PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal diseases of mutated hematopoietic stem cells (HSC), with more than 30,000 new cases being diagnosed each year in the Unites States. Given limited therapeutic options, long-term survival is less than 5% and new therapies are urgently needed. Genes encoding components of the cohesin complex are commonly mutated in human myeloid diseases, including 11% of patients with MDS and 21% of patients with secondary acute myeloid leukemia (sAML) arising in the context of MDS. Mutations in the cohesin subunit STAG2 have been recently identified as MDS-defining driver lesions. It is not understood how these mutations lead to MDS or leukemia, or whether cells with these mutations have vulnerabilities that can be exploited therapeutically. Using quantitative immunoprecipitation followed by mass spectrometry in STAG2 knockout cells, we identified incorporation of STAG1 into the mutant cohesin complex and loss of its interaction with splicing and RNA binding proteins. We also identified a synthetic lethal interaction between STAG1 and STAG2 loss, and CDK4 and STAG2 loss, the latter of which we corroborated by demonstrating selective killing of STAG2 knockout cells with CDK4/6 inhibitor LEE011 in vitro. Lastly, we demonstrated that STAG2 and STAG1 containing cohesin complexes have differential chromatin binding patterns. In light of these preliminary data, we hypothesize that STAG2 mutations alter cohesin complex formation and its chromatin association, with subsequent effects on gene expression through changes in critical long-range DNA looping interactions. To further define the mechanistic basis of mutant STAG2 mediated transformation in MDS, we propose the following Specific Aims: (1) Examine the effect of STAG2 loss on the composition of the cohesin complex; (2) Determine the transcriptional and epigenetic consequences of STAG2 inactivation; and (3) Validate CDK4 as a novel therapeutic target in STAG2 deficient cells in vivo. Mechanistic understanding of the STAG2 mutant cohesin complex promises to offer biological insight into MDS and identify new therapeutic opportunities. The applicant Dr. Zuzana Tothova is mentored by Dr. Benjamin Ebert, a physician scientist and expert in MDS, and co-mentored by Dr. Richard Young, a leader in the field of chromatin biology. Dr. Tothova has outlined a four-year career development plan to meet her goal of becoming an independent investigator in translational hematology, and she has assembled an Advisory Committee of internationally recognized experts to provide career and scientific mentorship. Dana Farber Cancer Institute is the ideal environment for completion of her scientific and career goals, given its outstanding research community and substantial record of training independent physician scientists.