WP-1034

Cytokine stimulation induces proliferation and growth of acute myeloid leukemia (AML) blasts and high levels of cytokines have been associated with poor prognosis in AML. The Jak-Stat pathway constitutes a major mediator of cytokine activity. We investigated whether WP-1034, a novel Jak-Stat inhibitor, is active against AML blasts. OCIM2 and fresh AML cells were incubated with 1 to 6 microM WP-1034 to determine its effect on proliferation. WP-1034 effectively inhibited proliferation of OCIM2 cells and fresh AML samples. We then analyzed the expressions of Stat 1, 3, and 5, as well as Phospho-Stat 1, 3, and 5 by Western immunoblotting after incubation of OCIM2 cells without and with 1 to 10 microM WP-1034 for 2 hours, and at 5 microM from 20 minutes up to 4 hours and found that WP-1034 blocked Stat 3 and 5 activation. Analysis of cell cycle status by PI staining and flow cytometry showed that WP-1034 caused cell cycle arrest of OCIM2 cells in sub-Go phase. We then evaluated the induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3 to 6 microM by annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western immunoblotting. We found that WP-1034 induced apoptosis of OCIM2 cells and that induction of apoptosis involved cleavage of caspase 3 and the DNA repair enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP). Taken together, our data suggest that WP-1034 is a potent inhibitor of AML cell proliferation by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.

Cytokine stimulation induces proliferation and growth of acute myeloid leukemia (AML) blasts and high levels of cytokines have been associated with poor prognosis in AML.The Jak-Stat pathway constitutes a major mediator of cytokine activity.We investigated whether WP-1034, a novel Jak-Stat inhibitor, is active against AML blasts.OCIM2 and fresh AML cells were incubated with 1 to 6 μM WP-1034 to determine its effect on proliferation.WP-1034 effectively inhibited proliferation of OCIM2 cells and fresh AML samples.We then analyzed the expressions of Stat 1, 3, and 5, as well as Phospho-Stat 1, 3, and 5 by Western immunoblotting after incubation of OCIM2 cells without and with 1 to 10 μM WP-1034 for 2 h, and at 5 μM from 20 min up to 4 h and found that WP-1034 blocked Stat 3 and 5 activation.Anal. of cell cycle status by PI staining and flow cytometry showed that WP-1034 caused cell cycle arrest of OCIM2 cells in sub-G₀ phase.We then evaluated the induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3 to 6 μM by annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western immunoblotting.We found that WP-1034 induced apoptosis of OCIM2 cells and that induction of apoptosis involved cleavage of caspase 3 and the DNA repair enzyme poly (ADP [ADP]-ribose) polymerase (PARP).Taken together, our data suggest that WP-1034 is a potent inhibitor of AML cell proliferation by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.