Figure 5.

HRI is a critical mediator of ISR induced by the inactivation of the BIRC6 complex. A and B, Blockade of BIRC6 depletion–induced ISR activation and loss of viability by ISRIB, an ISR inhibitor. HCC202-Cas9 and SNU503-Cas9 cells were transduced with the indicated sgRNA and maintained in either vehicle- or ISRIB-containing medium. In A, lysates were harvested 4 days later and subjected to immunoblotting. In B, cell viability was scored with an ATP-based viability assay 7 days later. Positive controls include sgRNAs targeting two common essential genes (POLR2D, SF3B1). ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (vs. corresponding ISRIB [-] sample). C, Schematic of the genome-scale screen to identify enhancers and suppressors of BIRC6 dependency. HCC202-Cas9 and SNU503-Cas9 cells were engineered to express a shRNA targeting BIRC6 in a doxycycline (DOX)-inducible manner. These cells were subsequently transduced with a genome-scale sgRNA library (Brunello) and subjected to DOX treatment 7 days after the library transduction. Cells were harvested after 7 days of DOX treatment and the relative abundance of individual sgRNAs in the genome of these cells was analyzed. D and E, Identification of genes whose knockout rescue or enhance the viability effect of BIRC6 knockdown. The significance of the change in sgRNA abundance between the genomic DNA (gDNA) of DOX-treated cells and the plasmid DNA (pDNA) of the library was scored using the hypergeometric distribution method and aggregated to the gene level and plotted together with the average LogFC (post-DOX sgDNA/pDNA) of the sgRNAs against the respective gene. HRI was among the strongest hits in both cell lines screened (HCC202 and SNU503; D). Correlation of the screen results between the two dependent cell lines is also plotted (E). The four genes that comprise the EIF2AK family of kinases are indicated by orange dots, while the genes with statistically significant (adjusted P value < 0.01) depletion/enrichment of corresponding sgRNAs are indicated by the green dots (in E, only genes with significant depletion/enrichment in both cells lines are indicated by the green dots). F, Blockade of BIRC6 depletion–induced ISR activation by the concomitant knockout of HRI. HCC202-Cas9 and SNU503-Cas9 cells were engineered to express either an sgRNA against HRI or PERK or a control sgRNA (sgCh2-2). These cells were subsequently transduced with a control sgRNA (sgAAVS1) or an sgRNA targeting BIRC6, and 4 days later, their lysates were harvested and analyzed. G, Rescue of the viability effect of BIRC6 knockout by the concomitant knockout of HRI. The cells expressing sgCh2-2, sgHRI, or sgPERK, used in F, were transduced with sgAAVS1 (negative control gene), an sgRNA against positive control genes, or an sgRNA against BIRC6, and their viability was scored 7 days later. ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (vs. corresponding sgCh2-2 sample). In A and F, values represent the intensity of the p-eIF2α band relative to that of the corresponding t-eIF2α band. In B and G, values = means ± SD [n = 3 (sgCh2-2 (B), sgAAVS1 (G)), 6 (positive ctrl, sgBIRC6)]. All the experiments were performed twice except for the genome-scale modifier screen (D and E), which was conducted once.