ABSTRACT
Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.
Subject(s)
Cytokines/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Hematopoietic Stem Cells/metabolism , Neoplastic Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Amino Acid Substitution , Animals , Cells, Cultured , Cytokines/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Hematopoietic Stem Cells/pathology , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , MAP Kinase Signaling System/genetics , Mice , Mutation, Missense , Neoplastic Stem Cells/pathology , Phosphatidylinositol 3-Kinases/genetics , Phospholipase C gamma/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Second Messenger Systems/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative neoplasm (MPN) that is refractory to conventional chemotherapy. Conditional inactivation of the Nf1 tumor suppressor in hematopoietic cells of mice causes a progressive MPN that accurately models JMML and chronic myelomonocytic leukemia (CMML). We characterized the effects of Nf1 loss on immature hematopoietic populations and investigated treatment with the MEK inhibitor PD0325901 (hereafter called 901). Somatic Nf1 inactivation resulted in a marked expansion of immature and lineage-committed myelo-erythroid progenitors and ineffective erythropoiesis. Treatment with 901 induced a durable drop in leukocyte counts, enhanced erythropoietic function, and markedly reduced spleen sizes in mice with MPN. MEK inhibition also restored a normal pattern of erythroid differentiation and greatly reduced extramedullary hematopoiesis. Remarkably, genetic analysis revealed the persistence of Nf1-deficient hematopoietic cells, indicating that MEK inhibition modulates the proliferation and differentiation of Nf1 mutant cells in vivo rather than eliminating them. These data provide a rationale for performing clinical trials of MEK inhibitors in patients with JMML and CMML.
Subject(s)
Benzamides/pharmacology , Diphenylamine/analogs & derivatives , Erythropoiesis/drug effects , Hematopoiesis, Extramedullary/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelomonocytic, Juvenile/drug therapy , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neurofibromin 1 , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation/drug effects , Child , Child, Preschool , Diphenylamine/pharmacology , Disease Models, Animal , Erythropoiesis/genetics , Hematopoiesis, Extramedullary/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelomonocytic, Juvenile/etiology , Leukemia, Myelomonocytic, Juvenile/genetics , Leukemia, Myelomonocytic, Juvenile/metabolism , Mice , Mice, Mutant Strains , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Neurofibromatosis 1/complications , Neurofibromatosis 1/drug therapy , Neurofibromatosis 1/geneticsABSTRACT
The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.
