Intracellular Signals as Molecular Biomarkers for Therapeutic Responses in Kras Mutant Myeloid Cells.

Somatic RAS mutations are found in many human cancers, including lung, pancreas, and colon carcinomas, melanoma, and myeloid malignancies. Most of these diseases respond poorly to current treatments. Mutant RAS alleles found in leukemia and other cancers encode proteins that accumulate in a constitutively active GTP-bound conformation, and aberrantly activate downstream effectors such as Raf/MEK/ERK, PI-3K/Akt, and Ral-GDS pathways. To investigate the mechanisms by which hyperactive Ras deregulates myeloid growth, we generated Mx1-Cre , LSL-Kras^G12D mice, which harbor a conditional oncogenic Kras^G12D allele and uniformly develop a fatal myeloproliferative disease (MPD) after induction of Kras^G12D expression (Braun et al., PNAS 10, 597, 2004). In contrast to acute myeloid leukemia (AML), which typically demonstrates multiple mutations, MPD are caused by fewer and even single mutations narrowing the window for targeted therapies. We are exploiting multiparametric FACS analysis as a general strategy to obtain a comprehensive biochemical understanding of mutant signaling networks and to identify and interrogate relevant populations mediating disease initiation and progression. This technique allows us to simultaneously measure intracellular phosphoproteins in phenotypically characterized subpopulations of cells. We have focused on comparing c-kit-positive, lineage-negative (c-kit+/lin-) cells in Kras^G12D and normal marrow. This population, which comprises only 2–3% of total bone marrow cells, accounts for 85–90% of progenitors cells. Primary bone marrow from normal and Kras^G12D mice with MPD were extracted, serum starved, and stimulated at different time points with granulocyte-macrophage colony stimulating factor (GM-CSF). Kras mutant cells exhibited hyper-phosphorylation not only of MAP kinase effectors (e.g. ERK and Mnk) but also of downstream components of the PI3 kinase cascades such as Akt and S6. We also detected elevated levels of phosphorylated STAT3 and 5 at baseline and after exposure to GM-CSF. In addition, Kras^G12D mutant cells showed a more sustained activation of pERK and pS6 over time. Paradoxically, Kras mutant cells showed an intriguing attenuation of the same effectors upon SCF stimulation. Parallel studies showed that inhibiting PI3 kinase interferes with ERK activation, thus revealing crosstalk among pathways. These effects are most evident in defined subsets of stem/progenitor cells that exhibit aberrant proliferation and differentiation in vitro. Finally, we observe dramatic differences in Ras signaling networks with progression from MPD to AML. Our data suggest that oncogenic hyperactive Ras induces rewiring in cellular signaling networks that are not static, but are strongly modulated by secondary mutations. Multiparameter flow cytometry is a robust methodology for analyzing the biochemical profiles of primary cells that can be used to monitor biochemical responses to targeted therapeutics in disease-relevant populations of hematopoietic stem and progenitor cells.