Supplementary Figure 2 from PLK1 Inhibitors Synergistically Potentiate HDAC Inhibitor Lethality in Imatinib Mesylate–Sensitive or –Resistant BCR/ABL+ Leukemia Cells In Vitro and In Vivo

Dasmahapatra, Girija; Patel, Hiral; Nguyen, Tri; Attkisson, Elisa; Grant, Steven

doi:10.1158/1078-0432.22455911.v1

Supplementary Figure 2 from PLK1 Inhibitors Synergistically Potentiate HDAC Inhibitor Lethality in Imatinib Mesylate–Sensitive or –Resistant BCR/ABL⁺ Leukemia Cells In Vitro and In Vivo

journal contribution

posted on 2023-03-31, 18:15 authored by Girija Dasmahapatra, Hiral Patel, Tri Nguyen, Elisa Attkisson, Steven Grant

PDF file - 90K, Various PLK1 and HDAC inhibitors interact synergistically in CML models including those sensitive or resistant to IM. A. LAMA84 cells were treated with BI2536 (2.0 nM) � vorinostat (1.0 �M) for 48 hr. B. K562 cells were treated with BI6277 (7.5 nM) or GW843682(7.5�M) � vorinostat (1.0 �M) or SBHA (30�M) for 48 hr, C. Fractional Effect (FA) values were determined by comparing results obtained for untreated controls and treated cells following exposure to agents administered at a fixed ratio (GW843682: vor = 5:1), after which Median Dose Effect analysis was employed to characterize the nature of the interaction. D. Adult-T315I cells were treated with BI2536 (3.0 nM) � vorinostat (1.0 �M) for 48 hr. Inset: Fractional Effect (FA) values were determined by comparing results obtained for untreated controls and treated cells following exposure to agents administered at a fixed ratio (BI25362: vor = 1:3), after which Median Dose Effect analysis was employed to characterize the nature of the interaction. E. BV173/E255K cells were treated with BI2536 (2.5 nM) � vorinostat (0.75�M) F. Primary human CML CD34+ samples were isolated as described in Methods and resuspended in medium containing 10% FCS at a cell density of 0.75 x 106/ml cells. Cells were then treated with BI2536 (5.0 nM) � vorinostat (1.0 �M) G. BaF/3 cells expressing wild-type (WT) or mutant forms of BCR/ABL (e.g., E255K, M351T, T315I) were treated with BI2536 (2.0 - 4.0 nM) � vorinostat (0.5-1.25 �M) for 48 hr. For A-G: At the end of drug exposure, the percentage of apoptotic cells was monitored by 7AAD staining as described in Methods. Values represent the means � S.D. for three separate experiments. For all studies, values represent the means for 3 experiments performed in triplicate � S.D.

History

ARTICLE ABSTRACT

Purpose: To determine whether Polo-like kinase 1 (PLK1) inhibitors (e.g., BI2536) and histone deacetylase (HDAC) inhibitors (e.g., vorinostat) interact synergistically in the BCR/ABL+ leukemia cells sensitive or resistant to imatinib mesylate (IM) in vitro and in vivo.Experimental Design: K562 and LAMA84 cells sensitive or resistant to imatinib mesylate and primary CML cells were exposed to BI2536 and vorinostat. Effects on cell viability and signaling pathways were determined using flow cytometry, Western blotting, and gene transfection. K562 and BV173/E255K animal models were used to test in vivo efficacy.Results: Cotreatment with BI2536 and vorinostat synergistically induced cell death in parental or imatinib mesylate–resistant BCR/ABL+ cells and primary CD34+ bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat cotreatment triggered pronounced mitochondrial dysfunction, inhibition of p-BCR/ABL, caspase activation, PARP cleavage, reactive oxygen species (ROS) generation, and DNA damage (manifest by increased expression of γH2A.X, p-ATM, p-ATR), events attenuated by the antioxidant TBAP. PLK1 short hairpin RNA (shRNA) knockdown significantly increased HDACI lethality, whereas HDAC1–3 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, suggesting a functional role for DNA damage in lethality. Finally, BI2536/vorinostat cotreatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL+ leukemia xenograft models and significantly enhanced animal survival.Conclusions: These findings suggest that concomitant PLK1 and HDAC inhibition is active against imatinib mesylate–sensitive or refractory CML and ALL cells both in vitro and in vivo and that this strategy warrants further evaluation in the setting of BCR/ABL+ leukemias. Clin Cancer Res; 19(2); 404–14. ©2012 AACR.