Combined Effects of a Pan-ABL1 Kinase Inhibitor, Ponatinib and Dasatinib Against T315I Mutant Forms of BCR-ABL1: In Vitro and In Vivo Studies
Authors: Tauchi Tetsuzo , Katagiri Seiichiro , Okabe Seiichi , Ashihara Eishi , Kimura Shinya , Maekawa Taira , Ohyashiki Kazuma 
Published: October 2019

Abstract
Abstract 2482
Ponatinib is a third-generation ABL1 kinase inhibitor optimized using structure-based drug design to bind the inactive, DFG-out conformation of wild-type (WT)-BCR-ABL1 and T315I BCR-ABL1. Ponatinib provides an effective in minimizing resistance, however, certain BCR-ABL1 compound mutations such as Y235H/T315I and E255K/T315I are predicted to be recalcitrant to ponatinib. Combining ponatinib and dasatinib may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant compound mutations. In the present study, we investigated the combined effects of ponatinib and dasatinib in mutant forms of BCR-ABL1-expressing cells. Co-treatment with ponatinib and dasatinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to ponatinib and dasatinib including BaF3 cells expressing T315I. Initially, we performed random mutagenesis for BCR-ABL1 experiments (Blood, 109; 5011, 2007). Single-agent ponatinib completely suppressed outgrowth on resistant clones at 40 nM. As the immediate clinical application of ponatinib is in refractory BCR-ABL1 positive leukemia patients harboring T315I BCR-ABL1 mutation, we next performed resistant screens starting from T315I BCR-ABL1 BaF3 cells to identify BCR-ABL1 compound mutations. Compared with single agents, the combination of ponatinib and dasatinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of ponatinib (2 micro M), the compound mutation narrowed to Y253H/E225K/T315I in single cell by direct sequencing. To assess the mechanism of combination effect between ponatinib and dasatinib on T315I BCR-AB1L compound mutation-expressing cells, we used RNA interference to determine whether reduction of dasatinib off-target molecules such as KHS, Raf-1, B-Raf, ACK-1, FAK, EPHB4 may affect the growth inhibition. BaF3 cells expressing T315I BCR-ABL1 pretreated with Raf-1or EPHB4 siRNA showed enhanced growth inhibition with ponatinib. These results demonstrate that the enhanced growth inhibition by ponatinib and dasatinib in T315I-expressing cells may be mediated by Raf-1 and EPHB4. To assess the in vivo efficacy of ponatinib and dasatinib, athymic nude mice were injected s.c. with BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 5 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, ponatinib (30 mg/kg; q.d.), dasatinib (10 mg/kg; q.d.), ponatinib (30 mg/kg; q.d.) + dasatinib (10 mg/kg; q.d.). The ponatinib and dasatinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or ponatinib- or dasatinib-treated mice. Histopathologic analysis of tumor tissue from ponatinib+dasatinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. This study provides comparative drug combination profiling analysis of the effect of ponatinib and dasatinib, these two compounds are clinical active for BCR-ABL1-positive leukemia. Both agents commonly target a number of important molecular pathways that regulate cell growth and survival. A single proapoptotic or antiproliferative pathway may not be critical for the therapeutic effects of ABL1 kinase inhibitors. The present findings have important implications for the clinical use of ponatinib and dasatinib as anti-leukemia agent either alone or in combination with other agents.

Disclosures:
No relevant conflicts of interest to declare.