BRD9 is a component of the noncanonical SWI/SNF (ncSWI/SNF) complex, which is a chromatin-remodeling complex that modulates genomic architecture and DNA accessibility. Since BRD9 has been shown to play a critical tumor-promoting role in various cancers, we investigated its biologic impact in multiple myeloma (MM) cells. We found that BRD9 was highly expressed in patient MM cells compared to normal plasma cells (GSE39754, P = 0.045). Moreover, higher BRD9 expression was associated with poor overall survival (GSE39754, P = 0.049) in MM patients. We next confirmed that genetic (shRNA) and pharmacological (dBRD9-A) depletion of BRD9 significantly reduced growth in a panel of MM cell lines and patient MM cells. Furthermore, dBRD9-A induced G1 cell-cycle arrest and apoptosis in OPM2 and H929 cells, even in the presence of anti-apoptotic cytokines (IL-6, IGF-1), or bone marrow stromal cells. In a murine xenograft model, dBRD9-A treatment significantly inhibited OPM2 tumor growth compared with vehicle control (P = 0.001 and P < 0.001, respectively) without significant body weight loss. Kaplan-Meier curves and log-rank analysis showed a significantly prolonged overall survival in the dBRD9-A treatment cohort compared with the vehicle control cohort (P < 0.001). Having nominated BRD9 as a potential therapeutic target, we next investigated the cellular mechanisms impacted by BRD9 depletion by RNA-seq analyses. After BRD9 knockdown (shRNA) or dBRD9-Aapplication, a total of 162 and 197genes were commonly up- or downregulated, respectively, compared with DMSO-treated control cells (Padj < 0.05). Importantly, we identified that ribosome biogenesis and rRNA processing were the most significantly downregulated pathways. Notably, target genes of MYC, the master regulator of ribosome biogenesis, were also markedly downregulated. Furthermore, we observed that the expression of representative proteins (i.e., MYC, RRS1, and BOP1) was also consistently decreased by dBRD9-A in vitro and in vivo. Indeed, we observed a significant decrease in total pre-rRNA levels and nucleolar number by dBRD9-A treatment in OPM2 and H929 cells, indicating ribosome biogenesis was positively regulated, at least in part, by BRD9. OPM2 cells treated with dBRD9-A had fewer ribosomal subunits and fewer actively translating polysomes compared with DMSO-treated controls, as determined by polysome profiling of cytosolic extracts. Furthermore, AgNOR staining of tumors harvested from our murine xenograft model demonstrated that dBRD9-A treatment reduced the number of nucleolar organizer regions in vivo. To understand how BRD9 regulates the expression of ribosome biogenesis genes, we next performed ChIP-seq. We found that many of the genes downregulated by dBRD9-A had BRD9 occupancy at their promoters compared to other intact genes. Notably, genes with BRD9 signals in their promoter regions were enriched for ribosome biogenesis and rRNA processing and were downregulated by dBRD9-A treatment. Although GLTSCR1, another ncBAF-specific component, was still able to form a complex even after BRD9 depletion, ChIP-seq revealed a significant loss of GLTSCR1 binding throughout the genome after treatment with dBRD9-A. Thus, we hypothesized that BRD9 is required for the localization of the ncSWI/SNF complex to target gene regions. Indeed, BRD9 co-immunoprecipitated with the positive transcription elongation factors, RNAPII, BRD4, and MYC in MM cells. Interestingly, the interaction between BRD9 and BRD4 was enhanced by an HDAC inhibitor, while BRD9 depletion attenuated the interaction between GLSTCR1 and BRD4, suggesting that BRD9 interacts with the acetylated form of BRD4, stabilizing its binding to the ncBAF complex and thereby supporting the initiation of transcription of target genes. Indeed, ChIP-seq analysis demonstrated that BRD9-bound sites preferentially co-localized with ChIP peaks of BRD4, MYC, and RNAPII to the promoter regions of all ribosome biosynthesis genes. In summary, our data identify a novel function for BRD9 in promoting ribosome biogenesis in MM. BRD9 depletion impairs ncBAF localization to its genomic targets, thereby disrupting ribosome biogenesis machinery, resulting in significant cell growth inhibition. Our study therefore provides the rationale for further evaluation of BRD9 degraders as a novel treatment strategy in MM.
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May 13, 2023
María Cristina Acosta + 2
Open Access