Role of p21 Activated Kinase and Specific Guanine Exchange Factors of Rac Gtpases in Oncogenic KIT Induced Systemic Mastocytosis and Acute Myeloid Leukemia
Authors: Martin Holly , Ma Peilin , Chatterjee Anindya , Ramdas Baskar , Sims Emily , Vlaar Cornelis , Dharmawardhane Suraganie , Kapur Reuben 
Published: October 2019

Abstract
Abstract 810
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 KIT involving an aspartic acid to valine substitution is found in ∼90% of patients with systemic mastocytosis (SM) and in ∼40% of core binding factor acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. In mice, presence of this mutation is sufficient to recapitulate many cardinal features of human SM. This mutation changes the conformation of KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth, which is resistant to imatinib and shows little therapeutic efficacy in response to Dasatinib in most SM patients. As there are currently no efficacious therapeutic agents against this mutation, we sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells (HSC/Ps) in diseases such as AML and SM. Previously, we and others have demonstrated that the regulatory subunit of PI3K, p85α, is required for KITD814V (murine homolog) induced transformation. Although difficult to target, we hypothesized that perhaps the downstream effectors of the PI3K signaling pathway, in particular p21 activated kinase (PAK1) and its upstream effectors including guanine exchange factors (GEF) such as Tiam1, Trio and Vav as well as the Rho family of GTPases (Rac) contribute to gain-of-function mutant-mediated transformation. We show that KITD814V (mouse) and KITD816V (human) bearing leukemic cells exhibit constitutive activation of PAK, Rac GTPases, and GEF Vav. Importantly, treatment of KITD814V bearing murine cells or mastocytosis patient derived cells bearing the KITD816V mutation with an allosteric inhibitor of PAK1 (i.e. IPA-3) results in significant inhibition in growth due to enhanced apoptosis. Consistently, expression of a dominant negative form of PAK1 (K299R) in KITD814V bearing cells profoundly inhibited their growth but not the growth of normal cells. Upstream of PAK, we show that suppression of Rac GTPases by expression of a dominant negative form of Rac (RacN17) abrogates activating KIT-induced hyperproliferation, and activity of downstream effector, PAK1. Although both Rac1 and Rac2 are activated due to the presence of KITD814V in primary HSC/Ps; loss of Rac1 only modestly corrects the growth of KITD814V bearing cells and loss of Rac2 contributes to only 50% correction. In contrast, loss of both Rac1 and Rac2 in HSC/Ps resulted in 75% correction in KITD814V induced ligand independent growth in vitro. In vivo, Rac repression significantly delayed the onset of KITD814V induced myeloproliferative neoplasms (MPN). Although, all KITD814V bearing mice died around 20 days of transplantation due to splenomegaly, increased white cell counts and massive lung infiltration by leukemic cells; KITD814V bearing mice in which Rac was repressed showed prolonged survival, significantly reducted spleen size, white cell counts and myeloid cell infiltration in the lungs. Prior studies have shown that Rac GTPases can be activated by GEFs such as Tiam1, Trio and Vav. To assess the specific role of these GEFs in KITD814V induced transformation, we utilized small molecule inhibitors that uniquely target different GEFs. We synthesized and utilized a novel inhibitor of Rac, EHop-016, which is based on the structure of an existing GEF inhibitor, NSC23766. While NSC23766 targets Tiam1 and Trio, EHop-016 targets Vav. The IC50 of EHop-016 is ∼50 fold lower than that of NSC23766. Using these two drugs, we demonstrate that EHop-016 is 50-fold more potent in inhibiting the growth of both murine and human patient derived leukemic cells compared to NSC23766. These observations were confirmed utilizing mice and bone marrow cells deficient in the expression of Vav1 engineered to express the KITD814V mutation. Taken together, a series of experiments using knockout mouse models, mouse models of MPN, dominant negative approaches, and a novel allosteric inhibitor of PAK1 and a novel small molecule inhibitor of GEF Vav provide a mechanism of KITD816V induced transformation and provide potential novel therapeutic targets for treating oncogenic KIT bearing neoplasms.

Disclosures:
No relevant conflicts of interest to declare.