Hardwired synthetic lethality within the cohesin complex in human cancer cells

Recent genome analyses have identified recurrent mutations in the cohesin complex in a wide range of human cancers. Here we demonstrate that the most frequently mutated subunit of the cohesin complex, STAG2, displays a strong synthetic lethal interaction with its paralog STAG1. Mechanistically, STAG1 loss abrogates sister chromatid cohesion in STAG2 mutated but not in wild-type cells leading to mitotic catastrophe, defective cell division and apoptosis. STAG1 inactivation inhibits the proliferation of STAG2 mutated but not wild-type bladder cancer and Ewing sarcoma cell lines. Restoration of STAG2 expression in a mutated bladder cancer model alleviates the dependency on STAG1. Thus, STAG1 and STAG2 act redundantly to support sister chromatid cohesion and cell survival. STAG1 represents a hardwired, context independent vulnerability of cancer cells carrying mutations in the major emerging tumor suppressor STAG2. Exploiting synthetic lethal interactions to target recurrent cohesin mutations in cancer, e.g. by inhibiting STAG1, holds the promise for the development of selective therapeutics.

4 al., 2013; Kon et al., 2013), indicating that cohesin mutations may promote tumorigenesis through altering different cohesin functions such as genome organization and transcriptional regulation (Galeev et al., 2016;Mazumdar et al., 2015;Mullenders et al., 2015;Viny et al., 2015). Regardless of the mechanisms driving cohesin mutant tumors, the recent success of poly (ADP-ribose) polymerase inhibitors in the treatment of BRCA-mutated ovarian and prostate cancer demonstrates that exploiting tumor suppressor loss by applying the concept of synthetic lethality in defined patient populations can impact clinical cancer care (Castro, Mateo, Olmos, & de Bono, 2016;G. Kim et al., 2015;Mirza et al., 2016;Oza et al., 2015). The estimated half a million individuals with STAG2mutant malignancies would greatly profit from exploring specific dependencies of these cancers.
We hypothesized that STAG2 loss could alter the properties and function of the cohesin complex leading to unique vulnerabilities of STAG2 mutated cells. To identify factors whose inactivation would be synthetic lethal with loss of STAG2 function, we first used CRISPR/Cas9 to inactivate STAG2 in near-diploid, chromosomally stable HCT 116 colon carcinoma cells ( Figure 1A). Two clones, STAG2-505c1 and 502c4, harboring deleterious mutations in STAG2 and lacking detectable STAG2 protein expression were selected for analyses (Figure supplement 1). The isogenic parental and STAG2-HCT 116 cells were transfected with short-interfering RNA (siRNA) duplexes targeting 25 known cohesin subunits and regulators. After normalization to the non-target control siRNA (NTC), the effects of siRNA duplexes targeting individual genes were compared in parental and STAG2-cells. Depletion of the known essential cohesin regulator SGOL1, had a detrimental impact on viability of both parental and STAG2-cells. Remarkably, depletion of STAG1 strongly decreased cell viability in STAG2-cells, while being tolerated by the isogenic parental cells ( Figure 1B). The pronounced selective effect of STAG1 depletion on STAG2-cells was confirmed in individual transfection experiments and colony formation assays ( Figure 1C,D,E). Expression of an siRNA-resistant STAG1 transgene alleviated the anti-proliferative effect of STAG1 but not of SGOL1 siRNA duplexes in STAG2-HCT 116 cells demonstrating the specificity of the siRNA 5 treatment (Figure supplement 2). Double depletion of STAG1 and STAG2 by siRNA in parental cells confirmed their synthetic lethal interaction (Figure supplement 3A). Co-depletion of p53 and STAG1 indicated that the dependency of STAG2-cells on STAG1 was independent of p53 (Figure supplement 3B). To corroborate these findings using an independent strategy, we introduced Cas9 into parental and STAG2-HCT 116 and KBM-7 leukemia cells for competition assays ( Figure   1F; Figure supplement 1). Transduction of lentiviruses co-expressing mCherry and single guide RNAs (sgRNAs) targeting essential cohesin subunit genes, such as RAD21 and SMC3, resulted in the rapid loss of the infected and mCherry-positive cells from the population irrespective of STAG2 genotype ( Figure 1F). In striking contrast, transduction with sgRNAs targeting STAG1 caused the depletion of STAG2-HCT 116 and KBM-7 cells but not of their parental STAG2 proficient counterparts ( Figure 1F). Collectively, these experiments identify STAG1 as a vulnerability of STAG2 mutated cells in engineered solid cancer and leukemia models. STAG1 inactivation has little if any impact on the viability and proliferation of wild-type cells, but is essential for survival in the absence of STAG2.
