The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors

Abstract

The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34(+) bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases.

Figure 1

Pimozide inhibits STAT5 activity. (A) Chemical structure of pimozide. (B) Reporter cell lines were treated with the indicated doses of pimozide for 2 hours, after which cytokines were added to activate the appropriate transcription factor. Luciferase activity was quantitated by luminometry 6 hours later. (C) KU812 cells were treated with vehicle or pimozide (5μM) for 18 hours, after which RNA was harvested, and expression of the indicated genes was measured using quantitative RT-PCR and normalized to the expression of β-actin. (D) KU812 cells were treated with the indicated concentrations of pimozide for 3 hours. Immunoblots were performed using antibodies specific for tyrosine phosphorylated STAT5 and total STAT5.

Figure 2

Pimozide is not a direct inhibitor of BCR/ABL. (A) K562 and KU812 cells were treated with pimozide (10μM) or imatinib (1μM) for 3 hours, after which immunoblot analysis was performed using antibodies to the phosphorylated forms of ABL1 and STAT5, as well as total ABL1 and STAT5. (B) K562 and KU812 cells were treated with pimozide (10μM) or imatinib (1μM) for 3 hours, after which immunoblot analysis was performed using a pan-antiphospho-tyrosine antibody. (C) K562 cells were treated with pimozide and imatinib for 3 hours, and MAPK activation was measured by immunoblot analysis using an antibody to phosphorylated MAPK and a total MAPK antibody.

Figure 3

Pimozide induces apoptosis of CML cells. (A) KU812 and K562 cells were treated with the indicated concentrations of pimozide for 48 hours, after which viable cell number was measured using an ATP-dependent bioluminescence assay. Data are representative of 3 independent experiments. (B) Unfractionated bone marrow mononuclear cells harvested from 2 untreated CML patients were treated with pimozide at the indicated concentrations for 48 hours, and viable cell number was determined. (C) KU812 cells were treated with vehicle or pimozide (5μM) for 48 hours, after which annexin V/propidium iodide staining and flow cytometry were performed. Among vehicle-treated cells, 7.6% showed annexin V staining, whereas among pimozide-treated cells 17.3% were annexin V positive. (D) Peripheral blood mononuclear cells harvested from 7 healthy persons were treated with the indicated concentrations of pimozide for 48 hours, after which viable cell number was measured using an ATP-dependent bioluminescence assay. Data are mean ± SEM.

Figure 4

The combination of pimozide and imatinib leads to enhanced effects on STAT5 inhibition and apoptosis. (A) KU812 cells were treated with pimozide (5μM), imatinib (50nM), or both for 3 hours, after which immunoblot analysis was performed for phospho-STAT5 and total STAT5. (B) KU812 cells were treated with pimozide (5μM), imatinib (50nM), or both for 48 hours, after which viable cell number was measured using an ATP-dependent bioluminescence assay. (C) Isobologram analysis was performed based on change in viable cell number for 9 different combinations of pimozide and imatinib. Data points below the line indicate superadditive effects. (D) KU812 cells were treated with pimozide (5μM), imatinib (50nM), or both for 48 hours, after which flow cytometric analysis was performed. The percentage of cells staining with annexin V is shown. Data are representative of 3 independent experiments.

Figure 5

Inhibition of MAPK enhances the effects of pimozide on cell viability. (A) K562 cells were treated with pimozide (5μM), nilotinib (3nM), or both for 3 hours, and immunoblots were performed to phospho-MAPK and total MAPK (left); phospho-MAPK/total MAPK band intensity is shown in the right panel. (B) K562 cells were treated with pimozide (7.5μM), the MEK inhibitor U0126 (10μM), or both for 48 hours, after which viable cell number was quantitated by ATP-dependent bioluminescence. (C) Isobologram analysis was performed based on loss of cell viability for 9 different combinations of pimozide and UO126. Data points below the line indicate superadditive effects.

Figure 6

Pimozide is similarly effective against cells with unmutated BCR/ABL and kinase inhibitor-resistant T315I mutant BCR/ABL. (A) The 32d cells reconstituted with the T315I mutant form of BCR/ABL were treated for 3 hours with pimozide (5μM), after which RNA was harvested, and expression of the indicated genes was measured using quantitative RT-PCR and normalized to the expression of β-actin. (B) The 32d cells reconstituted with unmutated BCR/ABL (p210) or BCR/ABL with the kinase inhibitor-resistant T315I mutation were treated with pimozide at the indicated concentration for 48 hours after which viable cell number was measured by ATP dependent bioluminescence.