Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Deregulated expression of lineage-affiliated transcription factors is a major mechanism of oncogenesis. However, how deregulation of non-lineage affiliated TF impacts chromatin to initiate oncogenic transcriptional programmes is not well known. To address this, we studied the chromatin effects imposed by oncogenic MAF as the cancer-initiating driver in the plasma cell cancer multiple myeloma. We found that the ectopically expressed MAF endows myeloma plasma cells with migratory and proliferative transcriptional potential. This potential is regulated by activation of enhancers and super-enhancers, previously inactive in normal B cells and plasma cells, and in co-operation of MAF with the plasma cell-defining TF IRF4. Forced ectopic MAF expression confirms the de novo ability of oncogenic MAF to convert transcriptionally inert chromatin to active chromatin with features of super-enhancers, leading to activation of the MAF-specific oncogenic transcriptome and acquisition of cancer-related cellular phenotypes such as CCR1-dependent cell migration. These findings establish oncogenic MAF as a pioneer transcription factor that can initiate as well as sustain oncogenic transcriptomes and cancer phenotypes. However, despite its pioneer function, myeloma cells remain MAF-dependent thus validating oncogenic MAF as a therapeutic target that would be able to circumvent the challenges of subsequent genetic diversification driving disease relapse and drug resistance.

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Myeloma initiating genetic events define subgroups (MIE) and drivedistinct oncogenic transcriptomes that converge into a mutually exclusive overexpression of CCND1 and CCND2 oncogenes. Here, with reference to normal PC, we dissect how MIE impact the chromatin regulatory landscape of MM. We find that chromatin accessibility combined with transcriptome profiling classifies myeloma genetic subgroups, while in a topologically constrained manner, distal rather than proximal regulatory elements influence myeloma transcriptomes. Across and within MIE-defined subgroups, genes and pathways critical for myeloma biology can be linked to developmentally activated or de novo formed enhancers. We show that existing transcription factors, co-opted to organise highly ordered, aberrant regulatory networks, generate known and novel myeloma cell dependencies and help identify prognostic markers. Finally, we discover and functionally validate the critical enhancer that regulates ectopic expression of CCND2 in MM. 3MM is a common, genetically heterogeneous incurable cancer of the bone marrow plasma cells (PC), the terminally differentiated immunoglobulin-secreting B lineage cells (Palumbo and Anderson, 2011). Distinct transcriptome profiles in MM reflect two categories of myeloma initiating genetic events (MIE): Over-expression in up to half of cases of oncogenes such as CCND1, MAF and MMSET by their juxtaposition to the IgH enhancer in the t(11;14), t(14;16) and t(4;14) cytogenetic subgroups respectively; hyperdiploidy (HD) is the MIE driving oncogenic transcriptomes in the rest of cases (Manier et al., 2017;Morgan et al., 2012). High frequency secondary copy number aberrations, single nucleotide variants and indels further shape the distinct impact of the MIE and generate extensive genetic heterogeneity (Chapman et al., 2011;Lohr et al., 2014;Walker et al., 2015). This heterogeneity converges, in most cases, to a functionally dichotomous, mutually exclusive overexpression of the cell cycle regulators CCND1 and CCND2 to which myeloma PC remain addicted, irrespective of primary or secondary genetic events (Bergsagel et al., 2005;Ely et al., 2005;Tiedemann et al., 2008). As well as over-expression by juxtaposition to the IgH enhancer in 20% of MM, CCND1 over-expression is associated with chr11q25 gain in over half of HD cases (Shah et al., 2018). However, the transcriptional mechanisms that result in CCND2 overexpression, seen in nearly 50% of MM cases and spanning all genetic subgroups except t(11;14), are not known.Chromatin accessibility profiling by ATAC-seq has been used to characterise the regulatory landscape of hundreds of different solid tumour cancers and in combination with other datatypes, has demonstrated its utility in categorising cancer and in the discovery of distal regulatory elements such as candidate enhancers of critical oncogenes (Corces et al., 2018). Further, by means of transcription factor (TF) footprinting, ATAC-seq ...