To elucidate the mechanistic basis for this synthetic lethal interaction, we hypothesized that the combined loss of STAG1 and STAG2, in contrast to loss of either component alone, could severely impair cell division. Chromosome alignment and segregation during mitosis rely on sister chromatid cohesion, the central function of the cohesin complex (Peters & Nishiyama, 2012). Depletion of STAG1 resulted in an increase in the mitotic index and a prolongation of the duration of mitosis in STAG2-but not wild-type cells (Figure 2A and Figure supplement 4A). Immunofluorescence microscopy revealed a failure to align chromosomes at the metaphase plate upon STAG1 loss in STAG2-cells ( Figure 2B). In mitotic chromosome spread analysis STAG2 inactivation caused a partial loss of centromeric cohesion in HCT 116 cells as previously reported (Canudas & Smith, 2009; J. S. Kim et al., 2016;Remeseiro et al., 2012;Solomon et al., 2011) (Figure 2C). Depletion of the essential centromeric cohesin protection factor SGOL1 resulted in a complete loss of sister 6 chromatid cohesion in most chromosome spreads irrespective of STAG2 genotype. In striking contrast, STAG1 depletion selectively abrogated sister chromatid cohesion in STAG2-but not parental cells (Figure 2C, single chromatids). The severe mitotic defects observed upon loss of STAG1 in STAG2-cells were accompanied by the emergence of aberrantly sized and shaped interphase nuclei (Figure supplement 4B) and by a progressive increase in apoptosis ( Figure   2D). These results provide a mechanistic basis for the synthetic lethal interaction between STAG1 and STAG2. STAG1 inactivation abrogates sister chromatid cohesion exclusively in STAG2-cells resulting in catastrophic mitotic failure, abnormal cell division and apoptosis. To hold sister chromatids together, cohesin can tolerate the loss of either STAG1 or STAG2 alone but not the loss of both.
We next expanded our analysis to patient-derived STAG2 mutations and STAG2-mutant cancer cell lines in order to investigate the disease relevance of the observed synthetic lethality. STAG1 depletion by siRNA abrogated both cell viability and sister chromatid cohesion in HCT 116 cell clones, in which three patient-derived deleterious mutations had been engineered into the STAG2 locus (J. S. Kim et al., 2016), but not in parental HCT 116 cells (Figure supplement 5). Among solid human cancers, STAG2 mutational inactivation is most prevalent in urothelial bladder cancer and Ewing sarcoma. Therefore, we assembled a panel of 16 bladder cancer cell lines: 11 STAG2positive, 3 with deleterious STAG2 mutations (UM-UC-3, UM-UC-6 and VM-CUB-3), one in which STAG2 was inactivated by CRISPR/Cas9 (UM-UC-5 STAG2-505c6) (Figure supplement 1), and two with no detectable STAG2 expression (LGWO1 and MGH-U3) (Table supplement 1) (Balbas-Martinez et al., 2013;Solomon et al., 2013). The STAG2 protein expression status in the panel of bladder cancer cell lines was confirmed using immunoblotting ( Figure 3A). siRNA experiments revealed that STAG2 status represented a predictive marker for the sensitivity to STAG1 depletion across the bladder cancer cell line panel. Whereas all cell lines were highly sensitive to depletion of the key mitotic kinase PLK1, STAG1 siRNA reduced cell viability in STAG2-negative bladder 7 cancer cells but had little or no effect on STAG2-positive bladder cancer cell lines ( Figure 3B).

