Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kurzrock, R., Kantarjian, H. M., Druker, B. J. & Talpaz, M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Ann. Intern. Med. 138, 819–830 (2003)
Deininger, M., Buchdunger, E. & Druker, B. J. The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood 105, 2640–2653 (2005)
Druker, B. J. et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N. Engl. J. Med. 355, 2408–2417 (2006)
Hochhaus, A. et al. Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood 109, 2303–2309 (2007)
Hochhaus, A. et al. Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia. Leukemia 23, 1054–1061 (2009)
Branford, S. et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 99, 3472–3475 (2002)
Bradeen, H. A. et al. 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. Blood 108, 2332–2338 (2006)
Redaelli, S. et al. Activity of bosutinib, dasatinib, and nilotinib against 18 imatinib-resistant BCR/ABL mutants. J. Clin. Oncol. 27, 469–471 (2009)
Senior, M. FDA halts then allows sales of Ariad’s leukemia medication. Nature Biotechnol. 32, 9–11 (2014)
Gibbons, D. L. et al. Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy. Proc. Natl Acad. Sci. USA 111, 3550–3555 (2014)
Zabriskie, M. S. et al. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia. Cancer Cell 26, 428–442 (2014)
Pemovska, T. et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discov. 3, 1416–1429 (2013)
Yadav, B. et al. Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies. Sci. Rep. 4, 5193 (2014)
McTigue, M. et al. Molecular conformations, interactions, and properties associated with drug efficiency and clinical performance among VEGFR TK inhibitors. Proc. Natl Acad. Sci. USA 109, 18281–18289 (2012)
Rini, B. I. et al. Comparative effectiveness of axitinib versus sorafenib in advanced renal cell carcinoma (AXIS): a randomised phase 3 trial. Lancet 378, 1931–1939 (2011)
Solowiej, J. et al. Characterizing the effects of the juxtamembrane domain on vascular endothelial growth factor receptor-2 enzymatic activity, autophosphorylation, and inhibition by axitinib. Biochemistry 48, 7019–7031 (2009)
Azam, M., Seeliger, M. A., Gray, N. S., Kuriyan, J. & Daley, G. Q. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nature Struct. Mol. Biol. 15, 1109–1118 (2008)
Dixit, A. & Verkhivker, G. M. Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations. PLOS Comput. Biol. 5, e1000487 (2009)
Zhou, T. et al. Structural mechanism of the pan-BCR-ABL inhibitor ponatinib (AP24534): lessons for overcoming kinase inhibitor resistance. Chem. Biol. Drug Des. 77, 1–11 (2011)
O'Hare, T. et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell 16, 401–412 (2009)
Gontarewicz, A. et al. Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against imatinib-resistant BCR-ABL mutations including T315I. Blood 111, 4355–4364 (2008)
Chan, W. W. et al. Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036. Cancer Cell 19, 556–568 (2011)
Davis, M. I. et al. Comprehensive analysis of kinase inhibitor selectivity. Nature Biotechnol. 29, 1046–1051 (2011)
Gross-Goupil, M., Francois, L., Quivy, A. & Ravaud, A. Axitinib: a review of its safety and efficacy in the treatment of adults with advanced renal cell carcinoma. Clin. Med. Insights Oncol. 7, 269–277 (2013)
Bracarda, S. et al. Axitinib safety in metastatic renal cell carcinoma: suggestions for daily clinical practice based on case studies. Expert Opin. Drug Saf. 13, 497–510 (2014)
Verzoni, E. et al. Targeted treatments in advanced renal cell carcinoma: focus on axitinib. Pharmgenomics. Pers. Med. 7, 107–116 (2014)
Josephs, D. H., Fisher, D. S., Spicer, J. & Flanagan, R. J. Clinical pharmacokinetics of tyrosine kinase inhibitors: implications for therapeutic drug monitoring. Ther. Drug Monit. 35, 562–587 (2013)
Chen, Y. et al. Clinical pharmacology of axitinib. Clin. Pharmacokinet. 52, 713–725 (2013)
Shah, N. P. et al. Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 14, 485–493 (2008)
O'Hare, T. et al. Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis. Cancer Res. 73, 3356–3370 (2013)
Morrison, J. F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta 185, 269–286 (1969)
Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010)
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010)
le Coutre, P. et al. In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor. J. Natl. Cancer Inst. 91, 163–168 (1999)
Solowiej, J., Chen, J. H., Zou, H. Y., Grant, S. K. & Murray, B. W. Substrate-specific conformational regulation of the receptor tyrosine kinase VEGFR2 catalytic domain. ACS Chem. Biol. 8, 978–986 (2013)
Acknowledgements
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
Authors and Affiliations
Contributions
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
Ethics declarations
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.
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)
Rights and permissions
About this article
Cite this article
Pemovska, T., Johnson, E., Kontro, M. et al. Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation. Nature 519, 102–105 (2015). https://doi.org/10.1038/nature14119
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature14119
This article is cited by
-
Robust scoring of selective drug responses for patient-tailored therapy selection
Nature Protocols (2024)
-
Role of the ABL tyrosine kinases in the epithelial–mesenchymal transition and the metastatic cascade
Cell Communication and Signaling (2021)
-
Allogeneic haematopoietic stem cell transplantation improves outcome of adults with relapsed/refractory Philadelphia chromosome-positive acute lymphoblastic leukemia entering remission following CD19 chimeric antigen receptor T cells
Bone Marrow Transplantation (2021)
-
The effects of combination treatments on drug resistance in chronic myeloid leukaemia: an evaluation of the tyrosine kinase inhibitors axitinib and asciminib
BMC Cancer (2020)
-
Synthesis, in vitro, and in vivo evaluation of novel N-phenylindazolyl diarylureas as potential anti-cancer agents
Scientific Reports (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.