The PI3K Catalytic Subunits, p110a and p110d, Serve Redundant Functions in Activating PTPN11-Induced Hematopoietic Progenitor Hypersensitivity to GM-CSF.

Charles B Goodwin1, Rebecca J. Chan1,2

Departments of Medical and Molecular Genetics1 and of Pediatrics2, Indiana University School of Medicine, Indianapolis, Indiana

Noonan syndrome (NS) is a common (1 in 1500 to 2500 live births) autosomal-dominant disorder caused by somatic gain-of-function mutations in PTPN11 in approximately 40 – 50% of patients.  PTPN11 encodes Shp2, a non-receptor protein tyrosine phosphatase, that has been demonstrated repeatedly to play a positive role in growth factor signaling to Ras in a phosphatase-dependent manner.  Although the anomalies observed in NS are diverse, several of the complications can be attributed to the increased function or number of macrophages or monocyte-derived cells includingtransient and remitting myeloproliferative disorder and, rarely, development offull-blown juvenile myelomonocytic leukemia (JMML).  JMML is characterized clinically by overproduction of myelomonocytic cells and by the in vitrophenotype of hematopoietic progenitor hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF).We have previously demonstrated that Phosphoinositol-3-Kinase (PI3K) signaling is hyperactivated in the presence of gain-of-function mutant Shp2 and that it contributes to activating PTPN11-induced GM-CSF hypersensitivity, which can be reduced pharmacologically with the pan-PI3K inhibitor, LY294002.  To study the contribution of Class IA PI3K, we genetically disrupted Pik3r1, which encodes regulatory subunits p85α, p55α, and p50α, and found significant, but incomplete, reduction of GM-CSF-stimulated hyperproliferation in gain-of-function Shp2 E76K-expressing cells.  The relative contribution of the different catalytic subunits to mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity is not known, though we found that in the absence of the Pik3r1-encoded regulatory subunits, p110d expression is significantly higher in cells expressing gain-of-function mutant Shp2 E76K compared to WT Shp2.  To investigate this question further, we measured GM-CSF-stimulated proliferation of WT Shp2- and Shp2 E76K-transduced hematopoietic progenitors in the presence and absence ofGDC-0941, a PI3K inhibitor with high specificity for p110a and p110d, and found a dose-dependent decrease in GM-CSF-stimulated proliferation.  To distinguish the role of p110aand p110din Shp2 E76K-induced hyperproliferation, we performed genetic studies using mice bearing a conditional knockout of Pik3ca(encoding p110a) and a kinase-dead knock-in of Pik3cd (p110d D910A), respectively.  Following GM-CSF stimulation, we found that genetic disruption of p110a or expression of kinase dead p110d D910A partially normalized Shp2 E76K-mediated Akt hyper-phosphorylation; however, GM-CSF-stimulated proliferation was unchanged.  These findings indicate that p110a and p110d functionally perform redundant tasks in Shp2 E76K-expressing cells.  We are currently crossing these genetically modified animals to further define the functional contribution of p110a and p110d to mutant Shp2-induced Akthyperactivation and hypersensitivity to GM-CSF.

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