Fig 2: STAG2 deficiency mediates DNA damage and activation of the cGAS-STING signaling. a WT and STAG2−/− HT-29 cells were analyzed by western blot with the indicated antibodies. b Immunofluorescence analysis of WT and STAG2−/− HT-29 cells: nucleus (DAPI, blue), mitochondria (MitoTracker, red), and cytoplasmic DNA (green). In merged panels, mitochondrial DNA is shown in yellow and non-mitochondrial cytoplasmic DNA is shown in green. Scale bar: 8 μm. c WT, STAG2−/−, and STAG2−/−STING−/− HT-29 cells were infected with IAV, VSV, and RV (MOI = 1) and viral mRNA level was measured at 24 h.p.i. by RT-qPCR. IAV influenza A virus, VSV vesicular stomatitis virus. For all figures, experiments were repeated at least three times in triplicates. Data are represented as mean ± SEM (***p ≤ 0.001)

Fig 3: Identification of STAG1 as a genetic vulnerability of STAG2 mutated cells.(A) Engineering of an isogenic HCT 116 cell model by CRISPR/Cas9-mediated inactivation of STAG2. The position of the sgRNAs used to create deleterious insertion and deletion mutations in the STAG2 coding sequence is indicated. (B) Parental HCT 116 cells and two STAG2 mutant clones (STAG2-) were subjected to an siRNA screen. Pools of 4 siRNA duplexes targeting 25 known cohesin subunits and regulators were transfected into the three cell lines. Cell viability was measured 7 days after transfection. Following normalization to non-target control (NTC) values, the average cell viability of siRNA pools in parental HCT 116 cells and two STAG2- clones was plotted against each other (n = 2 or more independent experiments with 2 biological repeats each). (C) HCT 116 parental cells and STAG2- clones were transfected with the indicated siRNAs. Protein lysates were prepared 72 hr after transfection and analyzed by immunoblotting. (D) Cell viability was assessed 8 days after siRNA transfection using a metabolic assay (n = 4 biological repeats, error bars denote standard deviation) and (E) using crystal violet staining. (F) Cas9-GFP expressing isogenic parental and STAG2- HCT 116 and KBM-7 cells were transduced with a lentivirus encoding mCherry and sgRNAs targeting the indicated genes. The percentage of mCherry-positive cells was determined over time by flow cytometry and normalized to the fraction of mCherry-positive cells at the first measurement and sequentially to a control sgRNA (n = 1 experimental replicate).DOI: http://dx.doi.org/10.7554/eLife.26980.003

Fig 4: Model for the synthetic lethal interaction between STAG1 and STAG2.In wild-type cells, both cohesin-STAG1 and cohesin-STAG2 complexes redundantly contribute to sister chromatid cohesion and successful cell division. Loss of STAG1 is tolerated in these cells as cohesin-STAG2 complexes alone suffice to support sister chromatid cohesion for cell division. In cancer cells in which STAG2 is mutationally or transcriptionally inactivated, sister chromatid cohesion is now entirely dependent on cohesin-STAG1 complexes. Inactivation of STAG1 in STAG2 mutated cells therefore results in a loss of sister chromatid cohesion followed by mitotic failure and cell death.DOI: http://dx.doi.org/10.7554/eLife.26980.016

Fig 5: The synthetic lethal interaction between STAG1 and STAG2 is manifested in disease-relevant bladder cancer and Ewing sarcoma cell lines.(A) The indicated bladder cancer cell lines were analyzed for STAG2 expression by immunoblotting. (B) The indicated bladder cancer cell lines were transfected with NTC, STAG1 and PLK1 siRNA duplexes. Viability was determined 7 or 10 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 2 independent experiments with 5 biological repeats each, error bars denote standard deviation). (C) STAG2 wild-type UM-UC-5 and STAG2 mutated UM-UC-3 cells were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. Colony formation was analyzed 7 days after transfection by crystal violet staining. (D) 72 hr after siRNA transfection into UM-UC-5 and UM-UC-3 cells, Giemsa-stained chromosome spreads were prepared and analyzed for sister chromatid cohesion phenotypes (n = 100 spreads, error bars denote standard deviation of two independently analyzed slides). (E) The indicated Ewing sarcoma cell lines were analyzed for STAG2 protein expression by immunoblotting. (F) The indicated Ewing sarcoma cell lines were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. Viability was measured 6 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 3 independent experiments with 3 biological replicates each, error bars denote standard deviation). (G) STAG2 mutated UM-UC-3 cells were transduced with a lentivirus encoding a FLAG-STAG2 transgene. Stably selected cell pools were subsequently transfected with NTC, STAG1 or SGOL1 siRNA duplexes. Viability was measured 7 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 4 biological replicates, error bars denote standard deviation).DOI: http://dx.doi.org/10.7554/eLife.26980.012