Loperamide, pimozide, and STF-62247 trigger autophagy-dependent cell death in glioblastoma cells

Abstract

Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7-dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.

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

“…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 .…”

“…We previously reported that STF-62247 and pimozide increase the autophagic flux of a panel of GBM cell lines 30 . To further evaluate the effects of STF-62247 and pimozide on autophagy induction in MEFs, we initially investigated some of the major hallmarks of autophagy 19,20 .…”

“…Cytoprotective roles of autophagy have for example been illustrated in several models of neurodegenerative diseases (ND), such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD), where the selective removal of pathological protein aggregates or damaged organelles via selective autophagy can support cell survival 10,22-24 .Since the induction of autophagy offers novel options to modulate human diseases 25 , the identification and characterization of compounds that stimulate autophagy has attracted special interest. In recent years, several natural compounds, small molecules and Food & Drug Administration (FDA)-approved drugs have been reported to induce autophagy and ACD under different cellular conditions and in different cell types [26][27][28][29][30] . Furthermore, triggering compound-induced autophagy and ACD has attracted particular interest, since many cancer types acquire loss-of-function mutations in conventional cell death pathways, like apoptosis, rendering them resistant against conventional chemotherapies 31 .Recently, we demonstrated that STF-62247 and pimozide trigger autophagy in glioblastoma (GBM) cells leading to ACD 30 .…”

“…Since the induction of autophagy offers novel options to modulate human diseases 25 , the identification and characterization of compounds that stimulate autophagy has attracted special interest. In recent years, several natural compounds, small molecules and Food & Drug Administration (FDA)-approved drugs have been reported to induce autophagy and ACD under different cellular conditions and in different cell types [26][27][28][29][30] . Furthermore, triggering compound-induced autophagy and ACD has attracted particular interest, since many cancer types acquire loss-of-function mutations in conventional cell death pathways, like apoptosis, rendering them resistant against conventional chemotherapies 31 .…”

Control of the Endo-Lysosome Homeostasis by the Paracaspase MALT1 regulates Glioma Cell Survival

“…Recent reports also highlighted that lysosomal homeostasis is essential in cancer stem cell survival 18,21 . Additionally, it has been shown that targeting the autophagic machinery is an effective treatment against apoptosis-resistant GBM 22,23 . The autophagic flux inhibitor chloroquine can decrease cell viability and acts as an adjuvant for TMZ treatment in GBM.…”