Fig 2: Validation of BIRC6 dependency in vitro and in vivo.A, Consequences of CRISPR-mediated BIRC6 knockout on cell viability. Five putatively dependent cells and six putatively nondependent cells [as defined by Chronos score (see Methods)], all of which constitutively express Cas9, were analyzed using an ATP-based assay 7 days after transducing an sgRNA against BIRC6 (three different sgRNA sequences were tested). Viability scores relative to the average viability of cells transduced with cutting control sgRNAs and the average viability of cells with knockout (KO) of common essential genes are shown. Values = means ± SD (n = 9). ****, P < 0.0001 (dependent vs. nondependent; for each guide). B, Consequences of CRISPR interference (CRISPRi)–mediated BIRC6 knockdown on long-term cell fitness. Clonogenic growth of the cells was evaluated 14 days after the transduction of an all-in-one CRISPRi construct targeting the indicated gene. Two sgRNA sequences against BIRC6 were tested. Presented are the representative images of cells with crystal violet staining (left) and the mean staining intensities per sample (n = 3, right). *, P < 0.05; ****, P < 0.0001 (sgCiCh2-2 vs. sgCiBIRC6). C and D, Cell cycle (C) and cell death (D) analysis following BIRC6 knockout. Cas9-expressing derivatives of indicated cells were transduced with a cutting control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6-1, sgBIRC6-4). Cells were harvested 4 (C) or 7 (D) days later, stained, and analyzed by flow cytometry. In C, the proportion of cells in the S-phase was reduced upon BIRC6 knockout in the three dependent models, but not in the three nondependent models. In D, the proportion of dead cells (late apoptosis + nonapoptotic death + early apoptosis) was increased following the knockout of BIRC6 in all of the three dependent cell lines, but only one of the three nondependent cell lines. ns, P = 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (n = 3). E–G,In vivo validation of the BIRC6 dependency. In E, ZR751 breast cancer cells expressing a doxycycline (DOX)-inducible shRNA against BIRC6 (shBIRC6–2) were implanted into the mammary fat pads of NRG mice. Following tumor formation, some of these mice were treated with DOX, while others were left untreated. In F and G, KYSE450 esophagus cancer cells (F) and HCC95 lung cancer cells (G), both of which were engineered to express an sgRNA against BIRC6 in a tamoxifen (TAM)-inducible fashion, were implanted subcutaneously via intraperitoneal injection (IP) into the NSG (NOD-scid Il2rg-/-) mice. Following tumor formation, some mice were injected with TAM, while others were treated with vehicle control. In both cases, the tumor growth is plotted to compare the two different groups of mice. Data are represented as means ± SEM [n = 8 (Keep w/o TAM group, G), 9 (Keep w/o TAM and TAM(-) groups, F; TAM hereafter group, G), 10 (Keep w/o DOX and DOX(-) groups, E; TAM hereafter and TAM (+) groups, F; TAM(-) and TAM(+) groups, G), 12 (DOX hereafter and DOX (+) groups, E)]. ns, P = 0.05; ****, P < 0.0001 (for each of the last five time points for the tumor growth curves). All the experiments were performed twice except for A, which was conducted three times, and E–G, which were conducted once.

Fig 3: Cell type–specific role of the UBA6/BIRC6/KCMF1/UBR4 module revealed by the coessentiality analysis. A, Based on the significance of correlation and the variance of essentiality, we selected 50 top coessential gene modules, which included 42 modules for which the functional interactions of the constituent genes have already been reported (green dots) and eight modules that contain previously unassociated gene pair(s) (pink dots). B, Correlation of the essentiality of the four genes that comprise the BIRC6 module (UBA6, BIRC6, KCMF1, and UBR4). The Pearson correlation coefficients between the dependency profiles of the indicated gene pairs in both CRISPR (top) and RNAi (bottom) datasets (left) are shown. The correlations between UBA6 and BIRC6 (r = 0.714) as well as KCMF1 and UBR4 (r = 0.742) in the CRISPR dataset are also shown individually in the scatter plots (right). C, All these genes exhibited dependency profiles with both high variance (>89th percentile among all genes) and strong efficacy (>83rd percentile of all genes), the latter being defined by the minimum dependency score (Chronos) across all cell lines. D, The dependency profiles of the four genes constituting the BIRC6 module. UBA6 and BIRC6 were strongly essential (>90% probability of dependency) in a small subset of cell lines, while KCMF1 and UBR4 were strongly essential in the majority (>65%) of cell line models. E, Dependency on the BIRC6 module per tissue type. The mean Chronos (mChronos) scores of the four genes comprising the BIRC6 module were plotted per tissue type. The dependency on this module is enriched in epithelial tissue–derived cancer cells. F, Significance of the lineage/subtype enrichment of the BIRC6 module gene dependencies in the CRISPR and RNAi screens. The distribution of mChronos or mean DEMETER2 scores in the individual lineages/subtypes was compared with the corresponding distribution in all the other cell lines within the dataset. The effect size and significance, determined by the two-sample Kolmogorov–Smirnov test (KS), were plotted. ERpos, estrogen receptor positive; Her2Amp, Her2 amplified; TNBC, triple-negative breast cancer.

