Bisphosphonate Zoledronic Acid (ZOL) Inhibits the Abnormal Proliferation and Differentiation of Juvenile Myelomonocytic Leukemia (JMML) Cells.

Abstract

BACKGROUND: JMML is a clonal myeloprolifelative/myelodysplastic disorder of early childhood. The mortality rate in JMML patients is high, but so far, stem cell transplantation is the only therapy capable of producing durable remissions. JMML cells are characterized by the ability to spontaneously proliferate in vitro, giving rise to granulocyte-macrophage (GM) colonies. Deregulation of the GM-CSF receptor-RAS signal transduction pathway is thought to play a key role in the pathogenesis. Therefore, putative RAS-blocking compounds can be potential inhibitors for JMML cells. ZOL, the third generation bisphosphonate, is known to not only treat bone diseases but also act as an antiproliferative drug for some cancers by inhibiting the activation of RAS-related proteins. We then investigated the effect of ZOL on JMML cell growth in vitro. METHODS: After obtaining informed consent, bone marrow (BM) samples were obtained from children with JMML or normal healthy volunteers. The diagnosis of JMML was based on criteria of the international JMML Working Group, and confirmed by the demonstration of spontaneous colony formation, most of which were GM colonies consisting of both macrophages and granulocytes. In methylcellulose clonal and suspension cultures, BM non-adherent mononuclear cells (2 x 104 cells/mL) in the absence or presence of GM-CSF (10 ng/mL) were exposed to 0, 1, 10 and 100 μM of ZOL. In the former, colonies (>40 cells) were counted on days 12 to 14 of culture, and the cellular composition of each colony was analyzed on cytospin smears cytochemically stained. In the latter, the determination of number of cultured cells and their cellular composition by the microscopic observation and flow cytometry was carried out on days 0, 5, 10 and 15. FINDINGS: 8 JMML children were enrolled in this study. In clonal culture, ZOL did not affect the spontaneous colony formation by JMML cells at 1 μM, but reduced it at 10 μM. Few colonies were produced at 100 μM. Interestingly, the colonies formed at more than 10 μM ZOL contained a number of smaller colonies consisting of only granulocytes, but no macrophages. Furthermore, the size of bipotential GM colonies was smaller and the percentage of macrophages included in each colony was fewer at the concentrations of ZOL. On the other hand, ZOL had no effect on the colony formation induced by GM-CSF from normal BM cells at up to 10 μM, whereas few colonies were observed at 100 μM as well as spontaneous colony formation by JMML cells. In suspension culture, 10 μM ZOL also inhibited the spontaneous proliferation and differentiation on monocyte/macrophage lineage of JMML cells, but not the development of normal BM cells induced by GM-CSF. INTERPRETATION: The inhibitory effects of ZOL on the abnormal proliferation and differentiation of JMML cells may offer an approach to therapy in JMML.