Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance.

ABSTRACT

Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer.Abbreviations ABCD3/PMP70 ATP binding cassette subfamily D member 3; ACOX1 acyl-CoA oxidase 1; AP autophagosome; COX cytochrome c oxidase; CQ chloroquine; CRISPRi clustered regularly interspaced short palindromic repeats interference; DLBCL diffuse large B-cell lymphoma; GO gene ontology; dCas9 Cas9 endonuclease dead, or dead Cas9; HDACi histone deacetylase inhibitors; IHC Immunohistochemistry; LAMP2 lysosomal associated membrane protein 2; LCFAs long-chain fatty acids; LFQ-MS label-free quantitation mass spectrometry; LPC lysophoshatidylcholine; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 beta; MTOR mechanistic target of rapamycin kinase; PBD peroxisome biogenesis disorders; PTS1 peroxisomal targeting signal 1; ROS reactive oxygen species; sgRNA single guide RNA; VLCFAs very-long chain fatty acids; Vor vorinostat; WO wash-off.