constitutively active EZH2(Y641) mutant melanoma and assessed whether these regions were also sites of DNA methylation. The genes identified were validated by their re-expression after treatment with EZH2 and DNA methyltransferase inhibitors. The expression of putative EZH2 target genes was shown to be highly relevant to the survival of patients with melanoma in clinical datasets. To determine correlates of response to EZH2 inhibitors, we screened a panel of 53 melanoma cell lines for drug sensitivity. We compared RNA sequencing profiles of sensitive to resistant melanoma cells and performed pathway analysis. Sensitivity was associated with strong downregulation of IFN-γ and IFN-α gene signatures that were reversed by treatment with EZH2 inhibitors. This is consistent with EZH2-driven dedifferentiated invasive states associated with treatment resistance and defects in antigen presentation. These results suggest that EZH2 inhibitors may be most effectively targeted to immunologically cold melanoma to both induce direct cytotoxicity and increase immune responses in the context of checkpoint inhibitor immunotherapy.

Co-targeting bromodomain and extra-terminal proteins and MCL1 induces synergistic cell death in melanoma.

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.

Epigenetic inhibitors eliminate senescent melanoma BRAFV600E cells that survive long­term BRAF inhibition.

It is estimated that ~50% of patients with melanoma harbour B­Raf (BRAF)V600 driver mutations, with the most common of these being BRAFV600E, which leads to the activation of mitogen­activated protein kinase proliferative and survival pathways. BRAF inhibitors are used extensively to treat BRAF­mutated metastatic melanoma; however, acquired resistance occurs in the majority of patients. The effects of long­term treatment with PLX4032 (BRAFV600 inhibitor) were studied in vitro on sensitive V600E BRAF­mutated melanoma cell lines. After several weeks of treatment with PLX4032, the majority of the melanoma cells died; however, a proportion of cells remained viable and quiescent, presenting senescent cancer stem cell­like characteristics. This surviving population was termed SUR cells, as discontinuing treatment allowed the population to regrow while retaining equal drug sensitivity to that of parental cells. RNA sequencing analysis revealed that SUR cells exhibit changes in the expression of 1,415 genes (P<0.05) compared with parental cells. Changes in the expression levels of a number of epigenetic regulators were also observed. These changes and the reversible nature of the senescence state were consistent with epigenetic regulation; thus, it was investigated as to whether the senescent state could be reversed by epigenetic inhibitors. It was found that both parental and SUR cells were sensitive to different histone deacetylase (HDAC) inhibitors, such as SAHA and MGCD0103, and to the cyclin­dependent kinase (CDK)9 inhibitor, CDKI­73, which induced apoptosis and reduced proliferation both in the parental and SUR populations. The results suggested that the combination of PLX4032 with HDAC and CDK9 inhibitors may achieve complete elimination of SUR cells that persist after BRAF inhibitor treatment, and reduce the development of resistance to BRAF inhibitors.

Do innate killing mechanisms activated by inflammasomes have a role in treating melanoma?

Melanoma, as for many other cancers, undergoes a selection process during progression that limits many innate and adaptive tumor control mechanisms. Immunotherapy with immune checkpoint blockade overcomes one of the escape mechanisms but if the tumor is not eliminated other escape mechanisms evolve that require new approaches for tumor control. Some of the innate mechanisms that have evolved against infections with microorganisms and viruses are proving to be active against cancer cells but require better understanding of how they are activated and what inhibitory mechanisms may need to be targeted. This is particularly so for inflammasomes which have evolved against many different organisms and which recruit a number of cytotoxic mechanisms that remain poorly understood. Equally important is understanding of where these mechanisms will fit into existing treatment strategies and whether existing strategies already involve the innate killing mechanisms.

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo.

RAS is frequently mutated in various tumors and known to be difficult to target. NRASQ61K/R are the second most frequent mutations found in human skin melanoma after BRAFV600E . Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRASQ61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRASQ61K mouse melanoma that can be used in vitro and in vivo in an immune-competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRASQ61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.

Targeting DNA Methylation and EZH2 Activity to Overcome Melanoma Resistance to Immunotherapy.

Methylation of DNA at CpG sites is the most common and stable of epigenetic changes in cancer. Hypermethylation acts to limit immune checkpoint blockade immunotherapy by inhibiting endogenous interferon responses needed for recognition of cancer cells. By contrast, global hypomethylation results in the expression of programmed death ligand 1 (PD-L1) and inhibitory cytokines, accompanied by epithelial-mesenchymal changes that can contribute to immunosuppression. The drivers of these contrasting methylation states are not well understood. DNA methylation also plays a key role in cytotoxic T cell 'exhaustion' associated with tumor progression. We present an updated exploratory analysis of how DNA methylation may define patient subgroups and can be targeted to develop tailored treatment combinations to help improve patient outcomes.

