PROJECT SUMMARY Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by the accumulation of abnormal myeloid progenitors arrested in differentiation. It is the most common form of leukemia among adults and the second most common among children. Despite considerable advances in our understanding of this disease, prognosis for AML patients remains dismal, with five-year survival rates languishing at <30% despite the use of intensive chemotherapy. Thus, there is a compelling need to identify novel molecular vulnerabilities of AML to help guide the development of more effective targeted therapies. The RNA helicase DDX6 emerged as a prominent hit in our CRISPR dropout screens aimed at identifying specific AML dependencies. DDX6 represses the translation of target mRNAs by using its ATP-dependent helicase activity to sequester them in P-bodies, cytoplasmic ribonucleoprotein granules. Our preliminary data indicate that AML cells overexpress DDX6 and harbor more P-bodies compared to normal hematopoietic progenitors. We find that loss of DDX6, triggering P- body dissolution, attenuates self-renewal of AML cells and promotes their differentiation in vitro but has little effect on normal hematopoietic progenitors. Congruently, we also find that loss of DDX6 abrogates the expansion of AML cells in vivo. By purifying intact P-bodies from AML cells, we found enrichment of potent tumor suppressors (e.g., KDM5B, TLE4, TRAF6) among the mRNAs sequestered by DDX6 in P-bodies. Together, these data lead us to hypothesize that DDX6 is a specific AML dependency that sustains AML by sequestering and translationally repressing mRNAs encoding tumor suppressors. We will test this hypothesis through two Specific Aims. In Aim 1, we will define the functional requirement for DDX6 in AML using novel mouse models. We expect that Ddx6 loss or mutation of its helicase activity will prevent or delay AML onset in vivo. Conversely, we expect that Ddx6 overexpression will accelerate AML onset in vivo. In Aim 2, we will elucidate the molecular mechanisms by which DDX6 sustains AML. We predict that we will find mRNAs encoding tumor suppressors to be enriched in the P-bodies of AML cells and translationally repressed. We anticipate that loss of DDX6 will de-repress these mRNAs and promote their translation and protein expression. Collectively, these experiments will unveil the RNA helicase DDX6 as a potential therapeutic target in AML by establishing it as a specific AML dependency and deciphering the mechanistic basis for this dependency. This study will also provide the first evidence of an oncogenic role for P-bodies in the context of hematological malignancies and may uncover novel tumor suppressors among DDX6 targets that can potentially be exploited against AML.