Indiana University-Purdue University Indianapolis (IUPUI) / Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.