The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.cell death | mitochondrial permeabilization | protein-membrane topology | membrane pores | cysteine-scanning mutagenesis C ommitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1-4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2-associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7-10). These novel "symmetric" homo-dimers can multimerize via association of α6 helices (8,11,12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two...

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

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SUMMARY Drug resistance represents a major challenge to achieving durable responses to cancer therapeutics. Resistance mechanisms to epigenetically-targeted drugs remain largely unexplored. We used BET inhibition in neuroblastoma as a prototype to model resistance to chromatin modulatory therapeutics. Genome-scale, pooled lentiviral open reading frame (ORF) and CRISPR knockout rescue screens nominated the PI3K pathway as promoting resistance to BET inhibition. Transcriptomic and chromatin profiling of resistant cells revealed that global enhancer remodeling is associated with upregulation of receptor tyrosine kinases (RTKs), activation of PI3K signaling and vulnerability to RTK/PI3K inhibition. Large-scale combinatorial screening with BET inhibitors identified PI3K inhibitors among the most synergistic upfront combinations. These studies provide a roadmap to elucidate resistance to epigenetic-targeted therapeutics and inform efficacious combination therapies.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2–associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.

The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

KRASG12D is the most frequent KRAS mutation in human cancers, with the highest prevalence in pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). RMC-9805 is a first-in-class, oral, mutant-selective covalent inhibitor of the GTP-bound and active RAS(ON) form of KRASG12D. The formation of a stable, high affinity tri-complex between RMC-9805, KRASG12D and cyclophilin A results in the suppression of signaling downstream of KRASG12D(ON) by disrupting its interactions with downstream effectors such as RAF kinases. RMC-9805 treatment caused selective and persistent modification of KRASG12D leading to deep and durable suppression of RAS pathway activity, inhibition of cell proliferation, and apoptosis induction in KRASG12D human cancer cell lines in vitro and tumor models in vivo. In a mouse clinical trial with KRASG12D xenograft tumor models, RMC-9805 administered orally as a single agent was well tolerated and induced objective responses in 7 of 9 PDAC PDX and CDX models and 6 of 9 NSCLC PDX models, as assessed by mRECIST. In the few models that exhibited sub-optimal responses to RMC-9805 monotherapy, combination treatment with various RAS Companion Inhibitors improved depth and/or duration of anti-tumor response. In contrast to RASMUTANT NSCLC and PDAC, CRC tumors are less dependent on RAS driver mutations. For example, a more heterogeneous response to KRASG12C inhibitors has been reported in CRC than in NSCLC patients, suggesting combinations will be desired to achieve significant clinical benefit in CRC. Likewise, RMC-9805 monotherapy is less active in CRC models at doses that were highly active in KRASG12D NSCLC and PDAC models. However, combinations of RMC-9805 with vertical or parallel pathway RAS Companion Inhibitors such as SHP2, mTORC1 or RASMULTI(ON) inhibitors, achieved objective response rates up to 60% and delayed the onset of resistance in tumor models in vivo. RMC-9805 also synergized with anti-PD1 therapy in KRASG12D tumors in immune-competent animal models by shaping a favorable tumor immune microenvironment through cytokine modulation. In addition, RMC-9805 engaged the adaptive immune system by increasing presentation and recognition of tumor antigens, promoting a diversification of the TCR repertoire, and inducing immunological memory. Overall, RMC-9805 monotherapy elicited tumor regressions in most preclinical PDAC and NSCLC cancer models harboring KRASG12D. Furthermore, RMC-9805 combination therapies drove regressions in CRC models relatively less responsive to monotherapy. Supported by these findings, RMC-9805 is currently in IND-enabling development to permit clinical evaluation of single agent and combination strategies in patients with KRASG12D tumors. Citation Format: Lingyan Jiang, Marie Menard, Caroline Weller, Zhican Wang, Les Burnett, Ida Aronchik, Shelby Steele, Mike Flagella, Ruiping Zhao, James W W. Evans, Shook Chin, Kang-Jye Chou, Yunming Mu, Michael Longhi, Laura McDowell, John E. Knox, Adrian Gill, Jacqueline A. Smith, Mallika Singh, Elsa Quintana, Jingjing Jiang. RMC-9805, a first-in-class, mutant-selective, covalent and oral KRASG12D(ON) inhibitor that induces apoptosis and drives tumor regression in preclinical models of KRASG12D cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 526.