DNA Hypomethylation within B-Cell Enhancers and Super Enhancers Reveal a Dependency on Immune and Metabolic Mechanisms in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response mediated by epigenetic mechanisms. In this study, we sought to better characterize the complexities of the CLL transcriptional profile by conducting an integrative analysis between the B cell enhancer and super enhancer signatures defined from 3 B cell H3K27Ac ChIPseq samples (CD19+ B cell, GM12878, and MEC1), the DNA methylation signatures defined from reduced-representation bisulfite sequencing (RRBS) of 42 CLL patient and 8 healthy donor samples, and the mRNA expression signatures defined from RNA sequencing of 47 CLL patient and 5 healthy donor samples. From our analysis, we identified super enhancers (SEs) in each of the ChIPseq profiles (approximately 4% of called enhancers) and discovered 741 SEs in GM12878, 374 SEs in MEC1, and 523 SEs in the CD19+ B cell profiles, respectively. Based on MSigDB gene ontology analysis, many of the genes corresponding with SEs were involved in pathways regulating immune signaling activation (e.g. TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY RESPONSE) or metabolic homeostasis (e.g. MTORC1_SIGNALING, FATTY_ACID_METABOLISM). By further analyzing the corresponding expression level of SE-associated genes in CLL patients, we identified 190 transcripts associated with SEs that were significantly overexpressed in CLL patient B cells (Student's t-test p<0.05), and this overexpressed subset of SE-associated transcripts was enriched in genes involved in either immune signaling (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). Based on the differential expression of genes associated with enhancers occurring between CLL patient and healthy donor B cells, we then wanted to determine whether differential DNA methylation within enhancers corresponded with upregulation of CLL transcripts. Based on differential DNA methylation (DM) analysis (methylation difference +/- .25; Student's t-test p-value<0.05) from our RRBS samples, we discovered 744 DM CpG sites that overlapped within our identified B cell enhancers, and most of the DM CpG sites in CLL were significantly hypomethylated (avg. DM GpG difference: enhancer = -0.40; non-enhancer = -0.08). Examples of hypomethylated enhancers included super enhancers corresponding with overexpressed transcripts ENO2, SEPT9, RXRA, and CCR7 as well as a typical enhancer that corresponded with the overexpressed transcript PDCD1. Based on the derived information from our integrative analysis of B cell enhancers, we then compared the effects of preferentially targeting enhancer-mediated expression with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. Based on in vitro assays and RNAseq expression analysis comparing THZ1 and JQ1-treated CLL cell lines MEC1 and MEC2, we saw that JQ1 could inhibit CLL cell line proliferation, suppress IgM-mediated primary CLL proliferation, and differentially disrupt transcription of genes involved in immune signaling cascades. Contrastingly, we saw that THZ1 elicited a different response in CLL cell lines and primary cells by disrupting cell viability, inducing apoptosis, and differentially downregulating genes involved in metabolic homeostasis. The specific enhancer-associated genes disrupted by the respective treatments further highlight the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 selectively suppressed the B cell activation marker gene FCER2 and the PD-1 receptor gene PDCD1, whereas THZ1 selectively suppressed the glycolytic enolase gene ENO2 and the proto-oncogene FGR. Collectively, these results reveal how CLL DNA hypomethylation within B cell enhancers can mediate immune signaling and metabolic expression signatures in CLL and can differentially be disrupted by BET bromodomain or CDK7 inhibition.