PROJECT SUMMARY Acute Myeloid Leukemia (AML) is a lethal blood cancer, with a 5-year survival rate of only 25%. One driver of especially poor prognosis in AML is mutation of Colony Stimulating Factor 3 Receptor (CSF3R). The normal function of CSF3R is to promote the expansion of neutrophil precursors and their differentiation into mature neutrophils. In AML, mutant CSF3R is unable to drive differentiation. We hypothesize that this differentiation arrest is crucial to the aggressive biology of CSF3R-driven AML. In AML, differentiation arrest is often driven by genetic alterations in key hematopoietic transcription factors. Indeed, the vast majority of patients with CSF3R-mutant AML have co-occurring mutations in the transcription factor CEBPA, or translocations of the core binding factor (CBF) complex. These genetic alterations disrupt transcription factor function and perturb the epigenetic landscape of myeloid cells. Our data shows that the combination of mutant CSF3R with either mutant CEBPA or a CBF translocation produces an aggressive, poorly- differentiated myeloid leukemia. Furthermore, we find that mutant CEBPA alters the balance of signaling downstream of CSF3R through STAT proteins, to favor proliferative programs at the expense of pro- differentiation programs. Finally, CBF translocations suppress the expression of CEBPA, suggesting that CEBPA mutation or dysregulation is a common mechanism of differentiation arrest in CSF3R-driven AML. We hypothesize that CEBPA mutations and CBF translocations act through altered STAT signaling and epigenetic dysfunction to disrupt the transcription of differentiation-associated genes. We will test this hypothesis through two specific aims: 1) understand the functional significance of STAT dysregulation in CSF3R- mutant AML, and 2) identify a common mechanism of differentiation blockade in CSF3R-mutant AML. Successful completion of these studies will provide us with a mechanistic understanding of oncogene synergy in this poor prognosis CSF3R-mutant AML subgroup. This will enable the future development of rational therapeutic approaches to prevent disease relapse.