Project Summary/Abstract Chronic Myelomonocytic Leukemia (CMML) is a lethal subtype of leukemia characterized by cytopenias, marrow dysplasia, monocytosis, and a propensity for transformation to acute myeloid leukemia (AML). Until this R37 parent grant, no mouse model of CMML, to include genetically engineered mice harboring CMML mutations, had recapitulated the unique clinical or histological characteristics of the human disease. We have overcome this limitation and generated phenotypically and genetically accurate PDX models of 92 CMML patients through the use of modified NOD/SCID IL2R null (NSG) mouse strains expressing human cytokines that uniquely drive CMML. Moreover, we have utilized these PDX models to identify novel therapies targeting aberrant cytokine- signaling characteristics and mRNA splicing in specific genetic subsets of CMML. In this R37 extension we propose to leverage our CMML PDX model to study a previously unrecognized observation we have made that approximately 20% of CMML patients harbor a very small concomitant mast cell clone. Because these cases are clinically and genetically unique, we hypothesize that there may be an ongoing but unknown biologic cooperativity in these patients. However, to study this accurately, new in vivo systems are needed to model this clone-to-clone communication. We aim to achieve this with our CMML PDX models in two specific aims: In Aim 1 we will generate the first hybrid CMML PDX in which mast cells will be additionally heterotopically xenografted with the goal of modeling clone-to-clone communication. Because our preliminary data suggest that subclonal mast cells may be derived from the same ancestral stem cell as CMML, we will also retrospectively determine if CMML and de novo mast cells were previously xenografted in CMML PDX models already generated from patients with occult subclonal mast cells. Last, we will use these models to identify secreted factors and annotate the clonal architecture of these patients at single cell resolution. In Aim 2 we will determine if SF3B1 mutated clones have distinct biologic cooperativity and secreted factors. Our preliminary clinical data suggests that SF3B1 mutations are enriched in patients with subclonal mast cells. We hypothesize that this is due to a unique genotype specific cooperativity. We will evaluate this by xenografting isogenic leukemic cells with and without SF3B1 mutation in the context of a concomitant mast cell tumor. We will also compare CMML PDX models with and without concomitant mast cell tumors in the context of SF3B1 mutation generated in aim 1. Last, we will compare the secreted factors and clonal trajectories identified in Aim 1 in SF3B1 mutant versus wild type CMML PDX. Collectively, this extension will build on the parent grant by using our CMML PDX model to explore novel biology that could translate to a better understanding of the disease and novel therapeutic strategies.