Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth.
Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood disorder with few therapeutic options. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) promote JMML cell growth. A hyperactive function of the ras oncogene is a hallmark of JMML. We therefore targeted the protein kinase Raf-1 downstream of Ras using a DNA enzyme that degrades mRNA-Raf-1. Western blots of JMML cell lysates revealed phosphorylated Raf-1 protein, indicating constitutive activation. Addition of GM-CSF, but not TNF-alpha, increased phosphorylation of both Raf-1 and the mitogen-activated protein kinases (MAPKs) JNK-1 and ERK-1. Depletion of Raf-1 protein markedly impaired activation of MAPKs, induced substantial inhibition of JMML cell colony formation, and virtually abolished GM-CSF hypersensitivity in JMML cells. Exogenous TNF-alpha, but not GM-CSF, restored colony formation of JMML cells pretreated with the enzyme. We could not detect any effect of the enzyme on the proliferation of normal bone marrow cells, indicating its specificity and potential safety. When immunodeficient mice engrafted with JMML cells were treated continuously with the enzyme via a peritoneal osmotic mini-pump for 4 weeks, a profound reduction in the JMML cell numbers in the recipient murine bone marrows was found. We conclude that GM-CSF is a chief regulator of JMML growth and exerts its proleukemic effects primarily via the Ras/Raf-1 signaling cascade. TNF-alpha plays a permissive role, being dependent upon GM-CSF to induce JMML cell proliferation. The DNA enzyme efficiently catabolized mRNA-Raf-1 with subsequent inhibition of JMML cell growth, suggesting its potential as a mechanism-based therapy in this fatal leukemia.
Iversen
Per Ole
PO
Institute for Nutrition Research, University of Oslo, and Department of Immunology, Molecular Medicine Group, the Norwegian Radium Hospital, Oslo, Norway. poiversen@hotmail.com
Emanuel
Peter D
PD
Sioud
Mouldy
M
D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D001943:Breast Neoplasms; D002455:Cell Division; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D015479:Leukemia, Myelomonocytic, Acute / Q000473:pathology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019908:Proto-Oncogene Proteins c-raf; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D004259:DNA-Directed DNA Polymerase