Deregulated expression and/or activity of Protein Tyrosine Kinases (PTKs) and their downstream signaling proteins are now widely recognized as oncogenesis-driving mechanisms that are amenable to targeted therapies. Studies by the PI and others have established the Cbl family of ubiquitin ligases (E3s) as physiological negative regulators of PTK signaling. Cbl proteins interact with PTKs and their effectors to promote their ubiquitination resulting in degradation-dependent or degradation-independent negative regulation of PTK signaling. Consistent with the importance of this mechanism in PTK regulation, mutations of Cbl that cluster in regions (linker region and RING finger) essential for E3 activity have now been identified in a subset of patients with myeloproliferative neoplasms and leukemia. Notably, most patients show acquired Uniparental Disomy with mutant Cbl allele replacing the wildtype allele. These findings lead us to our working hypotheses that Cbl is a tumor suppressor and that mutant Cbl proteins attain transforming properties due dominant-negative inhibition of endogenous Cbl proteins together with a gain-of-function trait that is selected for in cancer. Based on biochemical studies in previous funding cycles of this grant and work by others, we hypothesize the following molecular mechanisms for oncogenesis by Cbl mutants: The E3-deficient Cbl mutants produce dominant-negative inhibition of Cbl and related Cbl-b by competing for binding to activated target PTKs. Mutant Cbl proteins also serve as supra- molecular scaffolds to juxtapose the associated hyperactive PTKs with mutant Cbl-bound signaling intermediates to create a gain of function oncogenic cascade. This combination of dominant-negative and GOF mechanisms is hypothesized to allow the mutant Cbl proteins to establish a PTK-dependent oncogenic signaling cascade. In this renewal application, we will leverage our vast expertise and unique resources to test this model using molecular, biochemical, cellular and animal-based analyses. We will use engineer oncogenic Cbl mutant proteins that cannot interact with upstream PTKs through the TKB domain or with other signaling effectors through their C-terminal regions. In vitro and in vivo oncogenesis studies of these engineered proteins in hematopoietic stem/progenitor cells derived from wildtype, Cbl-null, Cbl-b-null and Cbl/Cbl-b-null backgrounds (which are available) will help test the role of key structural domains of mutant Cbl in establishing dominant-negative and gain-of-function mechanisms to drive oncogenesis. The engineered mutants and hematopoietic stem/progenitor cells expressing these, together with cells from an available Cbl RING Finger Knock-In mouse model, will be used to identify PTK and signaling protein partners that are required for mutant Cbl oncogenesis. Multi-pronged overexpression, knock-down/knockout, dominant-negative protein expression and chemical inhibition approaches will validate the role of identified Cbl partners in oncogenesis. Successful accomplishment of the proposed goals will help bring together the conceptual frameworks established during the previous cycles of this grant together with recent advances in the field to elucidate the mechanisms by which Cbl mutations contribute to human cancer and provide a conceptual and experimental framework for novel options towards targeted therapy of patients with Cbl mutations and related malignancies.