Synergistic Cytotoxicity Between Panobinostat and Ponatinib In Imatinib-Resistant Chronic Myeloid Leukemia Cells
Authors: Matsuda Yasufumi , Yamauchi Takahiro , Uzui Kanako , Negoro Eiju , Morinaga Koji , Shigemi Hiroko , Nishi Rie , Kimura Shinya , Maekawa Taira , Ueda Takanori 
Published: October 2019

Abstract
Current first-line treatment options for chronic myeloid leukemia (CML) include imatinib (IM) and the second-generation agents nilotinib and dasatinib. Despite the effectiveness of these tyrosine kinase inhibitors, a small percentage of chronic-phase CML patients are primarily refractory or acquire secondary resistance to these agents. Moreover, the prognosis of patients in blast crisis is still poor despite the use of the current treatment modalities because of drug resistance. The major mechanism of drug resistance is the re-activation of ABL kinase either through mutations in the BCR-ABL gene or by BCR-ABLgene amplification.
A novel pan-histone deacetylase inhibitor, panobinostat (formerly LBH589), induces the acetylation of heat shock protein 90, thereby inhibiting its chaperone function in association with its client proteins BCR-ABL, leading to the degradation of BCR-ABL. A new pan–ABL tyrosine kinase inhibitor, ponatinib, is a promising therapeutic option in patients with all kinds of BCR-ABLmutation including T315I. It was thus hypothesized that the combination of panobinostat and ponatinib exerts synergistic cytotoxicity against IM-resistant cells through a mechanism of action different from that of each agent. To test this hypothesis, K562/IM-R1 and Ba/F3/T315I cell lines were evaluated for the cytotoxicity of panobinostat and ponatinib in vitro. K562/IM-R1 cells, established in our previous study, showed BCR-ABL overexpression due to BCR-ABL gene amplification. The Ba/F3/T315I cell line showed BCR-ABL with a T315I mutation.
The XTT proliferation assay revealed that K562/IM-R1 cells were 12-fold more IM-resistant (50%-inhibitory concentration (IC50), 7.6 µM) than K562 cells (IC50, 0.6 µM). Ba/F3/T315I cells were refractory to IM treatment (IC50, 34.1 µM) compared with Ba/F3 cells (IC50, 3.4 µM). Panobinostat overcame IM-resistance and inhibited similarly the growth of K562, K562/IM-R1, Ba/F3, and Ba/F3/T315I cells, with IC50 values of around 50 nM (40.0 - 51.0 nM). Ponatinib inhibited the growth of both K562/IM-R1 cells (IC50, 4 nM) and Ba/F3/T315I cells (25 nM) as potently as parental K562 cells (IC50, 2 nM) and Ba/F3 cells (IC50, 5 nM). Importantly, the combination of panobinostat and ponatinib exhibited enhanced growth inhibition effects on all cell lines. The IC50 values for this combination were 0.7 nM for K562 cells, 1.3 nM for K562/IM-R1 cells, 3.7 nM for Ba/F3 cells, and 10 nM for Ba/F3/T315I cells. The combination index clearly showed synergism, with values of 0.5 for K562 cells, 0.28 for K562/IM-R1 cells, 0.9 for Ba/F3 cells, and 0.4 for Ba/F3/T315I cells. When the cells were treated with 10 nM panobinostat or 10 nM ponatinib for 48 h alone or in combination, the combination of the 2 agents led to greater-than-additive apoptotic cell death than each agent alone in all cell lines, evaluated by annexin V-positivity.
Western blotting was used to evaluate the protein expression levels of BCR-ABL and phospho-BCR-ABL in cells after treatment with panobinostat or ponatinib or both in combination. BCR-ABL expression was greater in K562/IM-R1 than K562 cells. Phospho-BCR-ABL expression was not inhibited by IM in K562/IM-R1 or Ba/F3/T315I cells. However, ponatinib inhibited the autophosphorylation of BCR-ABL in these cell lines. Treatment with panobinostat reduced the BCR-ABL and phospho-BCR-ABL expression levels in K562/IM-R1 and Ba/F3/T315I cells. The combination of the 2 agents augmented inhibition of the autophosphorylation of BCR-ABL in these IM-resistant cell lines.
The activity of histone deacetylase, determined using the HDAC assay Kit (Active Motif, Carlsbad, CA, USA), was inhibited by panobinostat in all cell lines regardless of IM sensitivity. In comparison, IM did not alter cellular histone deacetylase activity. Upon treatment of K562 cells with panobinostat, the protein expression levels of acetylated histone H3 and H4 were increased, suggesting the consequence of the inhibition of histone deacetylase.
In conclusion, we firstly reported that panobinostat and ponatinib demonstrated synergistic cytotoxicity against IM-resistant cell lines not only due to BCR-ABL gene amplification but also BCR-ABL T315I mutation. The synergism is attributable to the greater inhibition of ABL kinase activity through a mechanism of action different from that of each agent.

Disclosures:
No relevant conflicts of interest to declare.