PI3 Kinase, Phospholipase C (PLC)-γ, and RasGRPs Act Cooperatively to Activate the Ras-Extracellular-Related Kinase (ERK) Pathway in Response to Cytokines in Normal and Kras Mutant Myeloid Cells.

Abstract 2512

Poster Board II-489

Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells.