Disruption of the m-SWI/SNF Complex Mediated By Recurrent Non-Coding Mutations in BCL7A Induces Tumor Cell Proliferation in Multiple Myeloma

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy, however, the mechanisms underlying this complexity are incompletely understood. By deep (70x) whole-genome sequencing (WGS) of 312 primary MM patients, we observed mutations in about 62% of patients in the 5' untranslated region and intron 1 of the 12q24.3 region which cytogenetically marks the BCL7A gene. Integration of WGS with RNA-seq data suggested a widespread loss of BCL7A expression in MM cells as compared to normal plasma cells (PC).

To understand the effect of noncoding mutations in BCL7A promoter we performed in-vitro luciferase reporter assay in MM cell lines (H929, MM1S and KK1) devoid of BCL7A mutation in 5'UTR and found that the normalized luciferase reporter activity indicated that the mutation in the promoter significantly reduced BCL7A promoter activity.

Next, we recapitulated the loss of BCL7A observed in MM patients, in a panel of MM cell lines using shRNA based genetic interference and observed the appearance of a more proliferative phenotype. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7A-KO (knock-out) cells had higher proliferative rate compared to wild type (WT) cells and lentiviral add back of BCL7A plasmid reversed this effect. This phenomenon was validated in a in vivo model, where we found that mice bearing BCL7A KO MM cells developed tumors faster than mice injected with control or add-back cells, suggesting that BCL7A loss increases tumorigenesis in vivo. On the other hand, ectopic expression of BCL7A significantly reduced cell viability and colony formation over time, inducing apoptotic cell death and impacting genes involved in chromatin and chromosome organization, DNA repair and cell cycle.

Interestingly, BCL7A is an important member of the m-SWI/SNF chromatin remodeling complex. Using comparative mass spectrometry analysis, we observed that BCL7A expression is essential for the formation of a functional canonical m-SWI/SNF complex in MM cells, controlling the incorporation of active sub-units into the complex. These data suggest that loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional program that leads to MM cell growth and viability. We therefore evaluated the transcriptomic changes in MM cells upon BCL7A perturbation in a panel of MM cell lines by whole genome RNA-seq. Integrated analysis of modulated genes identified a set of 23 genes significantly upregulated in the presence of BCL7A depletion and downregulated in presence of BCL7A ectopic expression. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF (loss-of-function) studies (si-RNA screen) using these genes in scrambled and BCL7A KD (knock-down) MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of RPS3A (V-Fos transformation effector protein) only in the context of BCL7A loss.

Our RNA-seq data revealed that RPS3A is highly expressed in primary MM cells, and its expression significantly correlates with low expression of BCL7A in MM patients. Importantly, we observed that RPS3A binds to the core m-SWI/SNF complex proteins; SMARCC2 and SMARCB1 in the absence of BCL7A. RPS3A gene encodes a ribosomal protein that is a component of the 40S subunit and its overexpression transforms NIH3T3 mouse fibroblasts and induces tumor formation in nude mice and also used as an expression marker in squamous cell carcinoma.

In conclusion, we here report biological consequences of a frequent non-coding mutation of BCL7A in MM and loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional process that leads to MM cell growth and viability. Altogether this study shed lights on a new pathogenic mechanism that may drive MM growth with potential translational implication, that may be applicable to other hematological diseases.