PROJECT SUMMARY Myelodysplastic syndromes (MDS) are the most common form of bone marrow failure disorders with more than 30,000 new cases diagnosed each year in the United States. These disorders are characterized by ineffective hematopoiesis as a result of mutations acquired in the hematopoietic stem and progenitor cells that clonally expand over time. Mutations in the cohesin complex, most commonly STAG2, are found in the high-risk subset of MDS patients with poor overall survival and for whom there are currently no targeted therapeutic options. Therefore, it is critical to gain a better understanding of the cellular mechanisms that drive cohesin-mutant MDS which can be therapeutically targeted. Our preliminary work has revealed that under normal conditions, the cohesin complex interacts with the spliceosome through an RNA-intermediate. Interestingly, this interaction is lost upon mutation of STAG2 in cells. Furthermore, STAG2-mutant cells are selectively killed compared to wild- type when treated with splicing modulators that target the SF3B complex, drugs that are currently undergoing clinical testing for splicing-factor mutant MDS. Based on our preliminary work, our central hypothesis is that regulatory RNAs mediate the interaction between cohesin and the spliceosome at enhancer-promoter loops that are either lost or altered in cohesin-mutant cells. Furthermore, we believe the loss of this interaction renders cohesin-mutant cells more sensitive to splicing modulation than normal, healthy cells. The overall objective of this work is to fully characterize the RNA and protein components that allow cohesin to interact with the spliceosome and test the in vivo efficacy of splicing modulation in cohesin-mutant MDS mouse models. In Aim1, we will determine the RNAs that mediate the interaction between cohesin and the spliceosome that are lost in cohesin-mutant MDS. We will perform enhanced cross-linking immunoprecipitation (eCLIP) on the cohesin complex to identify bound RNAs and use precision run-on sequencing (PRO-Seq) and total RNA-Seq to quantify both nascent and steady-state levels of bound RNAs in wild-type and STAG2-mutant cells. In Aim2, we will quantify the alternative splicing burden observed in cohesin-mutant MDS mouse models and determine the efficacy of splicing modulators to reverse disease phenotypes and expansion of mutant clones in vivo. Our in vivo model develops MDS phenotypes upon sequential acquisition of Tet2 and Stag2 mutations, a process that mimics disease progression in patients. Our long-term goal is to contribute to a mechanistic understanding of how the interaction between cohesin and the spliceosome is disrupted in disease and potentially offer cohesin- mutant MDS patients a new therapeutic option of splicing modulation. This work will be carried out under the guidance of Dr. Zuzana Tothova and Dr. Ben Ebert in the Dana-Farber Cancer Institute, with our local collaborators Dr. Karen Adelman and Dr. Chris Burge. Together...