Derebail, Sanika

Abstract Multiple myeloma (MM) is a complex hematological malignancy characterized by genomic changes and transcriptomic dysregulation. Initial exome sequencing approaches have failed to identify any single, frequent (>25%) mutation in the coding genome. However, using whole-genome sequencing, we found that one of the genomic regions most frequently mutated (62% of the patients with MM) was the 5′ untranslated region and/or intron 1 of the BCL7A gene. RNA-sequencing data from a large cohort suggest a loss of BCL7A expression in a large majority of patients with MM as compared with normal plasma cells. BCL7A loss of function in a panel of MM cell lines led to a highly proliferative phenotype in vitro and in vivo, whereas its ectopic expression significantly reduced cell viability, suggesting a tumor suppressor function for BCL7A in MM. We studied the cellular and molecular effects of BCL7A loss and observed that it endows myeloma cells with proliferative potential in cooperation with the plasma cell–defining transcription factor IRF4. BCL7A is involved in a direct protein-protein interaction with IRF4, limiting its DNA-binding activity. Loss of BCL7A thus enhances the expression of IRF4-associated cytokines and reduces mitochondrial metabolism and reactive oxygen species levels. Our study therefore suggests that BCL7A loss provides the necessary molecular change to allow IRF4-mediated transcriptional activity and MM cell growth and survival.

Therapeutic targeting of CDK7 has proven beneficial in pre-clinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in multiple myeloma (MM) patient cells; and its selective targeting counteracts E2F activity via perturbation of the CDKs/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression increasing survival in several MM mouse models including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

Abstract Integrated genomic analysis including whole genome and RNA sequencing in primary multiple myeloma (MM) cells have reported a universal loss of BCL7A gene in MM patients compared to normal plasma cells (PC). Our Genetic modulation in in-vitro and in-vivo MM models have also validated the loss of BCL7A as an oncogenic event responsible for acquisition of a more proliferative phenotype in MM cells. We performed a comparative mass spectrometric analysis confirming BCL7A as a member of the canonical m-SWI/SNF chromatin remodeling complex. We therefore performed the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess genome-wide changes in DNA accessibility upon BCL7A gain or loss. We found that loss of BCL7A in wild-type KMS12BM and NCI-H929 cells resulted in enrichment of important transcription factor motifs in the transcriptionally active sites of chromatin. We identified 36 de novo accessible and 1079 de novo inaccessible regions across the genome after BCL7A shRNA KD (knock-down). These genomic regions with altered accessibility were associated with genes involved in protein binding (FDR < 0.001), GTPase activation (FDR = 0.016), and GTPase regulation (FDR = 0.030). Candidate transcription factors for the genomic regions with altered accessibility were identified by querying a database of human ChiP-seq experiments (ReMap 2020). IRF4 was identified to be enriched in regions of accessible chromatin upon BCL7A loss. IRF4 is an oncoprotein transcription factor and is a direct target of myc, generating a feedback loop in MM cells. IRF4 dependency is central to myeloma cell proliferation. Most importantly, we found that in addition to functions within the m-SWI/SNF complex, BCL7A forms a protein complex with IRF4. Altogether these data suggest a role for BCL7A in driving IRF4 oncogenic activities in MM. To understand how BCL7A-dependent changes may influence IRF4 activity, we performed CHiP assay using IRF4 antibody in BCL7A KO (knock-out) and AB (add-back) KMS12BM and NCI-H929 cells. We observed increased binding of IRF4 to the promoter of its target genes like PRDM1, CDK6, STAG2, PIM2, SQLE, Myc, CANX, IRF4, SCD, ELL2, CASP3 in BCL7A KO cells, while the binding of these target genes was significantly decreased in AB cells. While, CHiP assay using IRF4 antibody in control and ectopically expressed BCL7A in AMO1 and KMS11 cells showed significantly low binding of IRF4 to the promoter of most of its target genes in BCL7A overexpressed cells compared to control. Integrated transcriptomic analysis following BCL7A KD and overexpression revealed the existence of a set of genes transcriptionally regulated by IRF4 to be significantly upregulated following BCL7A depletion and downregulated following ectopic expression of BCL7A. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF studies (si-RNA screen) in scrambled and BCL7A KD MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of EEF1B2, RPS3A, SOX2, DCC and NDUFA1 only in the context of BCL7A loss, implicating a role as critical effector molecules downstream of the IRF4-BCL7A transcriptional network. In conclusion, we observe that BCL7A binds to IRF4, functionally restricting its activity. The universally observed down regulation of BCL7A in MM provides the necessary molecular change to allow IRF4 to exert its required transcriptional activity to induce MM cell growth. Our results now provide the basis to understand the mechanism of development of IRF4 dependency in MM. Disclosures Anderson: Pfizer: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Pfizer: Consultancy.