Identification of a Novel Epigenetic Mechanism of MYC Deregulation in Smoldering and Newly Diagnosed Multiple Myeloma Patients

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM. To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9. To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such a