Activity of histone deacetylase inhibitors and an Aurora kinase inhibitor in BCR-ABL-expressing leukemia cells: Combination of HDAC and Aurora inhibitors in BCR-ABL-expressing cells
© Okabe et al.; licensee BioMed Central Ltd. 2013
Received: 7 September 2012
Accepted: 18 February 2013
Published: 4 April 2013
The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL-positive leukemia patients. However, resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment of these patients.
In the present study, we examined the activity of histone deacetylase (HDAC) inhibitors in combination with an Aurora kinase inhibitor in BCR-ABL-expressing cells.
We found the HDAC inhibitors vorinostat and/or pracinostat (SB939) induced apoptosis in BCR-ABL-expressing cells. Additionally, HDAC inhibitors reduced levels of Aurora A and B protein. An Aurora kinase inhibitor, tozasertib (VX-680), inhibited growth, promoted pro-apoptotic activity, reduced the phosphorylation of BCR-ABL and Crk-L, and activated caspase-3 and poly (ADP-ribose) polymerase (PARP) in BCR-ABL-positive cells. Moreover, after treatment with tozasertib, HDAC protein expression was decreased. Combination of vorinostat or pracinostat with tozasertib had a synergistic inhibitory effect on the proliferation of T315I cells. Phosphorylation of Crk-L decreased, and PARP activation increased after treatment with vorinostat or pracinostat and tozasertib. Moreover, combination of vorinostat or pracinostat and tozasertib significantly increased the extent of apoptosis in primary chronic myeloid leukemia cells.
This study demonstrated that combination of HDAC and Aurora inhibitors was highly effective against BCR-ABL-expressing cells.
Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by unregulated proliferation of predominantly myeloid cells in the bone marrow . BCR-ABL fusion proteins resulting from the chromosomal translocation t(9;22) (the Philadelphia chromosome: Ph) cause CML . BCR-ABL activity leads to uncontrolled cell proliferation, reduced apoptosis, and malignant expansion of hematopoietic stem cell populations. The ABL tyrosine kinase inhibitor (TKI) imatinib has dramatically improved the management and prognosis of patients with CML . However, some patients, particularly those with advanced-phase CML, have developed resistance to imatinib . More than 50 distinct point mutations in the kinase domain of BCR-ABL have been detected in patients with imatinib-resistant CML; point mutations in this domain are the most frequent cause of acquired imatinib resistance in CML patients [5, 6]. Second-generation TKIs, such as dasatinib and nilotinib, have shown promising results in imatinib-resistant CML patients, but dasatinib and nilotinib are not effective against CML clones with T315I mutations . Recently, ponatinib (also known as AP24534) was identified as a potent oral tyrosine kinase inhibitor and was shown to block native and mutated BCR-ABL. Ponatinib is highly active in patients with Ph-positive leukemias, including those with BCR-ABL T315I mutations . However, alternative strategies against point mutations (i.e., T315I) within the BCR-ABL kinase domain are still important to improve the prognosis of CML patients.
Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are enzymes that regulate chromatin structure and function . Modification of histones (e.g., via histone acetylation and deacetylation) plays an important role in the regulation of gene expression . Increased expression of HDACs and disrupted activities of HATs have been observed in several tumor types . HDAC inhibitors are emerging as potent antitumor agents that induce cell cycle arrest, differentiation, and apoptosis in many tumor cells of different origins. HDAC inhibitors represent a new and promising class of antitumor drugs . HDAC inhibitors influence gene expression by enhancing histone acetylation. Because HDAC inhibitors regulate many signaling pathways, cotreatment of HDAC inhibitors with molecular targeted drugs, such as Aurora kinase inhibitors, is a promising strategy against many types of tumors.
This study aimed to examine the activity of the HDAC inhibitors vorinostat and pracinostat (SB939) in vitro, both alone and in combination with an Aurora kinase inhibitor. This study also explored the molecular mechanisms underlying treatment-related cell growth inhibition and apoptosis in BCR-ABL-expressing cell lines with point mutations. We found that the combination of HDAC and Aurora kinase inhibitors significantly inhibited cell growth in BCR-ABL-expressing cells.
