Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation


The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30–50% of cases of adult acute lymphoblastic leukaemia1. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival2, but acquired drug resistance remains a challenge3,4,5. Point mutations in the ABL1 kinase domain weaken inhibitor binding6 and represent the most common clinical resistance mechanism. The BCR–ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib7,8, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR–ABL1-driven leukaemia. Axitinib potently inhibited BCR–ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

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Figure 1: Ex vivo drug sensitivity and resistance testing (DSRT) of primary leukaemic cells establishes axitinib as a selective BCR-ABL1(T315I) inhibitor.
Figure 2: Co-crystal structures of axitinib bound to ABL1, ABL1(T315I) and VEGFR2 demonstrating different binding conformations.
Figure 3: Axitinib suppresses BCR–ABL1(T315I) autophosphorylation and proliferation of Ba/F3 cells expressing BCR–ABL1(T315I).
Figure 4: Axitinib potently and selectively targets BCR–ABL1(T315I)-expressing patient cells.

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Protein Data Bank

Data deposits

X-ray crystallographic coordinates and structure factor files for axitinib–ABL1(T315I) and axitinib–wild-type-ABL1 complexes have been deposited in the Protein Data Bank under accession numbers 4TWP and 4WA9, respectively.


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We would like to thank the patients and their families for participating in this study and donating their samples for research. We acknowledge the High Throughput Biomedicine Unit at the Institute for Molecular Medicine Finland (FIMM) for technical assistance, the laboratory of C. Gambacorti-Passerini (University of Milano-Bicocca) for engineered Ba/F3 cell mutant panel profiling data, and T. Lundán (Department of Clinical Chemistry and TYKSLAB, Turku University Central Hospital, University of Turku) for the BCR–ABL1(T315I) transcript-level quantification. This work was supported by the Jane and Aatos Erkko Foundation (to K.W.), Academy of Finland (K.W. and O.K.), Finnish Cancer Societies (O.K. and K.P.), Sigrid Juselius Foundation (O.K.), Instrumentarium Foundation (M.K.), and FinPharma Doctoral Program-Drug Discovery section (T.P.).

Author information




T.P., K.P., B.W.M. and K.W. conceived the study, designed experiments and wrote the manuscript. T.P. performed the DSRT and ex vivo assays on patient samples and associated data analysis. E.J., C.C., M.M. and B.W.M. designed, performed and interpreted the crystallography experiments. M.K. and K.P. coordinated the sampling of patient material, collection of associated clinical data, and clinical translation. G.A.R. and O.K. contributed to study design and manuscript writing. J.C., P.W. and B.W.M. coordinated and performed the in vitro biochemical and cellular experiments. K.P., B.W.M. and K.W. supervised the experimental and clinical analysis. All authors discussed the results, commented and edited the manuscript.

Corresponding authors

Correspondence to Brion W. Murray or Krister Wennerberg.

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Competing interests

B.W.M., P.W., C.N.C., M.M. and E.J. are all Pfizer employees.

Extended data figures and tables

Extended Data Figure 1 Axitinib adopts significantly different binding conformation in the active site of ABL1(T315I) relative to VEGFR2.

Overlay of the bound conformation of axitinib from VEGFR2 (green) (PDB ID: 4AGC) and ABL1(T315I) (purple) co-crystal structures.

Extended Data Figure 2 Axitinib exhibits selective inhibition to BCR–ABL1 gatekeeper mutants in engineered Ba/F3 cells.

a, Bar graph depicting in vitro cell proliferation data are displayed as pIC50 values. b, The corresponding IC50 values are shown in table format.

Extended Data Figure 3 Ex vivo sensitivity to BCR–ABL1 inhibitors and axitinib in primary cells derived from a CML patient harbouring the T315I mutation (FHRB.1408).

a, Waterfall plot showing the sDSS of axitinib, ponatinib, dasatinib, imatinib and nilotinib. b, Dose-response data of all approved BCR–ABL1 inhibitors and axitinib.

Extended Data Figure 4 Comparison of publicly available target specificity profiles of axitinib14,38 and ponatinib20.

The target specificity profiles were evaluated at Ki/IC50 up to tenfold ABL1(T315I) potency (Ki = 0.1 nM axitinib; IC50 = 2 nM ponatinib) illustrating that axitinib is a much less promiscuous inhibitor than ponatinib and likely to have a better safety profile in Ph+ leukaemia patients. Green labelled kinases are targeted by both inhibitors.

Extended Data Table 1 X-ray data collection and refinement statistics
Extended Data Table 2 Clinical characteristics of patients
Extended Data Table 3 Drug sensitivity scores for axitinib in a panel of BCR–ABL1(T315I)-positive, CML, AML and healthy donor control samples

Supplementary information

Supplementary Table 1

This file contains the oncology drug collection, which depicts the anti-cancer inhibitors that have been used to phenotypically profile the CML and Ph+ ALL patient samples included in the study. The names, research codes, aliases, mechanisms of action, approval status, range of tested concentrations, suppliers and suppliers references of the inhibitors are provided. (XLSX 620 kb)

Supplementary Table 2

This file contains the drug sensitivity profile of Ph+ ALL patient cells (FHRB.1278) harbouring the T315I mutation, it shows the source drug sensitivity testing results of cancer cells derived from patient FHRB.1278. The four curve fitting parameters are provided (EC50, slope, minimum and maximum inhibition) along with the % survival at each of the tested concentration of a given compound. In addition, the calculated drug sensitivity score value is shown for each of the tested inhibitors. (XLSX 147 kb)

Supplementary Table 3

This file contains drug sensitivity profiles of CML and Ph+ ALL patient samples, it shows the source drug sensitivity testing results of the remaining CML and Ph+ ALL patient samples included in the study. The four curve fitting parameters are provided (EC50, slope, minimum and maximum inhibition) along with the % survival at each of the tested concentration of a given compound. In addition, the calculated drug sensitivity score value is shown for each of the tested inhibitors. (XLSX 228 kb)

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Pemovska, T., Johnson, E., Kontro, M. et al. Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation. Nature 519, 102–105 (2015).

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