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

Abstract 1403

Resistance to imatinib is most commonly explained by acquired point mutations in the kinase domain of BCR-ABL, which impair drug binding. The more potent ABL1 inhibitors nilotinib and dasatinib have proven largely successful in imatinib-resistant chronic myeloid leukemia (CML) patients with the key exception of the T315I BCR-ABL1 mutant. Ponatinib, also known as AP24534, is an oral, the multi-targeted tyrosine kinase inhibitor (TKI) and highly activity against ABL kinase point mutation included T315I. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial (PACE trial). However, ABL1 inhibitor-resistant patients already harboring mutations have a higher likelihood of developing further mutations under the selective pressure of ABL1 TKIs. The challenge for development of an effective Philadelphia chromosome (Ph) positive leukemia therapy is therefore to develop an alternative treatment strategy that does not rely solely on kinase domain inhibition but rather results in degradation of the offending BCR-ABL1 protein regardless of its mutation status. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. Several small molecule HDAC inhibitors such as vorinostat (suberoylanilide hydroxamic acid: SAHA) are also evaluated for significant clinical activity in hematological malignancies. Therefore, combination therapy using a ponatinib and an HDAC inhibitor, vorinostat may help prevent development of ABLTKI resistance and may improve their long-term outcome. In the present study, we analyzed the ponatininb and vorinostat efficacy by using the BCR-ABL positive cell line, K562 and murine Ba/F3 cell line which was transfected with imatinib resistant BCR-ABL random mutagenesis and T315I mutant cells. 72 hours treatment of ponatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We also found that ponatinib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation. We found that phosphorylation of BCR-ABL, Crk-L was decreased and poly (ADP-ribose) polymerase (PARP) was activated in a dose dependent manner in these cells. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib caused significantly more cytotoxicity than each drug alone. We investigated the intracellular signaling of ponatinib and vorinostat. Although phosphorylation of BCR-ABL, Crk-L was not reduced after vorinostat treatment for 24 hours, acetylation of histone H4 was increased. Caspase 3 and PARP activation were increased after combination of ponatinib and vorinostat. Moreover, an increase in phosphorylation levels of γ-H2A.X was observed after treatment with ponatinib and vorinostat, compared to ponatinib or vorinostat alone suggests that combination of ponatinib and vorinostat induced DNA damage against T315I mutant cells. We next established ponatinib resistant cells by using Ba/F3 BCR-ABL with resistant random mutants. In the ponatinib resistant cell lines, IC50 of ponatinib was 200nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence and invader analysis. Ponatinib resistant Ba/F3 cells were also resistant to imatinib (IC50: more than 10μM) or nilotinib (IC50: 7.5μM) and also resistant to dasatinib (IC50: more than 100nM). We investigated the efficacy between ponatinib and vorinostat by using these cell lines. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and vorinostat caused significantly more cytotoxicity. Moreover, acetylation of histone H4, caspase 3 and PARP activation were found after combination of ponatinib and vorinostat. Data from this study suggested that administration of the ponatinib and HDAC inhibitor, vorinostat may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of ponatinib in those BCR-ABL mutant cells.