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The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

SUMMARY Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors., In Brief Through an in vivo epigenome-focused pooled RNAi screen, Maitituoheti et al. identify KMT2D as a tumor suppressor in melanoma. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways by reduction of H3K4me1-marked active enhancers, conferring sensitivity to glycolysis and IGFR inhibitors in melanoma with KMT2D-inactivating mutations., Graphical Abstract

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. To determine chromatin state patterns of human metastatic melanomas, we defined epigenome of melanoma by profiling 6 histone modification marks (H3K4me, H3K27Ac, H3K4me3, H3K79me2, H3K27me3 and H3K9me3) in 48 metastatic melanoma tumors, cell lines and short-term cultures. Computation of chromatin states identified specific chromatin state aberrations in human tumors and their relationship with specific genotypes. Specifically, we defined patterns of enhancer elements, broad H3K4me3 domains and bivalent chromatin states enriched in specific genotypes of BRAF, NRAS, and WT groups of melanomas. Importantly, we identified switch from specific bivalent chromatin states in melanocytes to active states leading to upregulation of EMT genes in NRAS mutant melanomas. Similarly, we noted gains of broad H3K4me3 domains in particular classes of oncogenes in specific gentoypes as well as during transitions from melanocytes to melanomas. Finally, we identified clusters of enhancers that are specifically enriched in subset of melanoma samples defining intertumor epigenetic heterogeneity in melanoma tumors. In addition, to investigate chromatin state changes associated with early stages of melanoma progression, we systematically profiled 35 epigenetic modifications in a cell phenotypic model of non-tumorigenic and tumorigenic states (and verified in human nevi and melanoma samples). Specific chromatin state transitions featuring loss of histone acetylations and H3K4me2/3 were observed on specific promoter and enhancer elements targeting cancer-regulatory genes in important melanoma-driving cell signaling pathways. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Overall, our study reveals principles of epigenetic reprogramming in melanoma progression stages and how they may establish transcriptional programs that impart aggressive characteristics to melanomas. Citation Format: Kunal Rai, Christopher Terranova, Mayinuer Maitituoheti, Ming Tang, Kadir Akdemir, Elias Orouji, Lynda Chin, Petko Fiziev, Mayura Dhamdhere, Neha S. Samant, Amikhsha Shah, Sneha Sharma, Jason Ernst. Chromatin state alterations during melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4314.

Cancer cells utilize genetic and epigenetic aberrations for their excessive growth. Although we have sufficient understanding of the genomic alterations in colorectal cancer, we have incomplete knowledge of epigenomic aberrations and their impact on tumor growth. In order to comprehensively define the epigenetic patterns specific to colorectal cancer, we generated profiles for 6 histone modification marks, including H3K4me1 (enhancer), H3K27Ac (active enhancer), H3K9me3 (heterochromatin), H3K27me3 (polycomb repression), H3K79me2 (transcription) and H3K4me3 (promoter), using a high-throughput ChIP-Seq methodology developed in house applicable to frozen tumors. Chromatin state transitions specifically pointed to drastic changes in enhancer patterns, consistent with some prior studies. Furthermore, using similarity network fusion (SNF) we identified the best singular mark and are currently developing a tool to identify combinatorial chromatin states that could most efficiently distinguish and eventually predict CRC from normal colon. In a more detailed investigation into patterns of active enhancers using normal colon, adenomas and colorectal cancers, we identified specific changes in enhancers from normal tissue to these neoplastic lesions. Importantly, we noted gains of enhancers in a large number of genomic loci in colon cancer compared to adjacent normal tissues. These enhancers are enriched in important signaling components including Notch, Wnt, and stem cell regulators. We specifically noted changes in enhancer regions in the proximity of ASCL2 (intestinal cancer stem cell gene), SALL4 (early pluripotency marker) as well as FZD10 and CTNNB1 (Wnt signaling). A preliminary Cas9-based deletion study suggested these enhancers to be functional in regulating gene expression, and currently its effect on cell proliferation and other cancer properties is being investigated. Further, we reasoned that blocking aberrantly gained enhancers using BRD inhibitor in combination with pathway inhibitors could be a useful strategy in this cancer. Our data supported this hypothesis, and we are currently performing a systematic study to identify most effective combinations. In summary, we have identified aberrant enhancer gains as a major feature of colorectal cancer and propose this to be utilized as a therapeutic approach. Citation Format: Elias Orouji, Ayush Raman, Ming Tang, Mayura Dhamdhere, Kunal Rai. Colorectal cancer epigenomic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4315.