PROJECT SUMMARY Germline cancer predispositions are well established for solid tumors. Once thought to be a rare event, predispositions to myeloid malignancies are now included in the latest leukemia classification scheme from the World Health Organization. Thus, more patients are expected to be recognized as carriers which can create a constant fear of developing leukemia in their families. Given the best treatment is prevention, understanding the important factors in transitioning from pre-malignant to overt disease stage is fundamental in establishing guidelines for monitoring and treatment of such patients. Of many leukemia predisposition syndromes, we focus on familial platelet disorders (FPD) caused by monoallelic RUNX1 germline mutations in. RUNX1-mutations in FPD lead to bleeding disorders, lower counts and dysfunction of platelets. FPD is one of the most common forms of inherited myeloid malignancies, and patients have ~40% life-long chance of developing leukemia at median age of 33 years. The transition to overt leukemia is accompanied by acquisition of secondary mutations, though the complete mechanism of disease transformationis unknown. Intriguingly, FPD patients manifest with different allergies such as skin eczema, indicating the possible role of increased inflammation in driving leukemia development. Therefore, we propose that inflammatory stress facilitates leukemia initiation and progression in FPD. To test this, we performed series of experiments using primary FPD bone marrow cells. We observed that FPD hematopoietic stem and progenitors (HSPCs) show myeloid biased differentiation and increased colony formation ability, while fall behind in megakaryocyte differentiation. Single cell transcriptome analysis of healthy and FPD bone marrow cells confirmed the skewed differentiation of progenitors and identified enrichment of inflammatory response pathways in FPD compared to healthy stem cells. Consistently, we found upregulation of inflammatory cytokines in the bone marrow niche, including from mesenchymal stromal cells. Therefore, I hypothesize that a cross-talk between RUNX1-mutations mediated changes in HSPCs and inflammatory microenvironment promotes myeloid growth and differentiation defects in FPD stem and progenitor cells. To address this hypothesis, in Aim 1, I will evaluate the impact of inflammatory stress on growth and differentiation of FPD and healthy HSPCs. I will use the genetic or pharmacological inhibition approaches to dissect the mechanism by which inflammatory stress impacts the functions of FPD HSPCs. Then, I will identify whether these effects are directly regulated by RUNX1 using CUT&Tag experiments. In Aim 2, I will determine the effect of RUNX1-mutated bone marrow stromal cells on the function of FPD and healthy HSPCs using newly established 3D co-culture methods. Then, I will use a unique mouse model with germline Runx1-hetrozygous mutation to determine the role of bone marrow niche on stem cell growth and...