PROJECT SUMMARY/ABSTRACT CBL is an E3 ubiquitin ligase that plays an essential role in both the positive and negative regulation of tyrosine kinases and the downstream signaling cascades that drive myeloid proliferation and differentiation. Recurrent mutations in CBL have been identified in a number of myeloid malignancies, including juvenile and chronic myelomonocytic leukemia (JMML and CMML, respectively), and analysis of the distribution of the mutations within CBL has revealed that these mutations cluster in the RING domain responsible for its E3 ubiquitin ligase catalytic function. It has been hypothesized that these mutations lead to a loss of negative regulation of signal transduction, while the maintenance of CBL scaffolding function preserves positive regulation of signaling. Dysregulation of receptor tyrosine kinase (RTK) and non-receptor tyrosine kinase (nRTK) signaling by CBL mutations can promote leukemogenesis by disrupting the natural regulation of cell growth and differentiation. While the function of CBL mutations have been studied in the regulation of specific RTKs, nRTKs, and downstream signaling pathways, a comprehensive analysis of CBL function and the consequences of CBL RING domain mutations has not been executed. Most importantly, targeted therapies for CBL-mutated myeloid malignancies have not been developed. Preliminary phospho-proteomic experiments have shown the hyperactivation of Pi3k/Akt/mTor signaling in CBL-mutant cells. Here, I propose a series of experiments to 1) identify dysregulated signaling pathways and key protein-protein interactions in CBL RING domain mutant cells using IP-MS, 2) explore the effect of small molecule inhibition and genetic perturbation of Lyn and the Pi3k/Akt/mTor signaling pathway on cytokine hypersensitivity and hyperactivation of signaling in vitro. The proposed studies will be the first comprehensive analysis of mutant CBL function and will provide insight into therapeutic vulnerabilities in CBL-mutated myeloid malignancies.