Autophagy-dependent cancer cells circumvent loss of the upstream regulator RB1CC1/FIP200 and loss of LC3 conjugation by similar mechanisms.

Unlabelled: Macroautophagy/autophagy degrades proteins and organelles to generate macromolecular building blocks. As such, some cancer cells are particularly dependent on autophagy. In a previous paper, we found that even highly autophagy-dependent cancer cells can adapt to circumvent autophagy inhibition. However, it remains unclear if autophagy-dependent cancer cells could survive the complete elimination of autophagosome formation. We extended our previous findings to show that knockout (KO) of both the upstream autophagy regulator and the downstream regulator and mediator of LC3 conjugation, , strongly inhibits growth in highly autophagy-dependent cells within one week of editing. However, rare clones survived the loss of or and maintained growth even under autophagy-inducing conditions. Autophagy-dependent cells circumvent the complete loss of autophagy that is mediated by KO, similar to the loss of , by upregulating NFE2L2/NRF2 signaling. These results indicate that cancer cell lines could adapt to the complete loss of autophagy by changing their biology to adopt alternative ways of dealing with autophagy-mediated cellular functions.

Abbreviations: CGS: CRISPR growth score; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: Earl's balanced salt solution; EEF2: eukaryotic translation elongation factor 2; FOXO3/FOXO3a: forkhead box O3; GFP: green fluorescent protein; KEAP1: kelch Like ECH associated protein 1; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NLS: nuclear localization signal; PCNA: proliferating cell nuclear antigen; PE: phosphatidylethanolamine; POLR2A: RNA polymerase II subunit A; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SNARE: soluble NSF attachment protein receptor; SQSTM1: sequestosome 1; STX17: syntaxin 17; TBHP: tert-butyl hydroperoxide; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; WT: wild type.