Chromatin Accessibility Profiling Reveals Cis-Regulatory Heterogeneity and Novel Transcription Factor Dependencies in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Recurrent IgH translocations, copy number abnormalities and somatic mutations have been reported to participate in myelomagenesis; however no universal driver of the disease has been identified. Here, we hypothesize that transcriptional deregulation is critical for MM pathogenesis and the maintenance of the MM cell state. In order to capture signatures of transcription factor engagement with the myeloma epigenome, we performed the assay for transposase-accessible chromatin sequencing (ATAC sequencing), deep RNA sequencing in 23 primary myeloma samples and 5 normal plasma cell samples (NPC) from healthy donors along with whole genome sequencing and H3K27ac ChIP-seq in a cohort of these primary MM samples. We identified 22,603 variable accessible loci between MM and NPC and correlated impact of these on expression of associated genes using RNA-seq data. Together with robust differential analysis of open chromatin regions and nuclease-accessibility footprints to identify discrete transcription factor binding events, we have discerned the myeloma-specific open chromatin landscape, identified transcription factor dependencies and potential new myeloma drivers. In our dataset we observe a vast number of loci with heterogeneous chromatin states across the sample cohort, and the majority of the open chromatin sites identified are unique to a single sample. However, distinct variable chromatin accessibility signatures indicative of the MM chromatin state when compared to normal plasma cells were observed. Remarkably, we observed more frequent recurrent loss of variable accessible loci compared to gains. In addition, specific open chromatin profiles evident in hyperdiploid and non-hyperdiploid MM were also identified. Accessibility footprinting revealed MM-specific enrichment for transcription factors known to be essential for MM cell survival including Interferon Regulatory Factors (IRFs), Nuclear Factor Kappa B (NFkB), Ikaros, and Sp1. Interestingly, we also identify the myocyte enhancer factor 2 (MEF2) family of transcription factors as being specifically enriched in open chromatin regions in MM cells. Using a CRISPR-Cas9 knockout system, we identify the MEF2 family member MEF2C as essential for MM cell proliferation and survival. MEF2C is significantly overexpressed at the RNA level in our study as well as in several independent cohorts and is a central enhancer-localized transcription factor in MM core regulatory circuitry as determined by H3K27ac ChIP-sequencing profiles of primary MM samples. In order to evaluate MEF2C as a therapeutic target, we used small molecule inhibitors targeting MEF2C activity via inhibition of MEF2C phosphorylation using inhibitors of salt-induced kinases (SIK) and microtubule-associated protein/microtubule affinity regulating kinases (MARK). SIK/MARK have been described to specifically activate MEF2C. SIK and MARK inhibition resulted in both dose- and time-dependent inhibition of MM cell growth and survival in a panel of 12 MM cell lines with various genotypic and phenotypic characteristics, revealing a potential approach to targeting the dysregulated gene regulatory state of myeloma. To conclude, here we identify here an altered chromatin accessibility landscape in multiple myeloma that likely contributes to oncogenic transcription states through the activity of transcription factors such as MEF2C, representing a new MM dependency and potential therapeutic target.