Combining the ABL1 Kinase Inhibitor Ponatinib and the Histone Deacetylase Inhibitor Vorinostat: A Potential Treatment for BCR-ABL-Positive Leukemia

<div><p>Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR-ABL mutant cells.</p></div

Ponatinib and vorinostat increase cell growth inhibition and induce apoptosis in BCR-ABL-expressing cells.

<p>(A) Cells were cultured at a concentration of 8×10⁴/mL in the presence or absence of ponatinib and/or vorinostat for 72 h. The percentage of proliferating cells was evaluated, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089080#s2" target="_blank">Materials and Methods</a>. (B) K562 or Ba/F3 T315I cells were treated with ponatinib and/or vorinostat for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, Crk-L, γH2A.X, cleaved caspase 3, PARP, acetyl histone H4, and tubulin antibodies. (C) Primary samples were cultured in the presence or absence of ponatinib and/or vorinostat. The number of living cells was calculated. *<i>P</i><0.05 or **<i>P</i><0.01, ponatinib and vorinostat treatment versus ponatinib treatment. (D) Primary cells were treated with ponatinib and/or vorinostat at the indicated concentrations for 24 h. The cells were examined by immunoblot analysis.</p

Effect of ponatinib on BCR-ABL-expressing cells.

<p>(A) K562 cells were cultured at a concentration of 8×10⁴/mL in the presence of varying concentrations of ponatinib for 72 h. The number of viable cells was calculated. Results are representative of three separate experiments. *<i>P</i><0.05, ponatinib treatment versus control. (B) K562 cells were treated with ponatinib for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, anti-Crk-L, anti-cleaved caspase 3, anti-PARP, and anti-β-tubulin antibodies. (C) Ba/F3 T315I cells were cultured in the presence of varying concentrations of ponatinib for 72 h. The number of viable cells was calculated. Results are representative of three separate experiments. *<i>P</i><0.05, ponatinib treatment versus control. (D) Ba/F3 T315I cells were treated with ponatinib for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, anti-Crk-L, anti-cleaved caspase 3, anti-PARP, and anti-tubulin antibodies.</p

Effects of ponatinib and vorinostat on Ba/F3 T315I cells in a xenograft model.

<p>(A) <i>In vivo</i> studies were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089080#s2" target="_blank">Materials and Methods</a>. At various time points, tumor sizes were reported. Average tumor weight per mouse was calculated and used to calculate the group mean tumor weight ± s.d (n = 5). (B) Immunohistochemical staining for hematoxylin and eosin (HE) at a magnification of 40×, Ki67 (magnification, 400×), and TUNEL (magnification, 400×) in Ba/F3 T315I xenograft sections. (C) Tumor cells treated with or without ponatinib and vorinostat were examined by immunoblot analysis.</p

Analysis of ponatinib-resistant Ba/F3 BCR-ABL cells.

<p>(A) Ba/F3 ponatinib-resistant cells were treated with various concentrations of imatinib, nilotinib, or dasatinib, and cellular growth was analyzed. These experiments were performed in triplicate. (B) Ba/F3 ponatinib-resistant cells were treated with imatinib, nilotinib, or dasatinib for 24 h. Whole cell lysates were analyzed by immunoblotting with phospho-specific Abl and Crk-L Abs. β-tubulin was used as the loading control.</p

Lentivirus-based reporter system.

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without GM-CSF stimulation <i>in vivo</i>. *:<i>P</i><0.05, n = 3. Data are representative of two independent experiments. B. Schematic illustration of the lentivirus vector for reporter assays. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. C. Elongation factor-1 (EF-1) promoter activity in bone marrow cells revealed by the lentivirus-based reporter system. Bone marrow cells were analyzed by flow cytometry after two days incubation with GM-CSF following viral infection. Shaded histogram, promoter-less control; open histogram, EF-1 promoter.</p

Involvement of CREB-C/EBPβ pathway in candidemia-induced “emergency granulopoiesis.”

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without candidemia. *:<i>P</i><0.05, n = 3. B. Western blotting analysis of c-kit+ bone marrow cells with or without candidemia. C. Chromatin immunoprecipitation of CREB using c-kit+ bone marrow cells during candidemia induced emergency granulopoiesis. Data are representative of two independent experiments.</p

Body weights of apo E-knockout mice treated with PBS or 15d-PGJ₂ from 8 to 16 weeks of age.

<p>From the 8th week to 16th week, female mice were randomized to receive a Western-type diet and PBS or 1 mg/kg/day of 15d-PGJ₂ (n = 15 animals for each group). At 16th week, body weight of 15d-PGJ2 treated mice tended to be higher than controls, but it was not significantly different (21.6±4.2 g and 21.2±3.9 g, respectively, <i>p</i> = 0.6). Statistical analyses were performed with Student's t test.</p

Flow cytometric analysis of the C/EBPβ proximal promoter region using the lentivirus-based reporter system.

<p>A. Schematic illustration of the lentivirus vector. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. B and C. Bone marrow cells were transduced with the lentivirus vector containing the indicated fragment and were analyzed for Thy1.1 and d2EGFP expression after 48 hours stimulation with GM-CSF. D and E. Effects of the mutated CREs at −110 and −65 bp on the activity of the promoter fragment (−243 to +16 bp). Shaded histogram, promoter-less control; open histogram, promoter fragment. Wt, wild type; mut, mutated. *:<i>P</i><0.05, **:<i>P<0.01</i> (n = 3). Data are representative of three independent experiments.</p

Serum lipid levels in the controls and the 15d-PGJ₂ group.

<p>Blood was collected from the cardiac cavity of mice aged 16 wk and analyzed for the lipid profile. The plasma chylomicron (CM) (B), very low density lipoprotein (VLDL) (C), low density lipoprotein (LDL) (D), and high density lipoprotein (HDL) (E) levels were determined by use of a high-sensitivity lipoprotein-profiling system by high-performance liquid chromatography. The total serum cholesterol level (A) was significantly lower in the15d-PGJ₂ group than in the control group (795.5±39.31 mg/dl vs 944.1±49.04 mg/dl, <i>p</i> = 0.029). Especially LDL was significantly reduced in the 15d-PGJ₂ group (186.9±13.49 mg/dl vs 234.3±16.60 mg/dl, <i>p</i> = 0.0397). CM, VLDL and HDL were not different between the control and 15d-PGJ₂ groups, 36.96±4.999 mg/dl vs 68.13±23.98 mg/dl, 553.5±26.67 mg/dl vs 622.7±28.02 mg/dl, 18.14±1.264 mg/dl vs 19.01±2.562 mg/dl, respectively. *<i>p</i><0.05, with Student's <i>t</i> test.</p