Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.

Clinical and genetic risk factors are currently used in multiple myeloma (MM) to stratify patients and to design specific therapies. However, these systems do not capture the heterogeneity of the disease supporting the development of new prognostic factors. In this study, we identified active promoters and alternative active promoters in 6 different B cell subpopulations, including bone-marrow plasma cells, and 32 MM patient samples, using RNA-seq data. We find that expression initiated at both regular and alternative promoters was specific of each B cell subpopulation or MM plasma cells, showing a remarkable level of consistency with chromatin-based promoter definition. Interestingly, using 595 MM patient samples from the CoMMpass dataset, we observed that the expression derived from some alternative promoters was associated with lower progression-free and overall survival in MM patients independently of genetic alterations. Altogether, our results define cancer-specific alternative active promoters as new transcriptomic features that can provide a new avenue for prognostic stratification possibilities in patients with MM.

NOTCH1 is a recurrently mutated gene in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Functional studies to investigate its role have been hampered by the inability to genetically manipulate primary human lymphoma cells, attributed to low transduction-efficacy and procedure-associated toxicity. To overcome these limitations, we have developed a novel method to retrovirally transfer genes into malignant human B cells. We generated isogenic human tumor cells from patients with CLL and MCL, differing only in their expression of NOTCH1. Our data demonstrate that NOTCH1 facilitates immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH1 causes silencing of the entire HLA-class II locus via suppression of the transcriptional co-activator CIITA. These NOTCH1-mediated immune escape mechanisms are associated with the expansion of CD4+ T cells in vivo, further contributing to the poor clinical outcome of NOTCH1-mutated CLL and MCL.