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Supplementary Figure S1 shows the strategy and result of co-essentiality module identificationfrom the DepMap CRISPR screen dataset and further characterization of the BIRC6 module. Supplementary Figure S2 shows the viability effect of BIRC6 depletion in BIRC6-dependent and -nondependent cancer cells lines as well asin nontransformed cells. Supplementary Figure S3 shows in vitro confirmations of the inducible BIRC6 depletion (by RNAi and CRISPR) systems as well as the effect of BIRC6 depletion on metastasis in xenograft models. Supplementary Figure S4 shows detailed results for the allele competition assay to evaluate the role of the BIR and UBC domains of BIRC6 and further evidence for the physical assembly of BIRC6 ubiquitin ligase complex. Supplementary Figure S5 shows evidence for the selective activation of the p-eIF2alpha/ATF4 arm of UPR (or ISR) upon BIRC6 depletion. Supplementary Figure S6 shows evidence supporting the importance of HRI-mediated ISR activation as a mechanism underlying the loss of viability caused by BIRC6 complex suppression. Supplementary Figure S7 shows evidence for the direct regulation of HRI ubiquitination and degradation by the BIRC6 complex Supplementary Figure S8 shows elevated expression of HRI mRNA in the tumor samples compared to the normal samples in large-scale datasets (TCGA, TARGET and GTEx). Supplementary Figure S9 shows the inefficiency of the gene expression and copy number of the functionally related genes in predicting BIRC6 dependency. Supplementary Table S1 lists the top 50 co-essentiality modules identified in the analysis explained in Supplementary Figure S1A. Supplementary Table S2 lists the key DNA and RNA sequences used in this study.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.Significance:We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells.See related commentary Leli and Koumenis, p. 535.This article is highlighted in the In This Issue feature, p. 517