Abstract More than 3% of the population develops an enchondroma (ECA), a benign tumor in bone composed of cells derived from the growth plate that can cause pain, deformity, and can be responsible for pathologic fractures. Enchondromas can progress to malignant chondrosarcoma (CSA). Mutations in genes encoding isocitrate dehydrogenase (IDH1 and 2) were identified in a large proportion of ECAs and CSAs. In our prior work, we found that IDH mutations inhibit growth plate chondrocyte differentiation, and chondrocyte-specific conditional Idh1 mutant mice develop ECAs. Mutant IDH uniquely produces the metabolite 2-hydroxyglutarate (2-HG), but we and others found that blocking the production of 2-HG pharmacologically does not alter CSA cell viability. While 2-HG has epigenetic effects that are likely important in tumor initiation, tumor maintenance must rely on other factors. Since IDH plays an important role in in metabolism, associated metabolic changes could drive the observed phenotype. We found high levels of glycogen in cells expressing a mutant IDH. Glycogen is also found in proliferating and pre-hypertrophic cells of the growth plate. In our previous work, we found that intracellular cholesterol biosynthesis was activated in IDH mutant chondrocytes and that it is also regulated in the growth plate, and its activity corelates with glycogen levels. This raises the possibility that intracellular cholesterol biosynthesis, which is activated by Sterol regulatory-element binding proteins (SREBP) transcription, also regulates glycogen. Our premise is that glycogen is an important energy source for pre- hypertrophic and hypertrophic growth plate chondrocytes and that glycogen stores are required to maintain the neoplastic phenotype in ECA and CSA. We also propose that glycogen depletion can suppress the neoplastic phenotype. In this proposal we will study what regulates glycogen in the growth plate, ECA and CSA, and determine the function of glycogen in these growth plate and neoplastic chondrocytes. To determine what regulates glycogen in the growth plate, ECA, and CSA, we prioritized genes known to regulate glycogen that were differentially regulated in the growth plate and by IDH mutations. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is one such gene which is differentially and interestingly, contains SREBP binding sites in its promoter region. Our preliminary data suggest that SREBP regulates PPP1R3C which then regulates glycogen. Our studies will use cell lines from human tumors and genetically modified mice that develop enchondromas to define the function of glycogen and PPP1R3C in the growth plate, ECA, and CSA. In addition, we will study how SREBP regulates PPP1R3C and glycogen. Glycogen synthase will be deleted genetically, or we will cells with drugs that inhibit glycogen synthesis and breakdown. This data will provide pre-clinical information on which to base novel therapies for ECA and CSA.