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PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

ABSTRACT: 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.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.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 ?H2A.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.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.

SUBMITTER: Dasmahapatra G

PROVIDER: S-EPMC3548959 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

Dasmahapatra Girija G Patel Hiral H Nguyen Tri T Attkisson Elisa E Grant Steven S

Clinical cancer research : an official journal of the American Association for Cancer Research 20121130 2

<h4>Purpose</h4>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.<h4>Experimental design</h4>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 us ...[more]

PMID: 23204129

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Project description:BACKGROUND:Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi. RESULTS:We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski's "rule of five," in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib co-treatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib. CONCLUSIONS:Collectively, the present findings show that combinations HDAC inhibitor-imatinib are likely to overcome drug resistance in CML pathology.

Project description:BackgroundWith the widespread clinical application of tyrosine kinase inhibitors (TKIs), an increasing number of chronic myeloid leukaemia (CML) patients have developed resistance or intolerance to TKIs. BCR/ABL is the oncoprotein of CML. HSP90 is an essential chaperone of BCR/ABL and plays an important role in protein folding and the function of BCR/ABL. Therefore, inhibiting the chaperone function of HSP90 may be an effective strategy for CML treatment and to overcome TKI resistance.MethodsThe effect of KW-2478 on CML cell viability, apoptosis and cell cycle progression was detected by CCK-8 assay or flow cytometry. The levels of BCR/ABL, HSP90 and other signalling proteins were detected by western blots. The mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The interaction between BCR/ABL and HSP90α was detected by coimmunoprecipitation. The effect of KW-2478 on BCR/ABL carcinogenesis in vivo was investigated in CML-like mouse models.ResultsKW-2478 inhibited growth and induced apoptosis of CML cells. KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. This treatment also caused an increase in p27 and p21 expression and a decrease in cyclin B1 expression, which led to G2/M phase arrest. The mitochondrial pathway was primarily responsible for KW-2478-induced apoptosis. KW-2478 had a synergistic effect with imatinib in growth inhibition. Notably, KW-2478 had a stronger effect on growth inhibition, apoptosis induction and cell cycle arrest of K562/G01 cells than K562 cells. KW-2478 could effectively prolong the mouse lifespan and alleviate disease symptoms in CML-like mouse models.ConclusionsThis finding demonstrated that KW-2478 had anticancer properties in imatinib-sensitive and imatinib-resistant CML cells and illustrated the possible mechanisms. This study provides an alternative choice for CML treatment, especially for TKI-resistant patients with BCR/ABL amplification and TKI-intolerant patients.

Dataset Information

PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

Publications

PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

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