Abstract 856 Hematology-Oncology II Poster Symposium, Sunday, 5/2

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, is a GTPase activating protein (GAP) that negatively regulates the output of p21 ras (Ras) proteins. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML), and heterozygous mice containing a disruption of the homologue of NF1 (Nf1) spontaneously develop a myeloid disorder that resembles JMML. Homozygous Nf1 -/- embryos die in utero; however, Nf1 -/- fetal liver cells and JMML cells have a similar hypersensitive pattern of CFU-GM growth in response to low concentrations of GM-CSF. Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. Nf1 contains a GAP-related domain (GRD) that binds to Ras and stimulates GTP hydrolysis. We tested whether restoration of GRD sequences in trans would restore normal sensitivity of Nf1 -/- CFU-GM to GM-CSF. Recombinant retroviral constructs encoding Nf1 GRD sequences and the puromycin (pac) resistance gene (vMSCV-GRD-pac) or the pac resistance gene alone (vMSCV-pac) were developed. Fetal liver cells from individual Nf1 +/+ and Nf1 -/- d13.5 gestation embryos were transduced with either vMSCV-GRD-pac or vMSCV-pac supernatant and cultured in semisolid media containing puromycin to select for colony formation from transduced CFU-GM. Nf1 -/- CFU-GM transduced with the reporter gene only were hypersensitive to GM-CSF analogous to untransduced Nf1 -/- CFU-GM. However, Nf1 -/- cells transduced with vMSCV-GRD-pac had identical growth responsiveness to GM-CSF as Nf1 +/+ cells transduced with vMSCV-pac. To ensure that these results were not simply a result of overexpressing GAP domains nonspecifically, experiments were conducted in which the GAP domain of the related protein p120GAP was transduced into Nf1 -/- fetal liver cells using the recombinant retrovirus (vMSCV-p120 GAP-pac). Transduction of vMSCV-p120GAP-pac into Nf1 -/- fetal liver cells did not restore normal GM-CSF dose responsiveness to CFU-GM suggesting that there is a specific requirement for Nf1 GRD to down regulate GM-CSF induced Ras activation. Preliminary results indicate a similar correction in proliferation of Nf1 -/- murine embryonic fibroblasts (MEFs) following transduction with vMSCV-GRD-pac, but not vMSCV-pac. These cellular data were associated with decreased MAPK activation in Nf1 -/- MEFs transduced with Nf1 GRD sequences. These data indicate that Nf1 GRD are necessary and sufficient for restoration of normal in vitro growth of hematopoietic progenitors and murine embryonic fibroblasts.