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SUMMARYIntrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2., BAX and BAK are pro-apoptotic proteins whose activity is essential for the action of many anti-cancer drugs and to suppress tumorigenesis. Here, the authors perform a genome-wide CRISPR/Cas9 screen and identify VDAC2 as a promoter of BAX-mediated apoptosis that is important for an efficient chemotherapeutic response and to suppress tumor formation.

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.