All-trans retinoic acid corrects gm-csf hypersensitivity in NF-1 −/− Knock-out hematopoietic progenitor cells in vitro

Abstract

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive form of leukemia in children. JMML is refractory to conventional therapy with most survivors undergoing stem cell transplantation. 13-cis retinoic acid has been used therapeutically to control the disease but is not curative. The phenotypic hallmark of JMML is a hypersensitivity to GM-CSF and is likely mediated through alterations in the Ras signaling pathway. Ras gene mutations occur in 15–30% of JMML patients with another 10–15% having mutations in the neurofibromatosis type 1 tumor suppressor gene encoding for the neuofiromin protein. Homozygous deletion of Nf-1 in mice is lethal, however hematopoietic liver cells from d13.5 embryos exhibit the same GM-CSF hypersensitivity as JMML cells. Moreover, transplantation of Nf-1 −/− cells into irradiated mice will reconstitute hematopoiesis and results in a myeloproliferative syndrome similar to JMML. To investigate whether these cells might serve as a model for JMML for examining the action of retinoic acid, we utilized low density fetal liver cells from Nf-1 −/− knock-out mice and their wild type littermates. Methylcellulose progenitor assays performed with varying concentrations of GM-CSF and all-trans retinoic acid (ATRA) resulted in correction of the GM-CSF hypersensitivity phenotype. Additionally, low density bone marrow mononuclear cells were obtained from irradiated recipient mice transplanted with Nf-1 −/− or +/+ hematopoietic liver cells. ATRA in a concentration dependent manner inhibited long term culture initiating cell growth to a significantly greater extent in Nf-1 −/− compared to wildtype. Thus Nf-1 −/− knock-out cells may be useful in mechanistic studies of ATRA in JMML.