STAG1 depletion prevented colony formation and abolished sister chromatid cohesion selectively in
STAG2 mutated UM-UC-3 (F983fs) but not in STAG2 wild-type UM-UC-5 bladder cancer cells ( Figure 3C,D). In contrast, SGOL1 depletion abrogated cell growth and cohesion in both cell lines.
Consistent with the results obtained in bladder cancer cells, STAG2 mutation status also positively correlated with STAG1 dependency in a panel of four Ewing sarcoma cell lines ( Figure 3E,F and  (Solomon et al., 2011;Tirode et al., 2014). Lentiviral transduction of a FLAG-STAG2 transgene into STAG2 mutated UM-UC-3 bladder cancer cells resulted in the restoration of STAG2 expression, nuclear localization of the transgenic protein and its incorporation into the cohesin complex (Figure supplement 6A-C). Crucially, restoration of STAG2 expression alleviated the STAG1 dependency of UM-UC-3 cells providing a causal link between STAG2 loss and STAG1 dependency ( Figure 3G). These results demonstrate that the synthetic lethal interaction between STAG1 and STAG2 that we discovered in isogenic cell pairs is recapitulated in disease-relevant bladder cancer and Ewing sarcoma cell models.
We here identify STAG1 as a strong genetic vulnerability of cells lacking the major emerging tumor suppressor STAG2 (Figure supplement 6D). The synthetic lethal interaction between STAG1 and STAG2 is observed in isogenic HCT 116 and KBM-7 cells as well as in bladder cancer and Ewing sarcoma cell lines. Thus, the genetic interaction between STAG paralogs is context independent and conserved in three major human malignancies: carcinoma, leukemia and sarcoma. Importantly, the finding that cancer cells harboring deleterious STAG2 mutations remain exquisitely dependent on STAG1 demonstrates that this genetic vulnerability is maintained throughout the process of carcinogenesis and not bypassed by adaptive processes, such as the transcriptional activation of the germline-specific paralog STAG3 (Pezzi et al., 2000;Prieto et al., 2001).

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Our experiments strongly suggest that the loss of sister chromatid cohesion followed by aberrant cell division and cell death is the mechanistic basis underlying the synthetic lethality between STAG1 and STAG2 (Figure supplement 6D). Both paralogs associate with the cohesin complex in a mutually exclusive manner (Losada et al., 2000;Sumara et al., 2000). Although STAG1 and STAG2 may confer distinct functionalities to the cohesin complex (Canudas & Smith, 2009;Remeseiro et al., 2012), STAG1 and STAG2 containing complexes act redundantly to ensure sister chromatid cohesion and successful cell division in human somatic cells. While loss of one paralog is compatible with cell viability and proliferation, the loss of both paralogs abrogates cohesin's ability to hold sister chromatids together, which results in lethality. The fact that STAG1 inactivation has little or no effect on proliferation of STAG2 proficient cells indicates that selective targeting of STAG1 could offer a large therapeutic window. Potential approaches for therapeutic targeting of STAG1 include the inhibition of the interaction between STAG1 and the cohesin ring subunit RAD21 (Hara et al., 2014) and the selective degradation of STAG1 using proteolysis-targeting chimera (PROTAC) technology (Deshaies, 2015). The mechanisms by which mutations in STAG2 and other cohesin subunits drive tumorigenesis in solid and hematological tissues are not yet firmly established. Our work highlights the fact that such knowledge is not a prerequisite for the identification of selective vulnerabilities.
