Project Summary/Abstract Isocitrate dehydrogenase 1 and 2 (IDH1/2) alterations are gain-of-function mutations that drive the synthesis of the oncometabolite 2-hydroxyglutarate (2HG) which induces chromatin abnormalities, including globally altered histone and DNA methylation profiles in MDS (and AML) cells. However, IDH1 and IDH2 mutations are not by themselves sufficient to cause MDS. In addition, IDH1 and IDH2 mutations are detected in expanded clones in aging healthy humans, a phenomenon termed “clonal hematopoiesis” that confers increased risk for MDS and AML development. These observations suggest a) that IDH1/2 mutations require additional cooperative signals to become fully transforming; and b) that such signals may accumulate with aging and potentiate the transformation to MDS (or AML). We and others have demonstrated that the bone marrow stroma is a critical regulator of the development of hematological myeloid malignancies, actively promoting their development or progression. In investigating this possibility in IDH1/2-mutant MDS/AML, we found that 2HG is elevated in the bone marrow plasma of MDS patients carrying IDH mutations as compared to MDS patients without such mutations. Methylome analysis of bone marrow stromal cells from a cohort of 4 MDS/AML patients with and 4 without IDH mutations showed differential hypermethylation in the stroma of IDH1 and IDH2-mutant patients as compared to IDH1 and IDH2 wild type patients. Integrative analysis of these results with RNAseq data from the same subjects and preliminary in vivo analysis suggests stromal cell candidate genes that may affect IDH1/2- related transformations. We hypothesize that IDH1 and IDH2 mutations in hematopoietic cells cooperate with signals from the bone marrow niche that transform IDH-harboring HSCs to dysplastic cells and promote MDS development. Such malignant signals from the marrow stroma may contribute to the clonal hematopoiesis with aging and the transformation of pre-existing mutant IDH1/2 clones to dysplastic ones. We will examine whether epigenetically modified stroma interacts with IDH1/2 mutant HSCs to promote their leukemic potential in MDS/AML; and correlate with response to treatment; determine whether epigenetic modifications in the aging bone marrow stroma support transformation of IDH1/2 mutated hematopoietic cells to MDS/AML-initiating events; and, expand the methylome analysis of the stroma to allow for integrative analyses with expression and promoter binding profiling that may uncover new stromal components of IDH1/2 transformation. These studies may identify stroma-derived signals that may be targeted therapeutically in combination with current IDH1/2 inhibitors to improve treatment of IDH1/2 mutant MDS patients or, in the future, to prevent MDS development.