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Blood, 1 June 2002, Vol. 99, No. 11, pp. 4147-4153
NEOPLASIA
Targeting Raf-1 gene expression by a DNA enzyme
inhibits juvenile myelomonocytic leukemia cell growth
Per Ole Iversen,
Peter
D. Emanuel, and
Mouldy Sioud
From the Institute for Nutrition Research, University
of Oslo, and Department of Immunology, Molecular Medicine Group, the
Norwegian Radium Hospital, Oslo, Norway; and the Division of
Hematology/Oncology, University of Alabama at Birmingham.
Juvenile myelomonocytic leukemia (JMML) is an
aggressive childhood disorder with few therapeutic options.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor
necrosis factor- (TNF-) 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-, 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-, 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- 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.
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