Both deleterious STAG2 mutations and the loss of STAG2 expression are strong predictive biomarkers for STAG1 dependence and could be utilized for patient stratification. Our work demonstrates that unique genetic dependencies of cohesin mutated cancer cells exist. Such vulnerabilities hold the promise to develop selective treatments for patients suffering from STAG2 mutated cancer.   (Ban et al., 2014), and cultured for two additional days.
Viability was determined using CellTiter-Glo (Promega), and by staining with crystal violet (Sigma, HT901). For sgRNA competition assays, Cas9-GFP was expressed constitutively (HCT 116) or was induced by doxycycline addition (KBM-7). mCherry and sgRNAs were introduced by lentiviral transduction. The fraction of mCherry-positive cells was determined at the indicated time points using a Guava easycyte flow cytometer (Millipore) and normalized to the first measurement and sequentially to control sgRNAs (non-targeting for HCT 116 and STAG2_19 for KBM-7). Apoptosis was analyzed using the IncuCyte Caspase-3/7 Apoptosis Assay (Essen BioScience).       The indicated bladder cancer cell lines were transfected with NTC, STAG1 and PLK1 siRNA duplexes. Viability was measured seven or ten 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 seven days after transfection by crystal violet staining. (D) 72 hours 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 six 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 an siRNA-resistant FLAG-STAG2 transgene. Stably selected cell pools were subsequently transfected with NTC, STAG1 or SGOL1 siRNA duplexes. Viability was measured seven days after transfection and normalized to the viability of NTC siRNA transfected cells (n=4 biological replicates, error bars denote standard deviation).    Figure supplement 2 Figure supplement 2. Rescue of the synthetic lethal interaction between STAG1 and STAG2 by expression of an siRNA-resistant FLAG-STAG1 transgene. HCT 116 parental cells, a STAG2 wild-type clone (502wt), and two STAG2-clones (505c1 and 502c4) were transduced with a lentivirus encoding no transgene (empty vector) or an siRNA-resistant and 3xFLAG-tagged STAG1 transgene (FLAG-STAG1). Stably selected cell pools were used for the analysis. (A) Immunofluorescence analysis of FLAG-STAG1 transgene expression and nuclear localization in HCT 116 STAG2-505c1 cells. Scale bar, 20 µm. (B) Protein extracts prepared from HCT 116 STAG2-505c1 cells expressing no transgene (empty vector) or a FLAG-STAG1 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG1 expressing cells indicating specific incorporation of the transgenic protein into the cohesin complex. (C) Protein extracts prepared from the indicated cell lines that were transduced with an empty vector or a FLAG-STAG1 transgene were analyzed by immunoblotting (left panel) and transfected with NTC, STAG1 and SGOL1 siRNA duplexes (right panel). Cell viability was measured seven days after transfection and is plotted normalized to the viability of NTC siRNA transfected cells (n≥5 biological repeats, error bars denote standard deviation). HCT 116 STAG2 cancer mutations 0 % viability of NTC 100 50 NTC STAG1 SGOL1 C Figure supplement 5. Patient-derived STAG2 mutations cause STAG1 dependency in engineered isogenic HCT 116 cells. HCT 116 cell lines engineered to harbor the indicated deleterious patient-derived STAG2 mutations were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. A, Protein extracts were prepared 48 hours after transfection and analyzed by immunoblotting. B, Cell viability was measured seven days after siRNA transfection and plotted normalized to the viability of NTC-transfected cells (n=4 independent experiments with 5 biological repeats each, error bars denote standard deviation). C, Sister chromatid cohesion phenotypes were analyzed in Giemsa-stained mitotic chromosome spreads that were prepared 48 hours after siRNA transfection (n=100 chromosome spreads, error bars denote standard deviation between two independently analyzed slides).  Protein lysates prepared 72 hours after siRNA transfection were analyzed by immunoblotting. (C) Protein extracts prepared from UM-UC-3 cells expressing no transgene (empty vector) or a FLAG-STAG2 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG2 expressing cells indicating successful incorporation of the transgenic STAG2 protein into the cohesin complex. (D) 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.