The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib

*Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 19, 1090 Vienna, Austria;
^†Institute of Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria; and
^§Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

Edited by John Kuriyan, University of California, Berkeley, CA, and approved June 26, 2007 (received for review March 21, 2007)

Abstract

Dasatinib is a small-molecule kinase inhibitor used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). We have analyzed the kinases targeted by dasatinib by using an unbiased chemical proteomics approach to detect binding proteins directly from lysates of CML cells. Besides Abl and Src kinases, we have identified the Tec kinases Btk and Tec, but not Itk, as major binders of dasatinib. The kinase activity of Btk and Tec, but not of Itk, was inhibited by nanomolar concentrations of dasatinib in vitro and in cultured cells. We identified the gatekeeper residue as the critical determinant of dasatinib susceptibility. Mutation of Thr-474 in Btk to Ile and Thr-442 in Tec to Ile conferred resistance to dasatinib, whereas mutation of the corresponding residue in Itk (Phe-435) to Thr sensitized the otherwise insensitive Itk to dasatinib. The configuration of this residue may be a predictor for dasatinib sensitivity across the kinome. Analysis of mast cells derived from Btk-deficient mice suggested that inhibition of Btk by dasatinib may be responsible for the observed reduction in histamine release upon dasatinib treatment. Furthermore, dasatinib inhibited histamine release in primary human basophils and secretion of proinflammatory cytokines in immune cells. The observed inhibition of Tec kinases by dasatinib predicts immunosuppressive (side) effects of this drug and may offer therapeutic opportunities for inflammatory and immunological disorders.

Footnotes

^¶To whom correspondence should be addressed. E-mail: gsuperti{at}cemm.oeaw.ac.at
Author contributions: O.H., U.R., U.S., and T.B. contributed equally to this work; O.H., U.S., T.B., W.E., P.V., and G.S.-F. designed research; O.H., U.R., U.S., T.B., M.K., J.C., and K.L.B. performed research; U.R., G.S., J.C., K.L.B., W.E., and P.V. contributed new reagents/analytic tools; O.H., U.R., K.L.B., W.E., P.V., and G.S.-F. analyzed data; and O.H., T.B., and G.S.-F. wrote the paper.
↵ ^‡Present address: Forschungs, Nabriva Therapeutics, Brunner Strasse 59, 1235 Vienna, Austria.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0702654104/DC1.
Abbreviations:

CML,

chronic myelogenous leukemia;

LC-MSMS,

liquid chromatography tandem MS;

BMMC,

bone marrow-derived murine mast cells;

TAP,

tandem affinity purification.
Freely available online through the PNAS open access option.