3171 – IDENTIFYING STRESS GRANULES AS DETERMINANTS OF LEUKEMIA STEM CELL MAINTENANCE AND STRESS ADAPTATION

Development of novel leukemic stem cell (LSC)-targeted therapeutics is an urgent goal towards reducing the high, LSC-driven, relapse rates in acute myeloid leukemia (AML). Through a recent in vivo CRISPR dropout screen targeting RNA binding proteins (RBPs) highly expressed in LSCs we identified an enrichment of stress granule (SG) RBPs crucial for murine AML LSC-mediated leukemic growth. SGs are dynamic ribonucleoprotein complexes protective against a variety of stress conditions and gaining attention as contributors to cancer pathology, but their role in LSCs is virtually unexplored. To address this we focused on core SG nucleator, G3BP1, a molecular switch that guides SG assembly. We identified that a subset of patient AML cells contained G3BP1 SGs in the absence of added stress. In AML cell lines and patient AML we found that G3BP1 knockdown increased apoptosis and reduced colony forming capacity and competition in vitro. These results were echoed with a small molecule G3BP1 inhibitor validating the SG-forming capacity of G3BP1 as a driver of human AML cell function. Importantly, in vivo xenotransplantation showed that G3BP1 knockdown LSCs were significantly compromised in their capacity to generate leukemic grafts indicating a dependency on G3BP1 SGs extends to these most primitive AML-propagating cells. Towards determination of SG mechanisms essential for AML regulation, we performed RNA-seq in G3BP1 knockdown AML cells as well as G3BP1 eCLIP and BioID in AML subsets to uncover an AML-specific SG proteome and transcriptome. These analyses highlighted numerous potential SG mechanisms of cancer cell stress resistance and/or stem cell regulatory processes including regulation of apoptotic and immune signalling pathways. Overall, these results elucidate SGs as key AML LSC stress adaptive regulatory networks and highlights them as novel targets for AML therapeutics.