Ras proteins regulate cell fates by cycling between an inactive, GDP-bound and an active, GTP-bound state. Ras activation is controlled by the opposing effects of guanine nucleotide exchange factors, which promote GTP binding, and GTPase activating proteins (GAPs), which markedly accelerate the slow intrinsic Ras GTPase. The mutant RAS alleles found in leukemia and other cancers encode proteins that accumulate in the GTP-bound conformation due to defective intrinsic GTPase activity and resistance to GAPs. These mutant Ras proteins, in turn, aberrantly activate effector pathways such as Raf/MEK/ERK, PI3 kinase/Akt, and Ral-GDS. To investigate the mechanisms by which hyperactive Ras deregulates myeloid growth, we previously generated Mx1-Cre, LSL-KrasG12D mice, which harbor a conditional oncogenic KrasG12D allele and uniformly develop a fatal myeloproliferative disease (MPD) upon induction of KrasG12D expression (
Braun et al., PNAS 10, 597, 2004
). This robust in vivo phenotype is associated with hypersensitivity of myeloid progenitors to cytokines in methylcellulose assays and elevated levels of Ras-GTP. However, we surprisingly found no increase in basal or growth factor-induced levels of phosphorylated MEK, ERK, and Akt in bone marrow cells from KrasG12D mice with MPD. To investigate this apparent paradox, we analyzed the activation states of MEK, ERK, Akt, and STAT5 under various conditions. By increasing the stringency of serum starvation prior to stimulation with GM-CSF, serum, or both, we uncovered reproducible hyperphosphorylation of MEK, Akt, and STAT5 in KrasG12D marrow. We observed similar phosphorylation patterns in marrow from mice one week after activation of oncogenic KrasG12D expression and from mice with advanced MPD, suggesting that myeloid lineage cells do not adapt to oncogenic Kras by modulating activation of downstream effectors on this time scale. Finally, we used multiparameter flow cytometric analysis of intracellular phosphoproteins to directly assay Ras effector pathways in defined populations of primary cells. We demonstrated that the c-kit-positive, lineage-negative (c-kit+/lin−) fraction contains the colony-forming unit granulocyte-macrophage (CFU-GM) progenitors in control and KrasG12D marrow and that the GM-CSF hypersensitivity characteristic of KrasG12D marrow is retained in this fraction. This population comprises 0.2–0.9% of nucleated marrow cells, making analysis by traditional methods difficult. Cells in this population exhibited distinct patterns of ERK and STAT5 phosphorylation in comparison with whole bone marrow. Importantly, c-kit+/lin− cells from KrasG12D mice demonstrated marked hyperphosphorylation of multiple signal transduction molecules under both starved and stimulated conditions. We conclude that KrasG12D expression from the endogenous Kras locus deregulates a network of signaling molecules in hematopoietic cells. This is most evident in a defined subset of progenitors that exhibits aberrant proliferation and differentiation in vitro. Multiparameter flow cytometry is a robust methodology for analyzing the activation status of signaling networks in primary cells that can be used to monitor responses to targeted therapeutics in disease-relevant populations of cells.
