Abstract B43: Requirement for PI3 kinase interaction in K-RasG12D-driven leukemogenesis

Background: Somatic RAS mutations occur in approximately 30% of human cancers. Oncogenic Ras proteins activate multiple downstream effectors including Raf, PI3 kinase (PI3K), and Ral-GDS. The tyrosine 64 residue, which is located in the Switch II effector binding domain of Ras, is an important contact for binding PI3K catalytic subunits. To investigate the role of PI3K binding in hematopoeitic transformation in vivo, we created a “second site” Lox-STOP-Lox (LSL- KrasG12D/Y64G) knock in allele. Methods: LSL-KrasG12D and LSL-KRasG12D/Y64G mice on a 129/Sv background were crossed with C57BL/6 Mx1-Cre mice. These mice were injected with polyIpolyC (pIpC) at 21 days of age to excise the LSL casette, which was confirmed by QPCR. Colony growth potential of primary bone marrow cells was assessed by sacrificing six to ten week old Mx1-Cre;LSL-KrasG12D, Mx1-Cre;LSL-KrasG12D/Y64G, and wild type mice, isolating bone marrow, and plating 50,000 cells in methylcellulose media containing 0, 0.01, 0.1, 1, or 10 ng/ml GM-CSF. Activation of Ras effector pathways was assessed in macrophages derived from primary bone marrow cells harvested from 6-10 week old mice and grown in the presence of M-CSF. Results: As previously described, inducing oncogenic KrasG12D expression in the hematopoietic compartment of Mx1-Cre;KrasG12D mice results in death by three months of age from an aggressive myeloproliferative neoplasm (MPN) that accurately models chronic and juvenile myelomonocytic leukemias. MPN development is greatly attenuated in Mx1-Cre;LSL-KrasG12D/Y64G mice, which develop anemia beginning at six months of age, but do not display elevated blood leukocyte counts and have >90% survival at eight months. Bone marrow cells from Mx1-Cre;LSL-KrasG12D mice form myeloid progenitor colonies in methylcellulose without added cytokines and show profound hypersensitivity to GM-CSF. By contrast, G12D/Y64G doubly mutant progenitors require GM-CSF for colony formation, and display modestly elevated cytokine sensitivity compared to wild type progenitor cells. Whereas cultured Mx1-Cre;LSL-KrasG12D and Mx1-Cre;LSL-KrasG12D/Y64G macrophages contain markedly elevated levels of active Ras-GTP, they exhibit different patterns of effector pathway activation. In particular, basal levels of phosphorylated ERK and Akt (pERK and pAkt) are unexpectedly higher in Mx1-Cre;LSL-KRasG12D/Y64G macrophages, implicating the PI3K pathway in feedback responses to oncogenic Ras output. Conclusions: Our initial data su