Functional genomic screens identify positive and negative regulators of venetoclax resistance

Abstract Multiple myeloma (MM) is a heterogeneous plasma cell malignancy characterized by variable responses to standard therapies. During B cell differentiation into plasma cells, anti-apoptotic dependency shifts from BCL2 to MCL1. However, the clinical use of MCL1 inhibitors is limited by associated toxicities. Venetoclax, a selective BCL2 inhibitor, is a promising treatment for certain BCL2 dependent blood cancers like chronic lymphocytic leukemia with relatively low toxicity. In MM, response to venetoclax is also highly heterogeneous, being most effective in the subset of patients with a B cell like phenotype and the t(11;14) translocation. The molecular determinants underlying this variation in intrinsic sensitivity or resistance to venetoclax remain poorly understood. In order to examine the factors regulating BCL2 dependence, we previously performed RNA sequencing on a panel of venetoclax sensitive and resistant MM patient samples and cell lines, identifying 408 differentially expressed genes. We then tested the contribution of these genes to venetoclax response by conducting targeted CRISPR activation (CRISPRa) and interference (CRISPRi) screens in the venetoclax resistant MM cell lines, KMS11 and KMS18, using six sgRNAs/gene derived from the Calabrese and Dolcetto libraries. Controls included non-targeting sgRNAs as well as both pro- and anti-apoptotic members of the BCL2 family. Cells were treated with vehicle (DMSO) and either 500 nM or 2500 nM venetoclax. Genomic DNA was collected on days 0, 15, and 30, and sgRNAs were amplified using barcoded primers. Sequencing data were processed using the MAGeCK pipeline to calculate fold changes and p-values. We compared results from the two cell lines and CRISPRa versus CRISPRi screens to identify commonly enriched or depleted sgRNAs following venetoclax treatment. Between the two CRISPRa screens, 7-10 shared sgRNAs were detected to be commonly depleted or enriched in both cell lines. As expected, BCL2L1, which encodes for the anti-apoptotic protein BCL-XL and would be predicted to induce resistance to venetoclax, was the most enriched sgRNA in both cell lines, while the pro-apoptotics BAX and BID targeting sgRNAs were depleted. Notably, sgRNA to BCL2 was not depleted, suggesting that increasing expression of BCL2 alone does not sensitize to venetoclax. Among the sgRNAs for the differentially expressed genes, the top enriched sgRNAs targeted E74-like factor 3 gene (ELF3) and basic helix-loop-helix family member e41(BHLHE41), while the top depleted guides targeted Krüppel-like factor 3 gene, KLF3. Upon targeted gene activation, KLF3 activation led to increased sensitization to venetoclax, whereas ELF3 activation conferred enhanced resistance. In the CRISPRi screen, BCL2L1 was the top depleted sgRNAs, while BAK1 and BC2L11 targeting sgRNAs were among the most enriched, consistent with their anti-apoptotic and pro-apoptotic functions, respectively. Interestingly, sgRNAs targeting BHLHE41 were among the most significantly depleted in the CRISPRi screen, mirroring their strong enrichment in the CRISPRa screen. This reciprocal pattern suggests that BHLHE41 promotes resistance to venetoclax, as its activation confers protection while its inhibition sensitizes cells to treatment. ELF3, an ETS family transcription factor, is a known regulator of cell proliferation, differentiation, apoptosis, and migration through modulation of gene expression. In the context of MM and venetoclax response, ELF3's role may be potentially critical as it promotes proliferation and invasion while suppressing apoptosis, partly via pathways such as mTOR and β-catenin signaling. KLF3 is a transcriptional repressor involved in regulating B-cell maturation and immune regulation. Recent evidence suggests that BHLHE41 may be important for MM cell differentiation and growth. BHLHE41 expression increases as B cells differentiate to plasma cells and higher expression of BHLHE41 in MM patients correlates with poorer survival outcomes, indicating its potential role in disease aggressiveness. We are currently independently validating the role of these genes in venetoclax response as well as repeating these CRISPR screens in additional MM cell lines, both sensitive and resistant. Our findings provide novel insights into the molecular pathways regulating venetoclax response and may facilitate the development of predictive biomarkers and combination therapeutic strategies for multiple myeloma.