Role of Intracellular Tyrosine Residues in Oncogenic KIT- Induced Transformamtion.

Abstract 1435

Poster Board I-458

Gain-of-function mutations of the receptor tyrosine kinase KIT have been associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myelogenous leukemia (AML). A mutation of aspartic acid to valine (KITD814V) in the activation loop of KIT results in altered substrate recognition and constitutive tyrosine autophosphorylation. However, the intracellular mechanisms that contribute to promiscuous signaling via this mutation are poorly understood. We have previously shown that KITD814V is sufficient to induce ligand independent growth in primary hematopoietic stem and progenitor cells (HSC/P) in vitro as well as transformation in vivo. However, it is not known whether ligand induced (i.e. KITL) signals in vivo contribute to transformation. To assess this, we generated a chimeric receptor (CHR) in which the extracellular domain of KIT was replaced with the human-macrophage colony stimulating factor receptor (h-MCSFR) to inhibit endogenous binding of murine SCF. In vitro thymidine incorporation assay confirmed that the WTCHR is only functionally responsive to human (h)-MCSF stimulation, but not to murine (m)SCF nor murine MCSF stimulation, and that CHRD814V receptor maintains ligand independent growth potential similar to the KITD814V receptor. To determine if the myeloproliferative disease (MPD) could occur in the absence of endogenous ligand binding, a murine transplantation model was utilized to compare CHRD814V and KITD814V-induced transformation. In vivo results demonstrated no significant difference in the onset of MPD in mice bearing KITD814V vs. CHRD814V (median survival 52 days vs. 49 days, n=10 to 20 mice), with similar disease manifestation in the two groups including splenomegaly, hepatomegaly, myeloid cell infiltration and elevated white blood cell counts (n=10 to 20). Transforming potential in the absence of KIT extracellular domain strongly suggested that self-association of KITD814V was sufficient to induce transformation rather than influences from endogenous ligand. To further assess the relative contribution of intracellular tyrosine residues in transformation, a CHRD814V mutant was generated in which seven critical tyrosine residues including the binding sites for Src family kinases, Grb2, p85α regulatory subunit of class IA PI3Kinase, PLC-g, Ras-GAP, and Grb7 were mutated to phenylalanines (i.e.CHRD814V-F7). Our results show that CHRD814V-F7 bearing primary bone marrow cells lost ligand independent growth potential in vitro, suggesting that seven tyrosine induced signaling may play a critical role in KITD814V-induced ligand independent transformation. Consistently, transplantation studies demonstrated that mice bearing bone marrow cells expressing CHRD814V-F7 exhibited a notable delay in MPD development (median survival= 95 days, n=4 to 8). These results suggest that intracellular tyrosines are crucial for ligand independent proliferation and transformation. Next, we individually restored each of the seven tyrosine residues back into CHRD814V-F7 to determine the importance of pathways essential for ligand independent growth and transformation. We found that among the single tyrosine add back receptors, only restoring p85α subunit binding site (Y719) alone was sufficient to induce promiscuous cellular growth, proliferation, and survival. Transplantation studies revealed that restoration of Y719 was sufficient to induce transformation in vivo (median survival= 55 days, n=5). Furthermore, restoration of other individual tyrosine residues demonstrated a notable delay in CHRD814V-induced transformation in vivo (median survival= 83∼120 days, n=5 to 12) and a minimal contribution to ligand independent growth in vitro. Taken together, our results demonstrate: 1) KITD814V induced transformation in vivo occurs in the absence of ligand stimulation; 2) intracellular tyrosine residues play an essential role in KITD814V induced transformation in vitro and in vivo and; 3) of all the critical tyrosine residues present in KIT, presence of tyrosine at position 719 is sufficient to completely restore ligand independent growth in vitro and transformation in vivo.