Role of Downstream Effectors in Kras-Driven Myeloproliferative Disease.

Mutations that deregulate Ras signaling are highly prevalent in myeloid malignancies. Previous work has shown that expressing oncogenic Kras G12D in hematopoietic cells from the endogenous Kras promoter leads to growth factor-independent and hypersensitive myeloid progenitor colony formation, and causes a fatal myeloproliferative disorder (MPD) (Braun et al PNAS101:597, 2004; Chan et al JCI113:528, 2004). These characteristics mirror the human juvenile and chronic myelomonocytic leukemias (JMML and CMML) - diseases that are associated with Ras pathway mutations. Oncogenic Ras accumulates in its active GTP-bound form and constitutively activates a number of downstream effectors. However, it is unclear which effectors are necessary for the maintenance of disease as well as how they contribute to specific phenotypes such as enhanced proliferation and defective apoptosis. We constructed second site mutants - secondary mutations on a backbone of Kras G12D (G12D) that prevent K-Ras G12D from binding to a subset of effectors - to begin to elucidate the individual contributions of downstream effector pathways to hematologic disease. Murine fetal liver cells engineered to express Kras G12D,E37G (E37G) and Kras G12D,Y64G (Y64G) second site mutants maintain a hypersensitive pattern of colony-forming unit granulocyte-macrophage (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), but are no longer able to grow in the absence of GM-CSF. Interestingly, both mutant proteins that display hypersensitivity also hyperactivate two major Ras effector pathways, as opposed to other second site mutants tested which only hyperactivate one major Ras effector pathway and do not show growth factor hypersensitivity. Whereas E37G activates both PI3 kinase and Ral-GDS but not Raf, Y64G stimulates Raf/MEK/ERK and PI3 kinase but not Ral-GDS. Consistent with the idea that activation of at least two effector cascades is required for aberrant in vitro growth, expression of activated effectors Braf V600E , p110a-CAAX , and RalGDS-CAAX did not confer GM-CSF hypersensitivity on CFU-GM colonies. We transplanted bone marrow cells transduced with MSCV-E37G-IRES-GFP or MSCV-Y64G-IRES-GFP retroviruses into lethally irradiated Balb/c mice. Seven of 8 mice that received Y64G cells died of T lineage acute lymphoid leukemia/lymphoma (T-ALL/L) with a median survival of 112 days. Diseased animals showed very high levels of GFP in the thymus, spleen, peripheral blood and bone marrow. Four of 9 mice injected with E37G-expressing cells mice also died of T-ALL/L with a median survival of 106 days and three of 9 died of anemia. None of the MIG mice (n=8) have died of leukemia/lymphoma. The Y64G and E37G T ALL/Ls studied to date are transplantable into sublethally irradiated recipients. We conclude that second site mutations in the Kras G12D oncogene that are defective for activation of either PI3 kinase or Raf/MEK/ERK are able to deregulate the growth of primary hematopoietic cells in vitro and in vivo . These data argue that targeting a single effector pathway downstream of oncogenic Ras may not be effective in many hematologic malignancies. We are interrogating Ras signaling networks in T-ALL/L cells and cloning MSCV integration sites to further characterize the effects of these second site mutant alleles.