Abstract 588: PLK1 inhibitors interact synergistically with HDAC inhibitors in IM-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.
Authors: Dasmahapatra Girija Prasad , Patel Hiral , Nguyen Tri , Benson Elisa , Grant Steven 
Published: August 2014

Abstract
Purpose: Previous studies have shown that inhibitors of the serine/threonine kinase PLK1 potently induce cell death in human BCR/ABL+ leukemia cells both sensitive and resistant to imatinib mesylate (IM; Cancer Research 70:1513, 2010). Furthermore, HDACIs are known to potentiate tyrosine kinase lethality in such cells. The goal of the present studies was to determine whether PLK1 inhibitors (e.g. BI2536) and HDACIs interacted synergistically in BCR/ABL+ cells sensitive or resistant to imatinib mesylate in vitro and in vivo and if so, to identify the mechanism(s) underlying this interaction. Experimental
Design: K562 and LAMA84 cells, IM-resistant Adult-T315I and BV173/E255K cells, and primary CML cells were exposed to BI2536 with or without vorinostat (Merck) for various intervals. Effects on apoptosis and signaling pathways were determined by flow cytometry, western blotting, and gene transfection. K562 and BV173/E255K cells were used to test the in vivo efficacy of this drug regimen in animal models.
Results: Co-treatment with BI2536 and vorinostat synergistically induced cell death in parental or IM-resistant BCR/ABL+ cells and primary CD34+ bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat co-treatment triggered pronounced mitochondrial dysfunction, caspase activation, PARP cleavage, ROS generation, and DNA damage (manifested by increased expression of γH2A.X, p-ATM, p-ATR), all of which were significantly attenuated by the anti-oxidant TBAP. Combined treatment had little effect on T210 phosphorylation of PLK1, but clearly reduced S137 phospho-PLK1 without changing PLK1 expression. While the agents alone had little effect on BCR/ABL phosphorylation, combined treatment induced modest reductions in phospho-BCR/ABL but not total expression. Exposure of cells to BI2536 alone induced marked G2M arrest, whereas vorinostat co-administration abrogated this effect. PLK1 shRNA knockdown significantly increased HDACI lethality, whereas HDAC 1-3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, arguing for a functional role for DNA damage in lethality. Finally, BI2536/vorinostat co-treatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL+ leukemia xenograft models (including those resistant to IM), and significantly enhanced animal survival. Notably, the regimen was highly effective in a late stage tumor model which is classically resistant to most therapeutic interventions.
Conclusions: These findings suggest that concomitant PLK1 and HDAC inhibition is active against IM-sensitive or refractory CML cells both in vitro and in vivo, and that this strategy warrants further evaluation in the setting of BCR/ABL+ leukemias.
Citation Format: Girija Prasad Dasmahapatra, Hiral Patel, Tri Nguyen, Elisa Benson, Steven Grant. PLK1 inhibitors interact synergistically with HDAC inhibitors in IM-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2013-588