Results and discussion
Activity of HDAC inhibitors in BCR-ABL-positive cells
Analysis of the effects of an Aurora kinase inhibitor on intracellular signaling in K562 cells
Activity of the Aurora kinase inhibitor in wild-type and mutant BCR-ABL-expressing cells
Efficacy of cotreatment with HDAC and Aurora kinase inhibitors in BCR-ABL-expressing cells
Efficacy of cotreatment with HDAC and Aurora kinase inhibitors in BCR-ABL-positive primary CML cells
In the current study, HDAC inhibitors induced apoptosis in BCR-ABL-positive leukemia cells. In particular, profound inhibition of cell growth and induction of apoptosis were observed in response to HDAC inhibitors in BCR-ABL-positive K562 and mouse pro-B Ba/F3 cells with ectopic expression of wt and mutant T315I. This response was amplified by cotreatment with an Aurora kinase inhibitor. In this study, we also demonstrated that Aurora kinase proteins were degraded by vorinostat or pracinostat in a dose-dependent manner (Figure 1B). Although the levels of Aurora family proteins were not directly reduced by tozasertib treatment, tozasertib inhibited the expression of HDAC proteins (Figure 2A). As such, our data indicated that vorinostat or pracinostat and tozasertib affected the activities of both Aurora kinase and HDAC, in turn increasing antitumor activity in this system. Clinical trials using tozasertib have been discontinued. However, other pan-Aurora/BCR-ABL dual inhibitors may exhibit a similar profile, and these continue to be studied clinically. Our findings suggest that cotreatment with these compounds and specific molecular-targeted drugs could benefit patients with leukemic BCR-ABL cells that are resistant to more conventional treatments.
Reagents and antibodies
The HDAC inhibitors vorinostat (SAHA, suberoylanilide hydroxamic acid) and pracinostat (SB939) were provided by Selleck Chemicals LLC (Houston, TX). Tozasertib (VX-680, MK-0457) was kindly donated by Vertex Pharmaceuticals Inc (Cambridge, MA). Stock solutions of vorinostat, pracinostat, and tozasertib were dissolved in dimethyl sulfoxide and subsequently diluted to the desired concentration in growth medium. Anti-phospho-Abl, -phospho-Crk-L, -cleaved caspase 3, -PARP-HDAC1, -HDAC2, -HDAC5, -HDAC7, -Bim, and -Aurora A and B antibodies (Abs) were obtained from Cell Signaling Technology (Beverly, MA). Other reagents were obtained from Sigma (St. Louis, MO).
The human CML cell line K562 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Ba/F3 wt-BCR-ABL cells and Ba/F3 T315I cells were described previously . These cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum with 1% penicillin/streptomycin in a humidified incubator at 37°C.
Cell proliferation assay
Cell proliferation analysis was performed as previously described .
Cell signaling assays and western blot analysis
Panorama Ab microarrays (Sigma) were analyzed according to the manufacturer’s instructions. The arrays were scanned using a GenePix Personal 4100A microarray scanner, and normalization was carried out using the housekeeping protein included with the chip. The protein expression ratio was calculated using MS Excel. Western blot analysis was performed as previously described [26, 27].
DNA microarray and microarray data analysis
DNA microarray analysis was performed as previously described . In brief, K562 cells were treated with 1 μM tozasertib for 16 h. Following incubation at 37°C, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and collected immediately for RNA isolation. In this study, we used the Human Genome U133A Genechip (Affymetrix Santa Clara, CA), which contains more than 47,000 transcripts. Target preparation was carried out following the manufacturer’s expression analysis manual. All arrays were screened for quality by standard methods, and the mean fluorescent intensity for each probe set was determined.
This study was approved by the Institutional Review Board of Tokyo Medical University, and informed consent was provided by all patients in accordance with the Declaration of Helsinki. Primary samples were obtained from the peripheral blood of CML patients. Mononuclear cells were isolated from blood samples and separated by Lymphosepar (Immuno-Biological Laboratories Co., Gunma, Japan). The cells were cultured in RPMI1640 medium containing 10% fetal calf serum and analyzed as described.
Flow cytometory analysis
Cells were treated with the indicated concentrations of tozasertib for 48 h. Annexin V/propidium iodide apoptosis assays were performed according to the manufacturer’s instructions (Beckman Coulter, Inc., Brea, CA). The cells were gently mixed and immediately analyzed by flow cytometry.
Differences between treatment groups, in terms of dose response and apoptosis, were determined using Student’s t test. P values of less than 0.05 were considered significant.
This work was supported by a High-Tech Research Center Project for private universities, a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and by the University-Industry Joint Research Project for private universities, a matching fund subsidy from MEXT. This work was also supported by Grants-in-Aid for Scientific Research from MEXT.
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