PROJECT SUMMARY CBL mutations are common in chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), myelodysplastic syndrome, and clonal hematopoiesis of indeterminate potential (CHIP). CBL is an E3 ubiquitin ligase that recognizes phosphorylated receptor tyrosine kinases (RTKs) and mediates both downstream signaling and destruction of activated RTKs that are critical for leukemogenesis, including KIT, FLT3 and CSF2RB. (1, 2) There are no therapies that specifically address CBL-mutant leukemia, an ideal target for genotype-directed therapy. CBL is a central node for kinase signaling, and unlike most common mutations in leukemia, CBL mutations result in a gain-of-function, activating specific signaling pathways. CBL-mediated ubiquitination of activated tyrosine kinases triggers their proteasomal degradation, a negative feedback for ongoing signaling. In leukemia, CBL mutations specifically abolish the ubiquitin ligase activity, leaving CBL- mediated downstream signaling unchecked, resulting in prolonged kinase signaling and increased cellular proliferation. The central goal of this proposal is to understand the mechanistic basis of CBL mutations in order to identify the set of proteins and pathways that are dysregulated and can be targeted for the treatment of CBL- mutant leukemia. In Aim 1, we will determine the specific phosphorylated proteins that are degraded by CBL in leukemia cells and stabilized when CBL is mutated, leading to constitutive signaling. Mechanistically, we will examine how specific point mutations in CBL alter the binding and ubiquitination of substrates, and the initiation of downstream signaling. In Aim 2, we will investigate the combinations of kinase inhibitors with greatest therapeutic potential using both in vitro and in vivo models. Preliminary studies highlight the aberrant activation of LYN kinase and PI3K/AKT signaling pathways in cells with CBL RING domain mutations. We hypothesize that inhibiting these signaling pathways, and other kinases identified in Aim 1, will selectively target CBL-mutant cells and may have therapeutic activity in CBL-mutant myeloid malignancies. We will develop and characterize in vivo models of CMML with Cbl mutations, and test the efficacy of FDA approved drugs, dasatinib and dactolisib, alone and in combination. In Aim 3, we will investigate the biology of the CRL5CISH ubiquitin ligase. Preliminary studies identified CRL5CISH ubiquitin ligase as a potential reserve for CBL, regulating an overlapping set of signaling pathways, but also degrading key signaling molecules that are not degraded by CBL. We will examine the biology of this CRL5CISH ubiquitin ligase in CBL wild-type and mutant cells. Manipulation of the activity of this ubiquitin ligase could alter ubiquitination of substrates that are aberrantly regulated in CBL-mutant leukemias. Overall, this project will elucidate the biology and therapeutic vulnerabilities in CBL-mutant leukemia. The project will leverage the ex...