PROJECT SUMMARY/ABSTRACT It is well established that JAK/STAT signaling is dysregulated in most myeloid malignancies. Identification of JAK-activating mutations in myeloproliferative neoplasms (MPNs) prompted the development of inhibitors that target JAK for use in the clinical setting. While these agents provide a symptomatic response, they do not alter the underlying disease in that they do not target MPN-initiating malignant hematopoietic stem/progenitor cells (HSPCs). This observation has been interpreted as indicating that alternative signaling pathways contribute to the pathogenesis of MPNs. The present proposal is based on the premise that identification of hitherto unknown pathogenic mechanisms will be required to effectively treat these neoplasms and eradicate disease initiating MPN stem cells. Our long-term goal is to define signal transduction events responsible for systemic maintenance of HSPCs, with particular focus on key adapter proteins that modulate signals regulating HSPCs self-renewal, differentiation and their malignant transformation. Our direct objective is to elucidate the role of one such adapter protein, Abelson interactor-1 (Abi-1), in signaling cascades that define the HSPC phenotype. Our recent findings indicate that: (1) conditional deletion of the Abi1 gene in mouse bone marrow induces a myelofibrotic phenotype; (2) hematopoietic progenitors and granulocytes from patients with the most severe of MPNs - primary myelofibrosis (PMF) show decreased Abi-1 transcript and protein levels, and; (3) loss of Abi-1 positively affects the activity of Src Family Kinases (SFKs) and their downstream signaling to STAT3 and NF- κB, resulting in impairment of HSPC self-renewal and fitness. Our central hypothesis is that Abi-1 plays an important and potentially targetable role in HSPC self-renewal and differentiation via negative regulation of the SFK/STAT3/NF-κB inflammatory module, and that its acquired absence can promote malignant transformation and progression to PMF. Our central hypothesis will be tested in the following three Specific Aims. In Specific Aim 1 we will use our new hematopoietic cell-specific Abi-1 knockout mouse model to determine the effect of Abi-1 loss on HSPC signaling and self-renewal, differentiation and malignant transformation. In Specific Aim 2, we will use advanced proteomics combined with proximity-dependent labelling technology to define the Abi-1 interactome in HSPCs. This approach will be complemented by phosphoproteomic analyses of Abi-1-deficient HSPCs and human PMF stem/progenitor cells. In Specific Aim 3 we will use our bone marrow stroma-specific Abi-1 knockout mouse model to assess the effect of deletion of Abi-1 in the stromal component on the self-renewal, differentiation and malignant transformation of HSPCs. Completion of the proposed aims will advance our understanding of the mechanisms by which dysregulation of Abi-1/SFK/STAT3/NF-kB signaling contributes to the neoplasm-inducing capacities ...