Gleevec Resistant Activating Mutation of c-Kit (D816V) Demonstrates Ligand Independent Growth and Promiscuous Cooperation with Multiple Cytokine Receptors Via the p85α Subunit of Class IA PI-3Kinase.

Abstract

Gain of function mutations in c-Kit are associated with a number of cancers in humans, including gastrointestinal stromal tumors (GIST), mastocytosis and acute myeloid leukemia (AML). Although, gleevec is a potent inhibitor of c-Kit juxtamembrane mutants that are associated with GISTs, little is known about the drugs that inhibit the activity of c-Kit with mutations in the catalytic domain (i.e. substitution of valine for aspartic acid in codon 816: c-Kit D816V), which are associated with mastocytosis and/or AML. Although, in vitro expression of c-Kit D816V in transformed cell lines results in ligand independent survival due to constitutive phosphorylation, the functional manifestation of this mutation in primary stem/progenitor cells (HSC/Ps) or in mast cells is poorly understood. In addition, little is known about the signaling pathways that contribute to c-Kit D816V induced transformation in HSC/Ps or mast cells. Here, we assessed the role of c-Kit D814V in primary bone marrow derived HSC/Ps and mast cells. Consistent with prior reports, expression of c-Kit D814V in HSC/Ps resulted in constitutive autophosphorylation of the c-Kit receptor. To determine the affect of this mutation on HSC/Ps, we first assessed alteration in the differentiation of HSC/Ps by assessing the cell surface expression of Gr-1, Mac-1 and F4/80. Compared to HSC/Ps transduced with the empty vector, no significant perturbation in the expression of Gr-1, Mac-1 or F4/80 was observed in c-Kit D814V expressing HSC/Ps (n=3, p<0.5). Consistent with the constitutively active nature of c-Kit D814V, a 20 to 40 fold increase in the growth of c-Kit D814V expressing HSC/Ps was observed in the absence of ligand stimulation as assessed by thymidine incorporation and colony forming ability (n=3, p<0.005). The growth of c-Kit D814V expressing HSC/Ps was further augmented 3 to 5 fold in the presence of stem cell factor (SCF), the ligand for c-Kit, compared to controls. Interestingly, stimulation of c-Kit D814V expressing HSC/Ps with additional cytokines, including G-CSF, M-CSF or IL-3, either alone or in combination with SCF further augmented their growth compared to controls. In line with the promiscuous cytokine induced cooperation via c-Kit D814V in HSC/Ps, expression of c-Kit D814V in primary mast cells also resulted in a significant increase in their growth via IL-3, IL-4 and IL-10; three cytokines known to play an essential role in mast cell growth and differentiation. We and others have shown that wildtype c-Kit stimulation by SCF results in the activation of the PI-3Kinase pathway via the recruitment of p85α regulatory subunit of class IA PI-3Kinase to the receptor. Since p85α is constitutively bound to c-Kit D814V, we hypothesized that p85a may contribute to c-Kit D814V induced hyperactivation and cytokine induced promiscuous growth in HSC/Ps and mast cells. To test this, we expressed c-Kit D814V in p85α-/- HSC/Ps and mast cells. Remarkably, deficiency of p85α in the setting of c-Kit D814V rescued ligand independent growth mediated via c-Kit D814V to near wildtype levels (n=3). More importantly, the promiscuous and enhanced growth in combination with other cytokines seen in c-Kit D814V expressing HSC/Ps and mast cells was corrected to wildtype levels. Taken together, our results identify p85α regulatory subunit of class IA PI-3Kinase as a novel therapeutic target for the treatment of mastocytosis and AML involving the c-Kit D814V.