STF-62247 accumulates in lysosomes and blocks late stages of autophagy to selectively target von Hippel-Lindau-inactivated cells

Abstract

Autophagy is a highly conserved, homeostatic process by which cytosolic components reach lysosomes for degradation. The roles played by different autophagic processes in cancer are complex and remain cancer type and stage dependent. Renal cell carcinoma (RCC) is the most common subtype of kidney cancer and is characterized by the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Our previous study identified a small compound, STF-62247, as an autophagy-modulating molecule causing selective cytotoxicity for VHL-inactivated cells. This present study investigates the effects of STF-62247 specifically on the macroautophagic flux to better characterize its mechanism of action in RCC. Our results clearly demonstrate that this compound is a potent blocker of late stages of autophagy. We show that inhibiting autophagy by CRISPR knockouts of autophagy-related genes rendered VHL-deficient cells insensitive to STF-62247, uncovering the importance of the autophagic pathway in STF-selective cell death. By exploiting the autofluorescence of STF-62247, we pinpointed its cellular localization to lysosomes. Finally, in response to prolonged STF treatments, we show that VHL-proficient cells are able to surmount the block in late stages of autophagy by restoring their lysosome numbers. Conversely, an increase in autophagic vesicles accompanied by a time-dependent decrease in lysosomes was observed in VHL-deficient cells. This is the first mechanistic study investigating STF-62447’s effects on the autophagic flux in RCC. Importantly, our study reclassifies STF-62247 as a blocker of later stages of autophagy and highlights the possibility of blocking this process through lysosome disruption in VHL-mutated RCCs.

STF-62247 and pimozide induce autophagy and autophagic cell death in mouse embryonic fibroblasts

“…Moreover, we speculate that short-time as well as lower-dose treatment with pimozide triggers autophagy as well as selective degradation of mitochondria, but does not exceed the threshold required for lethal autophagy, as we did not detect a marked induction of cell death in these settings. Interestingly, in contrast to our findings that classify STF-62247 as an inducer of autophagic flux and ACD, STF-62247 has recently been reported to block late stages of autophagy by inducing lysosomal disruption in von Hippel-Lindau (VHL)-deficient renal cell carcinoma cells 80 . This study however emphasized that VHL-competent cells are capable to overcome this block of late stages of autophagy and to maintain high lysosome numbers, which is consistent with our data showing that STF-62247 enhances the autophagic flux of VHL-competent MEFs as well as GBM cell lines 30,80 .…”

“…Interestingly, in contrast to our findings that classify STF-62247 as an inducer of autophagic flux and ACD, STF-62247 has recently been reported to block late stages of autophagy by inducing lysosomal disruption in von Hippel-Lindau (VHL)-deficient renal cell carcinoma cells 80 . This study however emphasized that VHL-competent cells are capable to overcome this block of late stages of autophagy and to maintain high lysosome numbers, which is consistent with our data showing that STF-62247 enhances the autophagic flux of VHL-competent MEFs as well as GBM cell lines 30,80 . From a mechanistic perspective, this could be due to STF-62247-induced inhibition the master regulator of autophagy, mTORC1, which we demonstrated in our previous study in GBM cells 30,81 .…”

REV1 Inhibition Enhances Radioresistance and Autophagy

“…7), where the cells appeared flattened under an inverted microscope as previously reported 8 . However, we also observed a curious cellular morphology of enlarged cytoplasmic vesicles that prompted us to test whether the enlarged structures were lysosomes 15 . Enlarged lysosomes are typically discernible with the DIC (differential interference contrast) brightfield microscopy.…”

Genetic approaches to understand cellular responses to oxygen availability

“…For instance, Turcotte et al screened VHL-deficient and VHL-reconstituted RCC4 cells with a 64,000 drug compound library, and identified that STF-62247 selectively killed the VHL-deficient cells [87]. Cell death was HIF independent and thought to be mediated by STF-62247 accumulation in lysosomes, and inhibition of late stage autophagy [88]. Thompson et al also used RCC4 cells, and screened with a library of pharmacologically active compounds [89].…”