E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

Intrinsic 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 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.

Dual BCL-2/BCL-XL Inhibitor Pelcitoclax (APG-1252) Overcomes Intrinsic and Acquired Resistance to Venetoclax in Multiple Myeloma Cells

Multiple myeloma (MM) is marked by several genetic abnormalities, including chromosome translocation t(11;14). Overexpression of anti-apoptotic BCL-2 in t(11;14) MM promotes disease progression, prompting clinical use of the BH3 mimetic and BCL-2 inhibitor venetoclax in combination with proteasome inhibitor therapy. Despite high initial response rates and prolonged progression-free survival, patients commonly relapse. To delineate mechanisms contributing to acquired drug resistance we modeled responses to venetoclax in two highly sensitive MM cell lines (KMS27 and KMS-12PE). Colonies generated from a surviving cell were cultured in high-dose venetoclax to generate monoclonal drug-tolerant expanded persister (DTEP) clones. To determine whether venetoclax resistance in DTEP clones is mediated by transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprogramming, we conducted whole-genome sequencing (WGS) and RNA-seq of the clones. WGS analysis did not show significant differences between parental and resistant clones, but transcriptomic analysis showed shared and unique transcriptome signatures in DTEP clones. Gene set enrichment analysis of the common significantly modulated genes in resistant clones revealed that PKA-ERK-CREB and K-Ras pathway genes were significantly upregulated, whereas apoptotic genes were downregulated in resistant clones compared to parental cells. Importantly, ectopically expressed ERK in venetoclax-sensitive cells conferred a resistant phenotype that was rescued using two specific ERK inhibitors in DTEP clones. These data confirm a key role for ERK activation in acquired venetoclax resistance. Resistant clones were further characterized by reduced mitochondrial priming assessed by dynamic BH3 profiling, with altered expression of anti-apoptotic regulators including MCL-1, BCL-xL, and BCL-W and the replaced BCL-2: BIM complex by both MCL-1 and BCL-xL. Because these data suggested a functional substitution between anti-apoptotic BCL-2 family members in cells with acquired resistance to venetoclax, we next evaluated if MCL-1 or BCL-xL are codependent in MM cells that are insensitive or resistant to venetoclax. Simultaneous inhibition of MCL-1 (via S63845) or BCL-xL (via A155463) and BCL-2 (via venetoclax) increased BIM release and enhanced cell death in resistant clones (vs single agents), with combination index values < 0.3 in all doses. Upregulation of BCL-xL or MCL-1 in MM cells also mediated primary venetoclax resistance independent of genetic hallmarks (e.g. t [11;14]-translocated cells). Thus, simultaneous inhibition of MCL-1 or BCL-xL and BCL-2 triggered synergistic cytotoxicity in MM cell lines intrinsically resistant to venetoclax. These data suggest that combined inhibition of BCL-2 and BCL-xL may overcome venetoclax resistance. However, the dependence of BCL-xL in mature platelets had triggered thrombocytopenia for patients under therapy using BCL-xL inhibitor. To further explore the potential clinical application of targeting BCL-xL, we employed novel BCl-2/BCL-xL dual inhibitor, BH3 mimetic pelcitoclax (APG-1252). Using pro-drug strategy for design, pelcitoclax has limited cell permeability during circulation, and was converted to a more potent metabolite APG-1252-M1 in tumors/tissues. APG-1252-M1 was thus used for in vitro cell based assays. We discovered that APG-1252-M1 induced cytotoxicity in MM cell lines intrinsically resistant to venetoclax (regardless of genetic background or BCL-2:BCL-xL ratio) and also significantly reduced MM cell viability in clones with acquired venetoclax resistance, overcoming ERK activation and decreasing BIM sequestration by BCL-xL. In vivo study using pelcitoclax is ongoing and will be presented at the meeting. In conclusion, we report that venetoclax resistance in MM evolves from outgrowth of persister clones displaying activation of the ERK pathway and a shift in mitochondrial dependency towards BCL-xL, which can potentially be effectively targeted via the novel BCL-2/BCL-xL inhibitor pelcitoclax (APG-1252), which is currently in clinical investigation for solid tumors (NCT03080311). Disclosures Deng: Ascentage Pharma Group: Current Employment. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.

Severe cellular stress activates apoptosis independently of p53 in osteosarcoma

Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

Targeting the BCL-2-regulated apoptotic pathway for the treatment of solid cancers

The deregulation of apoptosis is a key contributor to tumourigenesis as it can lead to the unwanted survival of rogue cells. Drugs known as the BH3-mimetics targeting the pro-survival members of the BCL-2 protein family to induce apoptosis in cancer cells have achieved clinical success for the treatment of haematological malignancies. However, despite our increasing knowledge of the pro-survival factors mediating the unwanted survival of solid tumour cells, and our growing BH3-mimetics armamentarium, the application of BH3-mimetic therapy in solid cancers has not reached its full potential. This is mainly attributed to the need to identify clinically safe, yet effective, combination strategies to target the multiple pro-survival proteins that typically mediate the survival of solid tumours. In this review, we discuss current and exciting new developments in the field that has the potential to unleash the full power of BH3-mimetic therapy to treat currently recalcitrant solid malignancies.

Targeting of MCL-1 in breast cancer associated fibroblasts reverses their myofibroblastic phenotype and pro-invasive properties

Cancer associated fibroblasts (CAF) are a major cellular component of epithelial tumors. In breast cancers in particular these stromal cells have numerous tumorigenic effects in part due to their acquisition of a myofibroblastic phenotype. Breast CAFs (bCAFS) typically express MCL-1. We show here that targeting this regulator of mitochondrial integrity using a specific BH-3 mimetic promotes fragmentation of these organelles without inducing cell death. MCL-1 antagonism in primary bCAFs directly derived from human samples mitigates myofibroblastic features and decreases expression of genes involved in actomyosin organization and contractility, associated with a cytoplasmic retention of the transcriptional regulator, Yes-Associated Protein (YAP). Such treatment decreases bCAFs ability to promote cancer cells invasion in 3D co-culture assays. These effects are counteracted by an inhibitor of the mitochondrial fission protein DRP-1, which interacts with MCL-1 upon BH3 mimetic treatment. Our findings underscore the usefulness of targeting MCL-1 in breast cancer ecosystems, not only to favor death of cancer cells but also to counteract the tumorigenic activation of fibroblasts with which they co-evolve.

Constitutive BAK/MCL1 complexes predict paclitaxel and S63845 sensitivity of ovarian cancer

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.