In multiple myeloma (MM), a malignancy of the bone marrow plasma cells (BMPC), hyperdiploidy (HY) and oncogene over-expression via chromosomal translocation [including CCND1- t(11;14), MAF- t(14;16), MMSET-t(4;14)] are the primary myeloma initiating events (MIE) that drive distinct transcriptional programs. These are further shaped by secondary SNV and CNV events. This genetic heterogeneity converges, in most cases, to a functionally dichotomous state of CCND1 or CCND2 overexpression. The molecular mechanisms underlying each of the distinct myelomagenic transcriptomes and the CCND1 vs CCND2 dichotomy have not been defined. To address these questions, we obtained highly purified BMPC from 3 healthy donors and 30 MM patients (HY: 15; CCND1: 4; MMSET: 5; MAF: 2; other: 4), either at diagnosis or relapse, and mapped their chromatin accessibility and transcriptome profiles by ATAC-seq and RNA-seq, respectively. In total, we obtained ~300K regions with accessible chromatin in either MM or normal PC. Overall chromatin accessibility increased in myeloma compared to normal PC, particularly in MAF- and MMSET-translocated subtypes. Analysis of combined ATAC-seq/RNA-seq by Multi-Omics Factor Analysis (MOFA) resulted in a clearer samples distinction than either ATAC-seq or RNA-seq alone, with altered chromatin accessibility accounting for more of the variance than expression. Of the top five identified factors, the top two (one transcriptome driven, one accessibility driven) distinguished normal from MM samples, whilst two more separated MMSET, MAF and CCND1 subgroups. Ninety seven, 157, 256 and 348 overexpressed genes in the CCND1, HY, MMSET and MAF subgroups, respectively, were predicted to be regulated by differentially accessible enhancers. Twenty percent (165/858) of these genes were overexpressed in >1 subgroup suggesting a process of chromatin accessibility-based convergence evolution. Enrichment analysis suggested direct or indirect involvement of Polycomb and chromatin remodellers; significant enrichment was also found for genes involved in neurogenesis. ATAC-seq footprinting predicted binding sites for 250 expressed transcription factors (TFs), 116 of which displayed higher binding frequency in myeloma than in normal PC and included both known (e.g., XBP1, RELA, IRF4, PRDM1) and potentially novel regulators of myeloma biology (e.g., CXXC1 and NFE2L1). The remaining 134 TF were predicted to be present in at least one MM subgroup, but absent in normal PC. Amongst them, as expected, MAF was active in the MMSET- and more so in the MAF-translocated subgroups. DepMap database analysis suggested myeloma cell dependency on 181/250 TF (CRISPR/Cas9 CERES score < -0.1 in >3/14 MMCL analysed). In dissecting the regulatory basis of CCND2 vs CCND1 dichotomy, one MOFA factor completely separated MAF from CCND1 samples, placing extreme opposite weights on the expression of CCND2 and CCND1 respectively. Interestingly, the same factor identified open-chromatin clusters upstream of CCND2 and linked them to its over-expression. These clusters were also open in the MMSET group and in CCND2-expressing HY samples. Conversely, no accessibility was detected in the CCND1 group, the CCND1-expressing HY samples or in normal PCs. Further, super-enhancer calling using the H3K27ac histone mark in MAF-translocated JJN3 cells identified the region of interest as a bona fide super-enhancer. Chromatin long range interactions, as assessed by Capture-HiC, demonstrated high frequency interactions of the CCND2 promoter with the constituent elements of the putative super-enhancer. Experimental validation using a CRISPR/Cas9i system confirmed the functional role of all 4 super-enhancer constituents tested in the regulation of CCND2 expression, while TF footprinting predicted MAF binding to the super-enhancer in MAF-translocated PC. In conclusion, we show that distinct oncogenic transcriptomes in MM are underpinned by extensive chromatin changes, accompanied by TF activity 're-wiring' that does not necessarily require transcriptional deregulation of the TF themselves. We identify novel, non-oncogene TF dependencies that suggest therapeutic opportunities in MM and we discover and characterise the critical super-enhancer that drives overexpression of the CCND2 oncogene in MM. Disclosures Auner: Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Hatjiharissi:Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.