Mutations that deregulate Ras signaling are prevalent in hematopoietic malignancies. Previous work has shown that expressing oncogenic Kras ^G12D in the hematopoietic compartment of mice leads to growth factor-independent and a hypersensitive pattern of myeloid progenitor colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and a fatal myeloproliferative disorder (MPD). This mirrors human myelomonocytic leukemias that are associated with Ras pathway mutations. Oncogenic Ras proteins accumulate in the active GTP-bound form and constitutively activate numerous downstream effector pathways. However, it is unclear which effectors are necessary to initiate and maintain malignant hematologic disease as well as the contributions of specific effector pathways to disease phenotypes.

I performed functional studies utilizing second site K-Ras^G12D mutant proteins to explore the role of the three main effector pathways—Raf/MEK/ERK, Phosphotidyl-Inositol-3Kinase(PI3K), RalGDS—in Kras^G12D-driven oncogenesis. Using primary murine hematopoietic cells transduced with second site mutant constructs, I found that second site mutants maintaining hyperactive signaling down only one effector pathway were unable to confer hypersensitivity to GM-CSF in methylcellulose assays. However, second site mutants that engage two of the three major effector pathways, K-RasG12D,E37G and K-Ras^G12D,Y64G (which activate PI3K/RalGDS and Raf/RalGDS respectively), retain some growth factor hypersensitivity but do not exhibit cytokine-independent growth.

Transplantation of bone marrow cells transduced with K-Ras ^G12D,E37G or K-Ras^G12D,Y64G into irradiated recipient mice induced invasive, monoclonal, CD4-CD8 double-positive T-cell acute lymphoblastic leukemias (T-ALLs) that originated in the thymus. A majority of these leukemias show elevated levels of activated Notch1, due to mutations in the PEST domain of Notch1. Tumors expressing K-Ras^G12D,E37G demonstrated elevated levels of this mutant K-Ras protein but no basal activation of the Raf/MEK/ERK pathway. Interestingly, T-ALLs initiated by K-Ras^G12D,Y64G acquired elevated levels of phosphorylated Akt, which was associated with diminished or absent PTEN expression. The strong selective pressure to increase signaling through PI3K by alternative mechanisms, increasing K-Ras^G12D,E37G protein levels or decreasing PTEN expression, supports the hypothesis that this pathway plays a central role in T-lineage leukemogenesis. An important therapeutic implication of these data is that inhibiting a single major effector pathway may be insufficient to fully reverse the growth advantage conferred by oncogenic Ras.