Complete Suppression of in Vitro Resistance by AP24534, a Pan-BCR-ABL Inhibitor

Abstract

The BCR-ABL inhibitor imatinib is front-line therapy for chronic myeloid leukemia (CML). The second-line inhibitors dasatinib and nilotinib provide treatment options for controlling imatinib-resistant CML associated with BCR-ABL kinase domain mutations. However, the T315I mutant of BCR-ABL is resistant to all 3 clinical inhibitors, and is a frequent cause of salvage therapy failure. AP24534 is an oral, multi-targeted kinase inhibitor with activity against native and kinase domain-mutant BCR-ABL, including T315I. We have previously utilized an in vitro mutagenesis-screening assay to successfully predict the profile of mutations that confer resistance to imatinib, dasatinib, and nilotinib in patients. Here we use the in vitro mutagenesis screen to test whether BCR-ABL mutants can emerge in the presence of AP24534.Methods: To determine a resistance profile for AP24534, Ba/F3 cells expressing native BCR-ABL were mutagenized with ENU, washed, and plated in the presence of graded concentrations of AP24534 (5–80 nM). For each condition, 4.8×107 mutagenized cells were distributed into 480 wells and observed for growth for 4 weeks. Resistant clones were expanded in the continued presence of AP24534 and sequenced for mutations in the BCR-ABL kinase domain.Results: We first established IC50 values for inhibition of proliferation of Ba/F3 cells expressing native BCR-ABL (IC50: 0.5 nM) and an extensive panel of imatinib-resistant BCR-ABL mutants (IC50 range: 0.5 nM to 35.7 nM) including T315I (IC50: 11.4 nM) and E255V (IC50: 35.7 nM). Parental Ba/F3 cells were not inhibited up to a concentration of 1713 nM AP24534. Corresponding immunoblot analyses confirmed the same rank order for effective inhibition of CrkL phosphorylation in cells expressing native BCR-ABL, the T315I mutant, or the E255V mutant. Inhibition of CrkL phosphorylation was also demonstrated with primary hematopoetic cells from CML patients harboring native BCR-ABL or the T315I mutant. In the mutagenesis screen starting with Ba/F3 cells expressing native BCR-ABL, resistant clones recovered in 10 nM AP24534 expressed native BCR-ABL or one of several imatinib-resistant BCR-ABL mutants (168/1440 wells in 3 independent experiments). By contrast, when the screen was conducted in the presence of 20 nM AP24534, the frequency of outgrowth of escape mutants was extremely low and limited to cells expressing the T315I mutant (2/1440 wells) or the E255V mutant (1/1440 wells). Remarkably, outgrowth was completely suppressed by 40 nM AP24534.Conclusions: AP24534 is a potent inhibitor of native BCR-ABL and all tested BCR-ABL mutants, including T315I. Mutagenesis screening reveals that single-agent AP24534 (40 nM) completely suppressed outgrowth of escape mutants. This is in marked contrast to any of the BCR-ABL inhibitors previously profiled in this assay, where outgrowth was evident at the highest tested drug concentrations and complete suppression was observed only when dasatinib or nilotinib was combined with an investigational T315I inhibitor (PNAS 2008; 105: 5507). As sequential BCR-ABL kinase inhibitor therapy has been linked to selection of rare subclones in which 2 mutations occur in the same BCR-ABL molecule, compound mutations are potentially capable of thwarting any of the current clinical BCR-ABL inhibitors, even in combination. Front-line therapy with a pan-BCR-ABL inhibitor could improve the depth and durability of responses by preventing selection of drug-resistant kinase domain point mutants. Our pre-clinical profiling indicates that AP24534 is an important new option in controlling resistance in CML. A phase 1 clinical trial designed to evaluate AP24534 treatment in patients with refractory CML and other hematologic malignancies has recently commenced.