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We have previously shown that the arylating NO donor compound O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) has potent anti-leukemic activity in vitro and in vivo (Molecular Cancer Therapeutics 2:409-417,2003). JS-K’s in vitro 50% growth inhibitory concentration (IC50) for HL-60 (human myeloid leukemia) cells is 0.2 - 0.5 μM. The purpose of this study was to determine the effect JS-K on normal hematopoietic stem and progenitor cells. Bone marrow was obtained from C57BL/6 mice and enriched for c-kit-positive cells by micromagnetic separation. c-kit-positive marrow cells were incubated with 0.5 μM JS-K in IMDM/30%FBS in the presence of stem cell factor and interleukin-3. Control cells were incubated under the same conditions without JS-K. After 24 hours, c-kit-positive marrow cells were plated in semi-solid media for clonogenic progenitor assays. Ten days after plating, colony assay revealed equal numbers of colonies in control and JS-K-treated cells (10 ± 5.25 and 10 ± 5.75 colonies, respectively). To further characterize potential hematopoietic toxicity of JS-K, we injected similarly JS-K-treated c-kit positive bone marrow cells into lethally irradiated congenic C57BL/6 CD45.2 mice. All mice given JS-K-treated bone marrow were rescued from lethal irradiation. Thus, JS-K did not compromise radioprotection. We then studied donor engraftment by flow cytometry using markers to detect CD3-, B220-, and Mac1/Gr1-positive cells (T-cells, B-cells, and monocytic/granulocytic cells). At 77 days post-transplant, we found percent donor chimerism to be indistinguishable between mice given JS-K-treated bone marrow compared with control DMSO-treated or freshly harvested bone marrow. This indicates no effect of JS-K on durable multilineage hematopoietic differentiation. We conclude that JS-K has a remarkable lack of toxicity against normal hematopoietic stem and progenitor cells in spite of substantial anti-leukemic activity. JS-K is therefore a very promising agent for future clinical development for the treatment of leukemia and other malignancies.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA