K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

Abstract

Defining how cancer-associated mutations perturb signaling networks in stem/ progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic Kras G12D allele on phosphorylated signaling molecules in primary c-kit ؉ lin ؊/low hematopoietic stem/progenitor cells. Comparison of wild-type and Kras G12Dc-kit ؉ lin ؊/low cells shows that K-Ras G12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas Kras G12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/ progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. IntroductionRas proteins are signal-switch molecules that modulate cell fates by cycling between inactive GDP-bound and active GTP-bound conformations (Ras-GDP and Ras-GTP). When GTP-bound, Ras interacts productively with downstream effectors including the Ral-GDS, Raf/MEK/ERK, and phosphoinositol 3Ј (PI3) kinase/ Akt pathways. 1 Guanine nucleotide exchange factors promote Ras activation, and signaling is terminated when Ras-GTP is hydrolyzed to Ras-GDP by its intrinsic GTPase activity, which is markedly accelerated by GTPase-activating proteins (GAPs). [2][3][4] Somatic KRAS2, NRAS, and HRAS mutations are found in approximately 30% of human cancers and encode mutant proteins that accumulate in the GTP-bound conformation due to reduced intrinsic GTPase activity and resistance to GAPs. 5,6 Hematopoietic malignancies are tractable experimental systems for interrogating signaling networks in primary cells and for integrating biochemical, genetic, and cell biologic data. KRAS2 and NRAS are mutated frequently in myeloid malignancies, including acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML), which are classified as myeloproliferative disorders (MPDs). 7-9 An important role of hyperactive Ras in myeloid leukemias is further illustrated by the fact that many patients without RAS mutation harbor other genetic lesions that deregulate Ras signaling such as loss of the NF1 tumor suppressor, point mutations that activate the SHP-2 protein tyrosine phosphatase, and the BCR-ABL fusion protein. 10,11 However, despite the extensive evidence implicating hyperactive Ras in myeloid leukemogenesis, the biochemica...

Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells

“…34), Cbl, Cbl-b dKO bone marrow cells were hypersensitive to GM-CSF and able to form colonies even in the absence of exogenous cytokines. However, whereas both Kras G12D and Ptpn11 D61Y mutant mice show a reduction in the number of LSK cells in the bone marrow (35,36), this population is expanded in the bone marrow of Cbl-deficient (14) and Cbl/Cbl-b double-deficient mice (Fig. 3).…”

Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement

“…It can be noted that, a higher percentage of Mx1-Cre, Kras G12D cells phosphorylated STAT5 in response to GM-CSF, but the magnitude and duration of STAT5 phosphorylation was similar in cells with both mutant and wild-type genotype. 8 We found that human myeloid progenitors bearing N-RAS mutation hyperphosphorylate STAT5 in terms of both magnitude of molecules and proportion of cells. Although our series is limited, the results show that human bone marrow CD34 þ / CD33 þ cells, but not CD34 þ /CD33À or CD34À/CD33 þ cells from JMML patients, represent the subset in which STAT5 hyperphosphorylation occurs in response to low doses of GM-CSF, and these observations were not reported earlier.…”

“…More recently, Chou et al 7 published a novel TaqMan system for the absolute quantification of MRD with one common forward primer, a common probe and mutationspecific reverse primers. Scholl et al 8 additionally introduced methodological approaches for the rapid screening of NPM1 mutations by LightCycler assays based on the FRET principle, as well as MRD analyses of the most common mutations A and B with the LightCycler, using mutation-specific primers and SYBR Green.…”

“…[5][6][7] GM-CSF-RAS pathway in turn affects signaling cascades downstream of GM-CSF receptor. 8 Multiparametric flow cytometric analysis has demonstrated new potentialities for assaying intracellular levels of phosphorylated proteins. 9 Here, we report the flow cytometric analysis of intracellular phosphorylated signal transducer and activator of transcription (pSTAT5) and evaluate the possible impact of our findings in the diagnostic work-up of JMML.…”

Loss of mTOR complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell acute lymphoblastic leukemia cells

“…A mouse model with an oncogenic Kras mutation (Kras G12D ) develops MPN, followed by T-ALL (20)(21)(22)(23). We evaluated the effect of Raptor deficiency on oncogenic Kras-driven hematopoiesis, particularly on the development of leukemia.…”