Abstract

IntroductionMitochondrial apoptosis evasion, driven by overexpression of anti-apoptotic BCL-2 family proteins, remains a major obstacle in the effective treatment of colorectal cancer (CRC). While BCL-2 homology 3 (BH3) mimetics (such as venetoclax) have shown clinical efficacy in hematologic malignancies, their effectiveness in solid tumors is limited. Direct delivery of pro-apoptotic effectors, including BAX, offers an alternative strategy; yet, the therapeutic potential of non-human BAX orthologs and their delivery methods has not been explored.MethodsThe functional activity of Xenopus laevis (African clawed frog) BAX was evaluated in human CRC cell lines (HCT116, LOVO) and in cell line-derived xenograft (CDX) and AOM/DSS-induced CRC mouse models. Direct binding to human BCL-2 was quantified by microscale thermophoresis. The mechanism of action was elucidated via JC-10 staining, subcellular fractionation and liposome permeabilization assays.Structure-guided mutagenesis based on the BCL-2–Beclin-1 BH3 complex (PDB: 5VAU) was employed to generate a triple mutant (I86T, A102S, R109M) . Optimized BAX was packaged into engineered exosomes for targeted delivery, and their anti-tumor efficacy and safety were assessed in CDX, patient-derived xenograft (PDX), and AOM/DSS models.ResultsToad BAX directly bound human BCL-2 (Kd = 12.7 ± 1.9 µM), triggered mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and caspase-9/3 activation, thereby suppressing CRC cell proliferation and inducing apoptosis. The rationally designed triple mutant exhibited enhanced BCL-2 affinity and superior in vivo antitumor activity compared to wild-type toad BAX. Exosome-mediated delivery of the optimized BAX efficiently targeted CRC cells, inhibited tumor growth in PDX models, and extended overall survival in AOM/DSS-induced CRC without inducing overt toxicity.DiscussionThis study establishes structurally optimized cross-species BAX, delivered via engineered exosomes, as