Cancer‐Associated BCL‐2 Mutants Reveal Mechanisms Towards Venetoclax Resistance

BCL-2 is an anti-apoptotic protein frequently upregulated in cancer, enabling cell survival despite oncogenic stress. This BCL-2 dependence sensitizes tumor cells to venetoclax, an FDA-approved drug used against chronic lymphocytic leukemia. Yet, treatment-induced mutations in BCL-2 frequently result in resistance. While mutations G101V and D103Y were reported to disrupt BCL-2 interaction with venetoclax, it remains unclear how other mutations cause resistance. Here, we performed a comprehensive comparison of frequently occurring cancer-associated BCL-2 mutations inside and outside the venetoclax binding site. We find that, besides disrupting venetoclax interaction, G101V and D103Y also increased the sequestration and inhibition of pro-apoptotic proteins, revealing a double effect of these mutations. We also define V156D, A113G, R129L, and R139H as previously uncharacterized BCL-2 mutations conferring venetoclax resistance. Remarkably, V156D reduces venetoclax binding allosterically, without altering pro-apoptotic protein inhibition. Other BCL-2 mutants, like A113G, R129L, and R139H, do not exhibit alterations in venetoclax binding, but venetoclax cannot efficiently release their pro-apoptotic partners from inhibitory complexes in cells. Finally, sonrotoclax binds with high affinity to all venetoclax-resistant BCL-2 mutants, yet its efficacy against these BCL-2 mutants in cells was heterogeneous. Our findings uncover diverse mechanisms that reduce venetoclax efficacy with the potential to inform anticancer treatment.