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.

Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Nicotinamide Inhibits T Cell Exhaustion and Increases Differentiation of CD8 Effector T Cells.

One of the limitations of immunotherapy is the development of a state referred to as T cell exhaustion (TEx) whereby T cells express inhibitory receptors (IRs) and lose production of effectors involved in killing of their targets. In the present studies we have used the repeated stimulation model with anti CD3 and anti CD28 to understand the factors involved in TEx development and treatments that may reduce changes of TEx. The results show that addition of nicotinamide (NAM) involved in energy supply to cells prevented the development of inhibitory receptors (IRs). This was particularly evident for the IRs CD39, TIM3, and to a lesser extent LAG3 and PD1 expression. NAM also prevented the inhibition of IL-2 and TNFα expression in TEx and induced differentiation of CD4+ and CD8 T cells to effector memory and terminal effector T cells. The present results showed that effects of NAM were linked to regulation of reactive oxygen species (ROS) consistent with previous studies implicating ROS in upregulation of TOX transcription factors that induce TEx. These effects of NAM in reducing changes of TEx and in increasing the differentiation of T cells to effector states appears to have important implications for the use of NAM supplements in immunotherapy against cancers and viral infections and require further exploration in vivo.

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.

Lysine Demethylases: Promising Drug Targets in Melanoma and Other Cancers.

Epigenetic dysregulation has been implicated in a variety of pathological processes including carcinogenesis. A major group of enzymes that influence epigenetic modifications are lysine demethylases (KDMs) also known as "erasers" which remove methyl groups on lysine (K) amino acids of histones. Numerous studies have implicated aberrant lysine demethylase activity in a variety of cancers, including melanoma. This review will focus on the structure, classification and functions of KDMs in normal biology and the current knowledge of how KDMs are deregulated in cancer pathogenesis, emphasizing our interest in melanoma. We highlight the current knowledge gaps of KDMs in melanoma pathobiology and describe opportunities to increases our understanding of their importance in this disease. We summarize the progress of several pre-clinical compounds that inhibit KDMs and represent promising candidates for further investigation in oncology.

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.

Pretreatment Innate Cell Populations and CD4 T Cells in Blood Are Associated With Response to Immune Checkpoint Blockade in Melanoma Patients.

The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following in vitro stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14+CD16- monocytes were higher and the major subset of NK cells (CD16hiCD56+) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.

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.

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.

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.

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.

Somatic Copy Number Amplification and Hyperactivating Somatic Mutations of EZH2 Correlate With DNA Methylation and Drive Epigenetic Silencing of Genes Involved in Tumor Suppression and Immune Responses in Melanoma.

The epigenetic modifier EZH2 is in the center of a repressive complex controlling differentiation of normal cells. In cancer EZH2 has been implicated in silencing tumor suppressor genes. Its role in melanoma as well as target genes affected by EZH2 are poorly understood. In view of this we have used an integrated systems biology approach to analyze 471 cases of skin cutaneous melanoma (SKCM) in The Cancer Genome Atlas (TCGA) for mutations and amplifications of EZH2. Identified changes in target genes were validated by interrogation of microarray data from melanoma cells treated with the EZH2 inhibitor GSK126. We found that EZH2 activation by mutations, gene amplification and increased transcription occurred in about 20% of the cohort. These alterations were associated with significant hypermethylation of DNA and significant downregulation of 11% of transcripts in patient RNASeq data. GSK126 treatment of melanoma lines containing EZH2 activation reversed such transcriptional repression in 98 candidate target genes. Gene enrichment analysis revealed genes associated with tumor suppression, cell differentiation, cell cycle inhibition and repression of metastases as well as antigen processing and presentation pathways. The identified changes in EZH2 were associated with an adverse prognosis in the TCGA dataset. These results suggest that inhibiting of EZH2 is a promising therapeutic avenue for a substantial fraction of melanoma patients.