Abstract

Proliferation and growth of AML cells result from stimulation by cytokines and high levels of cytokines are associated with poor prognosis in AML. Cytokines act through cellular receptors that are associated with members of the Jak family of protein tyrosine kinases. Upon phosphorylation and activation of Jak, proteins bound to Jak initiate signaling pathways including those regulated by Stat proteins. Since constitutive activation of Jak-Stat has been associated with leukemogenesis, we hypothesize that inhibition of Jak-Stat inhibits proliferation of AML cells. To do this, we studied the effects of WP-1034, a novel and potent inhibitor of Jak-Stat, in the OCIM2 AML cell line and fresh samples from AML patients. OCIM2 cells were deprived of serum for 2 hours and then incubated with 1 to 5 μM WP-1034 to investigate its effect on OCIM2 cell proliferation. After incubation of the cells without and with 1, 2.5, 5, 7.5, and 10 μM WP-1034 for 1 hour, and at 5 μM for 0, 20 min, 40 min, and 1, 2, 3, and 4 hours, we determined expression of Stat 1, 3, and 5, as well as Phospho-stat 1, 3, and 5 in the cells by Western Immunoblotting. In addition, we analyzed cell cycle status by PI staining and flow cytometry. We further evaluated induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3, 5, and 6 μM using the annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western Immunoblotting. To demonstrate the effect of WP-1034 on marrow cells from AML patients and healthy volunteers we incubated marrow cells with WP-1034 at increasing concentrations and used the blast colony assay to measure inhibition of proliferation. Our results show that: i) WP-1034 effectively inhibits proliferation of OCIM2 cells and AML blast proliferation from patient samples; ii) WP-1034 blocks activation of Stat 3 and 5 by decreasing the amount of Phospho-stat 3 and 5 in OCIM2 cells; iii) WP-1034 causes cell cycle arrest in sub-G0 phase and is able to induce apoptosis in OCIM2 cells; and iv) WP-1034 induces apoptosis involving cleavage of caspase 3 and PARP. Our data suggest that WP-1034, a potent inhibitor of Jak-Stat, inhibits proliferation of AML cells by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.

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