STF-62247 induces a non-canonical, caspase-independent cell death in ccRCC associated with autophagic and lysosomal dysfunction

Clear cell renal cell carcinoma (ccRCC), the most prevalent form of kidney cancer, is driven by early chromosome 3p loss and VHL inactivation, frequently followed by secondary mutations in other 3p genes, such as SETD2, PBRM1, and BAP1 . The small molecule STF-62247 was found to target VHL -deficient ccRCC through autophagy-dependent cell death. We recently demonstrated that SETD2 inactivation in VHL -reconstituted ccRCC cells also confers vulnerability to STF-62247, accompanied by caspase-3 and gasdermin E (GSDME) cleavage. Here, we investigated the underlying cell death mechanisms that drive STF-62247 cytotoxicity across different ccRCC genetic profiles. Our results ruled out necroptosis, ferroptosis, and GSDMD-mediated pyroptosis. In VHL -deficient cells, signature markers of classical intrinsic apoptosis, including cytochrome c release, PARP, and caspase-3 cleavage, as well as the extrinsic apoptosis marker, caspase-8 cleavage, were induced with STF-62247. Conversely, SETD2 -inactivated cells did not display cytochrome c release or caspase-8 activation. CRISPR knockout of CASP3 or GSDME , or pharmacological inhibition of caspases, failed to rescue cell death, proving these pathways are secondary. Mechanistically, immunofluorescence for LAMP1, cathepsin L (CTSL), and Galectin-3 indicated the absence of lysosomal membrane permeabilization. Instead, STF-62247-mediated PIKfyve inhibition caused endolysosomal enlargement, trapping immature pro-CTSL within non-functional compartments. We hypothesize that cells may alter their autophagic machinery to cope with this degradative arrest, potentially leading to an atypical secretory autophagy-like response; this could account for the observed LAMP1 plasma membrane translocation in VHL-deficient cells and dysfunctional vacuolar accumulation in SETD2-deficient models. Genetic ablation of ATG5 or BECN1 or pharmacological inhibition of autophagy with SAR405 partially rescued viability in SETD2 -inactivated cells, confirming that this autophagy-linked pathway actively contributes to cell death. Collectively, we found that STF-62247 triggers a multifaceted cell death program associated with autophagic machinery deregulation. While GSDME cleavage does not dictate survival, its pro-inflammatory potential remains an intriguing perspective to be explored for potentially optimizing immunotherapy strategies in ccRCC.