Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants

doi:10.1073/pnas.0609239103

Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants

*Howard Hughes Medical Institute,
^†Department of Medicine,
^‡Crump Institute for Molecular Imaging,
^§Department of Molecular and Medical Pharmacology,
^¶Molecular Biology Institute,
^‖Department of Chemistry and Biochemistry,
**UCLA-DOE Institute for Genomics and Proteomics,
^††Department of Biomathematics, and
^‡‡Department of Urology, University of California, Los Angeles, CA 90095

Communicated by John Kuriyan, University of California, Berkeley, CA, October 19, 2006 (received for review August 3, 2006)

Abstract

The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr²⁵³ and Tyr²⁵⁷) in the ATP binding loop (P-loop) of BCR-ABL when Thr³¹⁵ is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr³¹⁵ mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.

Footnotes

^§§To whom correspondence should be sent at the present address:
Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
E-mail: sawyersc{at}mskcc.org
Author contributions: C.L.S. and T.G.G. contributed equally to this work; B.J.S., M.E.G., C.L.S., and T.G.G. designed research; B.J.S., M.E.G., A.R., Y.X., and L.M.B. performed research; B.J.S., A.R., M.R.B., Y.X., Y.H., E.K., E.M.L., and T.G.G. contributed new reagents/analytic tools; B.J.S., Y.X., Y.H., E.K., J.A.L., E.M.L., C.L.S., and T.G.G. analyzed data; and B.J.S., E.M.L., C.L.S., and T.G.G. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609239103/DC1.
↵ ¶¶ Shah, N. P., Nicoll, J., Branford, S., Hughes, T. P., Paquette, R., Talpaz, M., Nicaise, C., Huang, F., Sawyers, C. L. (2005) Blood 106:1093 (abstr.).
Abbreviation:

CML,

chronic myelogenous leukemia.
Freely available online through the PNAS open access option.