Resistance to current Bcr-Abl kinase inhibitors in CML patients is associated with the emergence of Bcr-Abl point mutations, including the T315I variant which represents 15–20% of clinically observed mutants and is resistant to all approved agents (e.g. imatinib and dasatinib). AP24534 is a novel, orally active Bcr-Abl inhibitor that also potently inhibits T315I and other clinically relevant mutants. We previously showed that daily oral administration of AP24534 can induce complete regression of T315I-expressing tumors in mice. In this study, we set out to characterize the antitumor activity of AP24534 through correlative in vitro and in vivo efficacy and pharmacodynamic studies. AP24534 inhibited the kinase activity of unmutated and T315I Abl enzymes with IC50s of 12 and 58 nM respectively, and inhibited the proliferation of their respective Bcr-Abl Ba/F3-derived cell lines with IC50s of 1 and 8 nM in a 3-day assay. Exposure to AP24534 for three hours led to the inhibition of Bcr-Abl phosphorylation with IC50s of 25 and 78 nM, respectively, with substantial apoptosis observed within 24 hours, suggesting that short term exposure to AP24534 is sufficient for efficacy. In mononuclear cells isolated from 3 patients with the T315I mutation (2 CML patients in blast crisis and a Ph+ ALL patient), treatment with 50 nM AP24534 resulted in a >50% decrease in phospho-CrkL levels. To further investigate the relationship between dose, exposure and response, we tested the activity of AP24534 on the human CML cell line K-562. In vitro, exposure of K-562 cells to 60 nM AP24534 for only 3 hr was sufficient to reduce cell proliferation by >90% when measured 3 days later. Oral administration of AP24534 to mice bearing K-562 xenografts inhibited tumor growth in a dose-dependent manner; a 2.5 mg/kg daily dose induced complete tumor regression. Bcr-Abl phosphorylation was only transiently inhibited at this dose, confirming that sustained target inhibition is not required for antitumor activity. Indeed, complete regression could also be achieved by intermittent (twice-weekly) dosing. Compared to AP24534, dasatinib required significantly higher transient exposure levels to achieve >90% K-562 cell killing in vitro (300–600 nM), and showed reduced efficacy in the xenograft model when dosed daily or intermittently. Pharmacokinetic analyses showed that the longer half-life of AP24534 versus dasatinib results in higher levels of exposure in mice. The in vitro kinase specificity profile of AP24534 suggests that Bcr-Abl inhibition is the dominant mechanism for its activity in CML models. Specifically, it is inactive against Aurora kinases (IC50 > 1000 nM). AP24534 potently inhibits a discrete subset of other kinases, including Flt3 and members of the Src, VEGFR and FGFR families (IC50s = 0.4–58 nM), activities which may contribute to its potency against CML and suggest the potential for efficacy in other tumor types. Together these data indicate that AP24534 has the potential to be an effective treatment for CML, including in patients refractory to current targeted agents. A phase 1 clinical trial is being planned.

Author notes

Disclosure:Employment: VMR, QX, FW, JS, JK, SL, YN, SW, KR, MB, WCS, JDI and TC are employees of ARIAD Pharmaceuticals. Consultancy: BJD is a consultant for ARIAD Pharmaceuticals, Breakthrough Therapeutics and SGX Pharmaceuticals. Ownership Interests: BJD has equity interests in Molecular MD (scientific founder) and Breakthrough Therapeutics (consultant).; VMR, QX, FW, JS, JK, SL, YN, SW, KR, MB, WCS, JDI and TC are employees of ARIAD Pharmaceuticals and have an ownership interest. Research Funding: BJD is currently the principal investigator on several Novartis and Bristol-Myers-Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab receive funds from these contracts.