Marked Global DNA Hypomethylation Is Associated with Constitutive PD-L1 Expression in Melanoma.

Constitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy.

SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling.

While multiple mechanisms of BRAFV600-mutant melanoma resistance to targeted MAPK signaling inhibitors (MAPKi) have been reported, the epigenetic regulation of this process remains undetermined. Here, using a CRISPR-Cas9 screen targeting chromatin regulators, we discover that haploinsufficiency of the histone deacetylase SIRT6 allows melanoma cell persistence in the presence of MAPKi. Haploinsufficiency, but not complete loss of SIRT6 promotes IGFBP2 expression via increased chromatin accessibility, H3K56 acetylation at the IGFBP2 locus, and consequent activation of the IGF-1 receptor (IGF-1R) and downstream AKT signaling. Combining a clinically applicable IGF-1Ri with BRAFi overcomes resistance of SIRT6 haploinsufficient melanoma cells in vitro and in vivo. Using matched melanoma samples derived from patients receiving dabrafenib + trametinib, we identify IGFBP2 as a potential biomarker for MAPKi resistance. Our study has not only identified an epigenetic mechanism of drug resistance, but also provides insights into a combinatorial therapy that may overcome resistance to standard-of-care therapy for BRAFV600-mutant melanoma patients.

Simulation of melanoblast displacements reveals new features of developmental migration.

To distribute and establish the melanocyte lineage throughout the skin and other developing organs, melanoblasts undergo several rounds of proliferation, accompanied by migration through complex environments and differentiation. Melanoblast migration requires interaction with extracellular matrix of the epidermal basement membrane and with surrounding keratinocytes in the developing skin. Migration has been characterized by measuring speed, trajectory and directionality of movement, but there are many unanswered questions about what motivates and defines melanoblast migration. Here, we have established a general mathematical model to simulate the movement of melanoblasts in the epidermis based on biological data, assumptions and hypotheses. Comparisons between experimental data and computer simulations reinforce some biological assumptions, and suggest new ideas for how melanoblasts and keratinocytes might influence each other during development. For example, it appears that melanoblasts instruct each other to allow a homogeneous distribution in the tissue and that keratinocytes may attract melanoblasts until one is stably attached to them. Our model reveals new features of how melanoblasts move and, in particular, suggest that melanoblasts leave a repulsive trail behind them as they move through the skin.

Anti-PD-1-induced high-grade hepatitis associated with corticosteroid-resistant T cells: a case report.

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4-CCR6- effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.

Epigenetic modulation in cancer immunotherapy.

The success of immune checkpoint inhibitors in cancer immunotherapy has been widely heralded. However many cancer patients do not respond to immune checkpoint therapy and some relapse due to acquired tumor resistance. Epigenetic targeting may be beneficial in cancer immunotherapy by reversing immune avoidance and escape mechanisms employed by cancer cells, as well as by modulating immune cell differentiation and function. In this manuscript we review recent findings suggesting how epigenetics may be used to improve cancer immunotherapy. We focus on the inhibitors of the CTLA4 and PD1 immune checkpoints and epigenetic modifiers of histone acetylation and methylation and DNA methylation.

EZH2 as a mediator of treatment resistance in melanoma.

Direct treatments of cancer such as chemotherapy, radiotherapy and targeted therapy have been shown to depend on recruitment of the immune system for their effectiveness. Recent studies have shown that development of resistance to direct therapies such as BRAF inhibitors in melanoma is associated with suppression of immune responses. We point to emerging data that implicate activation of the polycomb repressive complex 2 (PRC2) and its catalytic component-enhancer of zeste homolog 2 (EZH2)-in progression of melanoma and suppression of immune responses. EZH2 appears to have an important role in differentiation of CD4 T cells and particularly in the function of T regulatory cells, which suppress immune responses to melanoma. We review mechanisms of EZH2 activation at the genomic level and from activation of the MAP kinase, E2F or NF-kB2 pathways. These studies are consistent with activation of EZH2 as a common mechanism for induction of immune suppression in patients failing direct therapies and suggest EZH2 inhibitors may have a role in combination with immunotherapy and targeted therapies to prevent development of immunosuppression.