Comparison of Imatinib, AMN107 and Dasatinib in an Accelerated Cell-Based Mutagenesis Screen.
Authors: Deininger Michael W.N. , Bradeen Heather , Jia Taiping , O’Hare Thomas , Willis Stephanie G. , Lee Frank , Druker Brian J. 
Published: April 2020

Background. Mutations in the Bcr-Abl kinase domain (KD) are the leading cause of acquired imatinib (IM) resistance. Dasatinib (BMS354825) and AMN107 are potent alternate Abl inhibitors with activity at nanomolar levels against wild type Bcr-Abl and most KD mutants, with the exception of T315I. In a cell-line based mutagenesis assay we compared incidence and type of Bcr-Abl mutants emerging in the presence of IM, AMN107 and dasatinib.

Methods. BaF3-p210Bcr-Abl cells were mutagenized by 24 hours exposure to 0.42 μM N-ethyl-N-nitrosourea (ENU), a dose with minimal cytotoxicity. After ENU washout cells were seeded at 5 x 105/well in 96-well plates and observed for growth for up to 4 weeks. Cells from wells with growth were expanded and subjected to BCR-ABL KD sequencing.

Results: The frequency of wells with growth decreased with higher doses of all 3 inhibitors (table 1) and growth tended to occur later. Only isolated wells had growth without ENU exposure. At ≥2 μM IM (2-fold the IC90 in cell proliferation assays) 18 different mutations were seen, with highly resistant mutants prevailing at higher concentrations (table 2). At 50 nM AMN107 (2-fold the IC90) Y253H, G250E, F359C, E255K, L384M, L387F, E292V and T315I were detected, at 500 nM Y253H, E255V and T315I were recovered and only T315I at 2000 nM. At 5 nM dasatinib (2-fold the IC90), E255K, L284V, F317V were detected in addition to T315I, at 10 nM T315I, F317V/I and V299L were found and at 25 nM only T315I. All resistant clones growing out at ≥4 μM IM, 500 nM AMN107 or 10 nM dasatinib were KD mutant, suggesting that KD mutations were the sole cause of the observed resistance.

Conclusions: (i) At drug concentrations corresponding to 2-fold IC9018 different mutations were recovered with IM, 9 with AMN107 and 6 with dasatinib, suggesting that the conformational requirements for dasatinib binding to Abl may be least stringent. If free plasma trough levels ≥25 nM dasatinib or ≥2000 nM AMN107 are achievable, the only mutant predicted to emerge clinically is T315I. (ii) No additional mutations were observed with AMN107 compared to IM, suggesting the structural changes in AMN107 compared to IM did not generate novel vulnerable sites. (iii) At least in this in vitro model, resistance to Abl kinase inhibitors is entirely dependent on Bcr-Abl, despite the fact that ENU treatment is expected to induce multiple additional mutations. Thus a T315I inhibitor combined with AMN107 or dasatinib may be effective at preventing the emergence of resistance to Abl kinase inhibitors.

Table 1 Recovery of resistant clones (representative experiment) Imatinib (microM) Wells with mutations/wells sequenced/wells with growth Dasatinib (nM) Wells with mutations/wells sequenced/wells with growth AMN107 (nM) Wells with mutations/wells sequenced/wells with growth 2 62/62/82 5 8/24/96 10 0/24/96 4 74/74/74 10 38/38/56 50 20/24/96 8 27/27/42 25 22/22/24 500 45/45/46 16 12/12/12 100 20/20/21 2000 23/23/24

Table 2 Percentage of resistant clones with T315I mutations Imatinib (μM) number of different mutations/% T315I Dasatinib (nM) number of different mutations/% T315I AMN107 (nM) number of different mutations/% T315I 2 16/27.8 5 4/16.7 10 0/0.0 4 7/43.2 10 3/63.2 50 8/20.8 8 4/37.4 25 1/100 500 3/62.0 16 4/50.0 100 1/100.0 2000 1/100.0