Colony Stimulating Factor 3 Receptor (CSF3R) is activated by CSF3 (aka GCSF), the cytokine that promotes neutrophil proliferation and differentiation. We have recently identified CSF3R mutations in 59% of patients with chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). The most common of these mutations, T618I, promotes ligand-independent receptor dimerization through loss of an O-linked glycosylation site. This mutation also highly activates the Janus Activated Kinase (JAK) signaling pathway and JAK kinase inhibitors were effective in mouse models and a patient with this mutation. In Aim 1 we will investigate the regulation of normal CSF3R receptor function by O-linked glycosylation. Oncogenic point mutations in SETBP1 occur in combination with CSF3R mutations, and confer poor prognosis in these diseases. In Aim 2 we will sequence patient samples and our CSF3R T618I mouse model to identify novel genetic drivers that contribute to leukemogenesis and also investigate the order of acquisition of CSF3R and SETBP1 mutations by single cell RNA sequencing. In Aim 3 we will determine the functional consequences of having both CSF3R and SETBP1 mutations for cancer progression and kinase inhibitor sensitivity. These Aims will provide fundamental insight into the regulation of normal CSF3R function, the molecular evolution of CNL/aCML, and the prognostic consequence of having multiple genetic drivers expressed. My goal is to become a successful independent investigator and a leader in the field of molecular characterization of hematologic malignancies and identification of novel therapeutic targets. During the mentored phase I will continue to receive excellent mentorship from Dr. Brian Druker, a pioneer of targeted cancer therapy and leader of the Knight Cancer Institute. I will undertake additional training in the fiel of protein-glycosylation, with guidance from my advisory committee member, Dr. Carolyn Bertozzi at UC Berkeley, and also in single cell analysis methods, guided by Dr. Jerald Radich at the Fred Hutchinson Cancer Research Center. Working with Dr. Bertozzi and Dr. Radich will allow me to expand my training outside of my institution. The proposed research will also be enhanced by guidance from Dr. Jeffrey Tyner, an expert in cancer cell signaling and mouse models, and Dr. Shannon McWeeney who will guide me in bioinformatics analysis of sequencing data. Additionally, Dr. Druker and my advisory team will assist me in navigating the transition to an independent faculty position.