An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.

Network models of primary melanoma microenvironments identify key melanoma regulators underlying prognosis.

Melanoma is the most lethal skin malignancy, driven by genetic and epigenetic alterations in the complex tumour microenvironment. While large-scale molecular profiling of melanoma has identified molecular signatures associated with melanoma progression, comprehensive systems-level modeling remains elusive. This study builds up predictive gene network models of molecular alterations in primary melanoma by integrating large-scale bulk-based multi-omic and single-cell transcriptomic data. Incorporating clinical, epigenetic, and proteomic data into these networks reveals key subnetworks, cell types, and regulators underlying melanoma progression. Tumors with high immune infiltrates are found to be associated with good prognosis, presumably due to induced CD8+ T-cell cytotoxicity, via MYO1F-mediated M1-polarization of macrophages. Seventeen key drivers of the gene subnetworks associated with poor prognosis, including the transcription factor ZNF180, are tested for their pro-tumorigenic effects in vitro. The anti-tumor effect of silencing ZNF180 is further validated using in vivo xenografts. Experimentally validated targets of ZNF180 are enriched in the ZNF180 centered network and the known pathways such as melanoma cell maintenance and immune cell infiltration. The transcriptional networks and their critical regulators provide insights into the molecular mechanisms of melanomagenesis and pave the way for developing therapeutic strategies for melanoma.

Lysyl oxidase-like 3 is required for melanoma cell survival by maintaining genomic stability.

Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis. In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma. We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines. Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation. Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis. Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion. Moreover, LOXL3 is required for efficient DSB repair in melanoma cells. Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.

Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene.

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis.

Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis.

Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma.

Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.

Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy.

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.