Tozasertib treatment inhibited cell growth in mutant BCR ABL expressing cells in a dose dependent manner data not shown. Next, we used flow cytometry with annexin V to examine whether tozasertib could induce apoptosis in BCR ABL expressing cells. Tozasertib induced apoptosis in the BCR ABL ex pressing selleck compound cell line K562. We also examined intracellular signaling. The phosphorylation of Abl and Crk L was decreased after tozasertib treatment. Caspase 3 and PARP levels were significantly increased. Similarly, the phosphorylation of Abl and Crk L was decreased, while caspase 3 and PARP expression levels were increased in BCR ABL expressing Ba F3 cells. These results indicated that tozasertib was effective in cell expressing wt BCR ABL and BCR ABL mutants like T315I.

Efficacy of cotreatment with HDAC and Aurora kinase inhibitors in BCR ABL expressing cells Next, we examined the intracellular signaling of HDAC and Aurora kinase inhibitors. The expression of Aurora A and B was reduced after cotreatment with vorinostat or pracinostat and tozasertib. Survivin expression was also decreased, while PARP was activated after cotreatment with vorinostat or pracinostat and tozasertib. These results suggested that vorinostat or pracinostat affected Aurora kinase expression, while treatment with vorinostat or pracinostat and tozasertib regulated intracel lular signaling pathways in BCR ABL positive cells. An in creased frequency of BCR ABL point mutations has been found in advanced phase and recurrent cancers. T315I and P loop mutations, such as G250E, Y253F, and E255K, are highly resistant phenotypes.

Next, we investi gated whether cotreatment with vorinostat or pracinostat and tozasertib caused growth inhibition in Ba F3 T315I cells and wt BCR ABL positive K562 cells. Ba F3 T315I and K562 cells were treated with vorinostat or pracinostat and tozasertib, and cell proliferation was examined. We found that cotreatment with vorinostat or pracinostat and tozasertib significantly inhibited cell growth in both wt BCR ABL positive cells and T315I positive cells. We also performed statistical analyses to deter mine the combination index for vorinostat or pracinostat and tozasertib, which was calculated according to the method of Chou and Talalay. Combination of vorinostat or pracinostat with tozasertib resulted CI values of 0. 396 and 0. 765.

These results suggested that combin ation of vorinostat or pracinostat with tozasertib synergis tically enhanced the toxicities of these drugs in T315I positive Ba F3 cells. Thus, we demonstrated that tozasertib combined with vorinostat or pracinostat could potentially overcome imatinib resistance in mutant BCR ABL expressing cells. Although high Carfilzomib concentrations of compounds were used in these experiments, signifi cantly higher plasma concentrations of these com pounds have been reported in clinical trials.