Enchondromas (ECA) are a cartilage tumor, arising from growth plate chondrocytes that fail to undergo terminal differentiation, present in greater than 3% of the population causing pain, deformity, and can progress to chondrosarcoma. Enchodnromatosis is a condition in which there are multiple ECAs. There are currently no effective drug therapies for this condition. We identified a mutation in PTHR1 that results in constitutive activation of Hedgehog (Hh) signaling in Enchondromatosis, and found that constitutive Hh signaling is a common occurrence in ECAs. We generated a mouse in which constitutive Hh transcriptional activation causes ECAs, and used this to investigate the role of mediators in the Hh signaling cascade in normal and ECA chondrocytes. One mediator we investigated was Suppressor of fused (Sufu), and we unraveled important insight about its role as a negative regulator of Hh signaling, but found that it requires other, yet to be identified, interacting partners in its regulation of Hh signaling. The mutation we identified in enchondromatosis is present in about 5% of cases. Recently, mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), were found in a larger proportion of cases of enchondromatosis. We found that Idh mutations in mouse explant cultures inhibit chondrocyte differentiation and activate Hh signaling upstream of Hh ligand activation. Here we will expand on this work to identify the mechanism by which the mutant Idh regulates chondrocyte differentiation and Hh signaling using a mouse we generated that expresses a mutant Idh1 in chondrocytes and develops ECA. How the mutant Idh and Hh interact is important in developing novel targeted therapies for ECA. Our previous work shows that Sufu interacts with other proteins to regulate Gli signaling. We found that speckle-type Poz protein (Spop), a cullin 3 - based ubiquitin ligase, is expressed in an overlapping manner with Sufu. In our cell couture studies, Spop opposes the action of Sufu in the regulation of Gli transcription factors. We propose to use a Spop conditional knockout mouse generated though the North American Conditional Mouse Mutagenesis project to study its interaction with Sufu and other regulators of chondrocyte differentiation in the growth plate. We identified Hh target genes by ChIP-Seq using antibodies to the Hh activated Gli transcription factors. Using this technique, we identified mediators of cholesterol metabolism as Hh target genes in chondrocytes and in ECAs, and that Hh activation causes intracellular accumulation of cholesterol in chondrocytes. The functional role of these genes in chondrocytes will be studies in the growth plate and in ECAs, using genetically modified mice. Our ultimate goal is to use this information to develop novel therapies to treat ECAs and enchondromatosis. To this end, we also propose to test novel therapeutic approaches based on modulating Hh, Idh, and/or cholesterol.