Exploring the Mechanisms of Venetoclax Resistance Via Drug Screening and Genome-Wide CRISPR Screening

Introduction: Combining Venetoclax, a selective BCL-2 inhibitor, with hypomethylating agents has revolutionized frontline acute myeloid leukemia (AML) treatment. Despite its success, the occurrence of resistance remains a significant concern, limiting the range of available treatment alternatives. Little is known about the mechanism by which the cells escape the treatment. Recent studies have observed MCL1 upregulation in AML cells to promote resistance, and the rationale to use an MCL1 inhibitor became attractive. Although MCL1 inhibitors work exceptionally well in vitro their clinical trials were unsuccessful due to their toxicity. Here, we aim to identify other genes responsible for acquiring resistance and tackle the problem through screening for clinically approved drugs to rapidly benefit the patients with our study. Methods: First, we generated a model of the venetoclax resistance from 3 AML cell lines: MOLM-13, HL-60 and MV4-11. Resistance was achieved through chronic administration of the compound, inhibiting cell viability to 80-90% in multiple rounds by gradually increasing concentrations until the cells developed resistance. We have then characterized these Venetoclax-resistant (VeR) cells using quantitative RT-PCR and RNA sequencing to reveal the mechanisms behind their resistance. VeR cell lines were subjected to drug screening with a library of 859 FDA and EMA-approved compounds with various clinical indications. Cell viability in response to the drug library was assessed after 72h by Cell-Titer Glo and evaluated against the wild-type counterparts. Top-performing compounds were validated in dose-response curves, and synergy scores were identified from combinational treatments. Simultaneously, we performed genome-wide CRISPR/Cas9 knockout screening using Brunello CRISPR knockout library on both MOLM-13 wild-type (WT) and VeR cell lines to identify genes that maintain the resistance to venetoclax and genes that may potentially synergize with targeting BCL-2. Results: Results from the CRISPR screen pointed out multiple genes whose loss of function may contribute to venetoclax resistance in MOLM-13 cells, among which BAX, NOXA, ELAVL1 and TP53 ranked as top hits. On the contrary, we also identified several genes whose loss of function sensitized the cells to venetoclax (e.g., MCL1, OPA1, CDK2, MCAT), suggesting them to be promising therapeutic targets. Among these hits, CDK2 correlates with our drug screening data indicating Flavopiridol as an e