PROJECT SUMMARY/ABSTRACT The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem cell expansion and increased risk of transformation to frank leukemia. The hematopoietic stem cell compartment in MPN is heterogeneous with the presence of both wild-type and JAK2V617F mutant cells in most patients with MPNs. Despite mutant cells bearing an in vitro proliferative advantage because of constitutive kinase activity, in some patients, there is little or no change in the mutant/wild-type cell ratio over long term follow up; while in others, MPN can evolve to acute leukemia and patients experience high relapse rates following allogeneic stem cell transplantation, the only curative treatment for these patients. JAK2V617F is also one of the common mutations associated with clonal hematopoiesis of indeterminate potential and most individuals with such clonal hematopoiesis do not convert to advanced disease. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we demonstrated that the presence of wild-type cells can prevent the expansion of co- existing JAK2V617F mutant cells in a normal microenvironment. Delta-like ligand 1 (Dlk1), a noncanonical Notch ligand important in stem cell maintenance, was significantly inhibited in JAK2V617F mutant hematopoietic stem/progenitor cells with competition compared to mutant cells without competition. We also found that a mutant microenvironment can promote mutant cell expansion over wild-type cells. CXCL12, a chemokine critical for both hematopoietic stem cell maintenance and immune suppression, was up-regulated in the mutant microenvironment. Critically, both upregulated Dlk1 expression and dysregulated CXCL12 signaling have been reported in patients with MPNs. Based on these observations, we hypothesize that, in the hematopoietic system, competition between normal and neoplastic stem cells provides an essential mechanism to protect against cancer development. The objective of the proposed work is to determine whether normal cells could potentially be used as a therapeutic approach to control mutant clone expansion and the evolution of MPN to leukemia, in combination with approaches targeting the MPN tumor microenvironment. In particular, we propose the following two specific aims: 1) To elucidate the mechanisms by which wild-type cells prevent the expansion of JAK2V617F mutant cells in a normal hematopoietic microenvironment. The roles of Dlk1 deregulation in cell competition- induced JAK2V617F mutant stem cell suppression will be determined. 2) To study how the tumor immune microenvironment alters the competition between wild-type and JAK2V617F mutant cells. The roles of tumor- specific T cells and CXCL12 signaling in tumor microenvironment-induced mutant clonal expansion will be defined. We expect these studies will expand our understanding of the molecular mechanisms controlling the competitive interactions between normal and neoplastic cells, and how these mechanisms break down during cancer progression and relapse.

PROJECT NARRATIVE Human tissues show high levels of genetic mutations, yet such mutant tissues rarely develop frank tumors. The goal of this research proposal is to understand the molecular mechanisms controlling the competitive interactions between normal and mutant cells, and how these mechanisms break down during cancer progression. While accumulation of mutations is unavoidable during aging, results from this study will have the potential to identify new strategies to control neoplastic cell expansion and prevent the development of a full-blown malignancy.