Fig 4: Biochemical demonstration of the BIRC6 complex assembly. A, Competition assay to evaluate the essentiality of each of the two functional domains of BIRC6 using a strategy to repair a CRISPR-mediated cleavage of the genomic locus corresponding to each of these domains (BIR and UBC) via homologous recombination. We show the two different donor DNAs that were introduced: one harboring a damaging mutation and the other containing a silent mutation. This assay scores the relative abundance of alleles with damaging versus silent mutations. ssDNA, single-strand DNA. B, Relative abundance of the damaging versus silent mutations in each of the two functional domains of BIRC6. Plotted is the change in the ratio of damaging over silent mutations at day 7 after the transduction of the Cas9/crRNA ribonucleoprotein complex relative to the corresponding ratio at day 3, normalized against the doubling time of the cells. Values = means ± SD (n = 4). ns, P = 0.05; **, P < 0.01. C–E, Protein–protein interactions between the components of the BIRC6 module. In C, endogenously expressed BIRC6 was immunoprecipitated (IP) from the lysate of SNU503 cells that were engineered to have the 3xFLAG tag–encoding sequence inserted at the N-terminus of the BIRC6-encoding sequence. In D and E, exogenously expressed, V5-tagged UBA6 (D) and V5-tagged KCMF1 (E) were immunoprecipitated from the lysates of HCC202 and SNU503 cells. In all these cases, eluates, crude (input) lysates, and cleared (sup) lysates were analyzed by immunoblotting. F, The BIRC6 module is composed of an E1 enzyme (UBA6), an E2 enzyme (BIRC6), and two E3 enzymes that have been shown to work cooperatively (KCMF1 and UBR4). All the experiments were performed twice except for B, which shows the summary of four independent experiments.

Fig 5: Ubiquitination and stability of HRI are governed by the BIRC6 complex. A, Proteomic changes following BIRC6 depletion in the presence and absence of ISRIB. HCC202-Cas9 cells were transduced with either a control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6-4). Four days later, cells were harvested and subjected to LC/MS-MS. The magnitude [LogFC (sgBIRC6/sgCh2-2)] and significance [-log10 (P)] of the difference in protein expression between the control and BIRC6 knockout samples were plotted. Here and in B, the products of the genes that are transcriptionally regulated by ISR are indicated by the orange dots, while HRI is indicated by the green dot. B, Comparison of the BIRC6 depletion–induced proteomic changes in the presence and absence of ISRIB treatment. C, Elevated expression of HRI protein after depleting individual components of the BIRC6 complex. HCC202-Cas9 and SNU503-Cas9 cells were transduced with the indicated sgRNA, and their lysates were harvested 4 days later. Lysates of the cells treated with MG132 (10 µmol/L) or a vehicle control for 6 hours were also analyzed by immunoblotting. D, Stabilization of HRI following BIRC6 depletion. HCC202-Cas9 cells, transduced with either sgCh2-2 or sgBIRC6-4, were transiently transfected with a plasmid expressing HRI-V5. These cells were subsequently treated with cycloheximide (CHX; 50 µg/mL) and harvested at the indicated time points. Changes in the relative intensity between V5 and ß-actin signals were plotted (right). Values = means ± SEM (n = 4). ****, P < 0.0001. E, Reduced HRI ubiquitination following BIRC6 depletion. HCC202-Cas9 cells that constitutively express HA-tagged Ubiquitin (HA-Ubiquitin) were further engineered to express HRI-V5 in a doxycycline (DOX)-inducible manner and then transduced with sgCh2-2 or sgBIRC6-4. These cells were subsequently treated with DOX (1 µg/mL, 48 hours), ISRIB (1 µmol/L, 48 hours), and/or MG132 (10 µmol/L, 6 hours), and their lysates were immunoprecipitated (IP) with anti-V5 followed by immunoblotting. The ubiquitin chains attached to HRI-V5 were clearly detected in the control (sgCh2-2) sample treated with all the three reagents (DOX, ISRIB, MG132) but was less clear in the BIRC6 knockout (sgBIRC6-4) sample. The relative intensity between HA(-ubiquitin) and (HRI-)V5 signals for the samples cotreated with DOX, ISRIB, and MG132 was plotted (right). Values = means ± SD (n = 5). F, A physical interaction between UBR4 and HRI. HCC202-Cas9 cells were engineered to express HRI-V5 in a DOX-inducible manner. Following treatment with DOX (1 µg/mL, 48 hours), ISRIB (1 µmol/L, 48 hours), and/or MG132 (10 µmol/L, 6 hours), cells were harvested, and the lysates were subjected to anti-V5 IP and analysis by immunoblotting. G, Analysis of HRI phosphorylation status using a Phos-tag gel. HCC202-Cas9 cells, transduced with either sgCh2-2 or sgBIRC6-4, were transiently transfected with a plasmid expressing HRI-V5. HCC202-Cas9 cells without sgRNA transduction were also transfected with an HRI-V5–expressing plasmid and subsequently treated with either arsenite (300 µmol/L, 3 hours) or vehicle control (mock). Lysates of these cells were either treated with lambda phosphatase (+?PP) or left untreated (+?PP) and analyzed by immunoblotting using a Phos-tag gel and a standard protein (regular) gel. The knockout of BIRC6 resulted in the upregulation of phosphorylated and nonphosphorylated forms of HRI. H, Changes in expression of ISR markers upon HRI depletion. The Cas9-expressing derivatives of the indicated cells were transduced with either an sgRNA against HRI or a control sgRNA (sgCh2-2). Four days later, their lysates were harvested and analyzed for the expression levels of various ISR marker proteins. Relative intensity of the ATF3 and SESN2 bands, both of which were normalized to the intensity of the corresponding ß-actin band, between sgCh2-2 and sgHRI samples were plotted. Values = means ± SD (n = 3). ****,P < 0.0001 (dependent vs. nondependent). The experiment shown in A and B was conducted once, the experiments shown in C and F were conducted twice, the experiments shown in G and H were conducted three times, the experiment shown in D was conducted four times, and the experiment shown in E was conducted five times.