Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug combinations

Abstract

BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to F317V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.

Figure 1.

Prevalence of KD mutations in resistant cell clones. ENU-treated Ba/F3-p210^BCR-ABL cells were cultured in the presence of graded concentrations of nilotinib, dasatinib, or imatinib mesylate. (A) At higher drug concentrations, only KD mutant clones were detected. (B) T315I is the single mutation that persists in highest concentrations of nilotinib and dasatinib.

Figure 2.

Spectrum of point mutations in patients with imatinib mesylate resistance compared with mutations recovered from in vitro assay in the presence of imatinib mesylate. Ba/F3-p210^BCR-ABL exposed to ENU and cultured in the presence of imatinib mesylate at graded concentrations. Bars represent the frequencies of mutations that occur in patients with imatinib mesylate resistance, as summarized by Hughes et al. Asterisks denote mutations that appeared in the screen but that have not been described clinically.

Figure 3.

Outgrowth of resistant cells in the presence of inhibitor combinations. (A) Number of wells with resistant growth in the presence of imatinib mesylate 1 μM and 2 μM combined with graded concentrations of nilotinib (50 nM, 500 nM, 1000 nM, and 2000 nM). (B) Number of wells with resistant growth in the presence of imatinib mesylate 1 μM and 2 μM combined with graded concentrations of dasatinib (5 nM, 10 nM, 25 nM, and 100 nM). (C) Number of wells with resistant growth in the presence of graded concentrations of nilotinib (50 nM, 500 nM, 1000 nM, and 2000 nM) and dasatinib (5 nM, 10 nM, 25 nM, and 100 nM).