PROJECT SUMMARY/ABSTRACT The goal of this project is to define vulnerabilities that can be leveraged to comprehensively eradicate MDS stem cells (MDS-SCs). This objective is based on the concept that MDS-SCs lie at the root of disease and are not effectively targeted by current therapies. A significant challenge in achieving this goal is the heterogeneity of the MDS-SC population, as recent studies have identified distinct subsets of MDS-SC with varying responsiveness to therapy. Thus, the premise of our work is that understanding and targeting the unique properties of heterogenous MDS-SCs is critical to improve outcomes in MDS. The foundation of our project is built upon laboratory studies coupled with an active clinical trial (NCT03564873) evaluating the clinical impact of targeting protein synthesis with the global translation inhibitor omacetaxine (oma) in MDS-SC from individuals with high- risk MDS. While the results from our trial using oma in combination with the hypomethylating agent azacytidine (aza) are indeed promising and represent a substantial improvement over standard of care (aza alone), instances of disease progression in patients suggest oma may not eradicate all MDS-SC subsets. These outcomes, alongside toxicities associated with global protein translation inhibition have motivated us to better characterize the unique biological and functional properties of heterogenous MDS-SC subsets. With the goal of developing next-generation approaches that more effectively target MDS-SC while minimizing dose-limiting toxicities to patients, here we will address two primary questions: 1) what is the overall efficacy of protein synthesis inhibition amongst varying MDS-SC subtypes, 2) what are the optimal therapeutic strategies to eradicate MDS-SC based on their sensitivity to protein synthesis inhibition? Hence, we will leverage comprehensive CITE-seq based analyses of serial pre- and post-treatment specimens from our oma/aza clinical trial alongside PDX-based functional studies to identify the link(s) between features such as protein synthesis activity, MDS-SC genotype and clonal behavior with oma response at single cell resolution. We will define the mechanisms governing reliance on protein synthesis in MDS-SC, focusing on pharmacological and molecular genetic-based targeting of key pathways linked with oma sensitivity. Lastly, we will develop improved approaches to comprehensively target heterogenous MDS-SC populations, with focus on next-generation strategies that more selectively target aberrant metabolic features of MDS-SC uncovered by our ongoing CITE-seq characterization of patient specimens. Taken together, the proposed studies will use advanced functional models and single-cell resolution analyses to identify key vulnerabilities of MDS-SC that can be leveraged to design rational therapeutic approaches to improve outcomes for MDS patients.