SRSF2 mutation induces BH3 mimetics sensitivity via BCL2L2 mis-splicing

Abstract Recent advances in next-generation sequencing have unveiled the complex mutational landscape of acute myeloid leukemia (AML), leading to the development of molecularly targeted therapies such as FLT3, BCL2, and IDH inhibitors. However, the clinical benefits of these agents remain limited to specific genomic subsets, emphasizing the need for expanded therapeutic strategies based on individual molecular features. Among these features, mutations in serine/arginine-rich splicing factor 2 (SRSF2), which encodes a critical RNA splicing regulator, represent the most frequent splicing-related alterations in AML, occurring in approximately 10% of cases. Although previous studies have reported enhanced sensitivity of SRSF2-mutant AML cells to the BCL2 inhibitor venetoclax, the mechanistic basis of this phenomenon and its uniformity across AML subtypes have remained unresolved. To elucidate the mechanisms of BCL2 inhibitor sensitivity in SRSF2-mutant AML, we generated isogenic human AML cell lines by introducing the SRSF2 P95H mutation via lentiviral transduction. Following venetoclax treatment, SRSF2-mutant derivatives of THP1, MV4;11, and NB4 displayed significantly increased drug sensitivity relative to their SRSF2 wild-type counterparts. In contrast, other AML cell lines, including MOLM13, Kasumi1, Kasumi3, HEL, and CMK11-5, showed no change in venetoclax sensitivity despite overexpression of SRSF2 P95H. Interestingly, SRSF2 mutation conferred increased sensitivity to the MCL1 inhibitor (S-63845) or the BCL2L1 inhibitor (A-1331852) in AML cell lines that were otherwise insensitive to venetoclax, in a manner dependent on their intrinsic BH3 family protein expression profile. These findings suggest that SRSF2 mutations induce a shift in apoptotic dependency, with variable consequences shaped by the intrinsic anti-apoptotic landscape of the cell. To uncover the molecular basis of this shift, we analyzed the expression of BCL2 family members in SRSF2-mutant and wild-type cells. Immunoblotting revealed a consistent and marked downregulation of BCL2L2 (BCL-w), a relatively understudied anti-apoptotic BCL2 family member, in SRSF2-mutant cells. Restoration of the canonical BCL2L2 isoform by ectopic expression reversed the enhanced sensitivity to BH3 mimetics, while knockdown of BCL2L2 in parental wild-type cells sensitized them to BH3 mimetics in a manner that reflects the phenotype observed in SRSF2-mutant cells. These results implicate BCL2L2 as a key mediator of survival in AML cells, whose loss may redirect dependency to other anti-apoptotic proteins such as BCL2, BCL2L1, or MCL1. We then performed RNA sequencing and RT-PCR to determine whether the downregulation of BCL2L2 in SRSF2-mutant cells was due to transcriptional or post-transcriptional mechanisms. These analyses revealed aberrant splicing of BCL2L2 transcripts specifically in SRSF2-mutant cells, with reduced expression of the canonical isoform and a corresponding increase in a mis-spliced variant lacking both the BH2 and transmembrane domains—regions essential for anti-apoptotic function and membrane localization. The resulting protein was unstable and rapidly degraded, leading to a functional loss of BCL2L2. Importantly, this splicing defect was also detected by RT-PCR in primary AML specimens harboring SRSF2 mutations, confirming its clinical relevance and supporting a model in which SRSF2 mutation drives mis-splicing-mediated dysfunction of BCL2L2 and a compensatory shift in apoptotic dependency toward other BCL2 family members. Collectively, our findings refine the current understanding that SRSF2 mutations uniformly sensitize AML cells to BCL2 inhibition. Rather, SRSF2 mutations induce BH3 mimetics sensitivity through context-dependent reprogramming of apoptotic dependency, mediated by aberrant splicing and functional loss of BCL2L2. This shift may enhance susceptibility not only to venetoclax, but also to MCL1 or BCL2L1 inhibition depending on the cellular expression pattern of BCL2 family proteins. Our results uncover a mechanistically defined and therapeutically actionable vulnerability in SRSF2-mutant AML and provide broader insight into how splicing factor mutations reshape apoptotic regulation in leukemia. These findings may inform the rational design of combination therapies and support the development of precision medicine strategies targeting splicing-derived vulnerabilities in AML.