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 wildtype 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 support sister chromatid cohesion to redundantly ensure cell survival. STAG1 represents a vulnerability of cancer cells carrying mutations in the major emerging tumor suppressor STAG2 across different cancer contexts. 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.

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together with the use of γ-secretase inhibitors, scRNA-Seq confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.

. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/155309 doi: bioRxiv preprint first posted online Jun. 25, 2017; 3 Main textCohesin is a highly conserved ring-shaped protein complex that is thought to topologically embrace chromatid fibers (Peters & Nishiyama, 2012), which is essential for sister chromatid cohesion and chromosome segregation in eukaryotes. In addition, cohesin participates in DNA repair, genome organization and gene expression (Losada, 2014). The cohesin subunits SMC1, SMC3 and RAD21 (also called SCC1) comprise the core ring of the complex. A fourth universally conserved subunit, a HEAT repeat protein of the Scc3/STAG family, peripherally associates with the core cohesin ring by binding to RAD21 (Toth et al., 1999), and is required for the dynamic association of cohesin with chromatin (Hu et al., 2011;Murayama & Uhlmann, 2014). Human somatic cells express two paralogs of this protein, called STAG1 and STAG2 (Losada, Yokochi, Kobayashi, & Hirano, 2000;Sumara, Vorlaufer, Gieffers, Peters, & Peters, 2000).Recent cancer genome studies identified recurrent mutations in cohesin subunits and regulators in approximately 7.3% of all human cancers (Lawrence et al., 2014; Leiserson et al., 2015;Solomon et al., 2011). STAG2, the most frequently mutated cohesin subunit, emerges as one of only 12 genes that are significantly mutated in 4 or more major human malignancies (Lawrence et al., 2014). STAG2 mutations have been reported in ~6% of acute myeloid leukemias and myelodysplastic syndromes (Kon et al., 2013; Thota et al., 2014; Walter et al., 2012), 15-22% of Ewing's sarcomas (Brohl et al., 2014; Crompton et al., 2014;Tirode et al., 2014), and in up to 26% of bladder cancers of various stages and grades (Balbas-Martinez et al., 2013; Guo et al., 2013;Solomon et al., 2013;Taylor, Platt, Hurst, Thygesen, & Knowles, 2014). The deleterious nature of most STAG2 mutations strongly suggests that the gene represents a new tumor suppressor . STAG2 mutations were initially thought to promote tumorigenesis due to defects in sister chromatid cohesin leading to genome instability (Barber et al., 2008;Solomon et al., 2011). However, the vast majority of cohesin-mutated cancers are euploid (Balbas-Martinez et . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/155309 doi: bioRxiv preprint first posted online Jun. 25, 2017; 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 o...