ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis

The <i>VPS37A</i> gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that <i>VPS37A</i>-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in <i>VPS37A</i>-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon <i>VPS37A</i> depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with <i>VPS37A</i> loss. <b>Abbreviations:</b> ATG: autophagy related; BMS: BMS-345541; CASP: caspase; CHMP: charged multivesicular body protein; DKO: double knockout; Dox: doxycycline; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; gRNA: guide RNA; GSEA: gene set enrichment analysis; GSK157: GSK2656157; iDISC: intracellular death-inducing signaling complex; IKK: inhibitor of NFKB kinase; IPA: ingenuity pathway analysis; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-kB: nuclear factor kappa B; OZ: 5Z-7-oxozeaenol; RNA-seq: RNA sequencing; UPR: unfolded protein response; TFT: transcription factor target; THG: thapsigargin; TUN: tunicamycin; VPS: vacuolar protein sorting.