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

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.

Fig. 1.

Dasatinib binds Btk and Tec, but not Itk. (A) Immobilized dasatinib was incubated with K562 total cell extract and bound proteins eluted by boiling in SDS sample buffer, resolved on a 4–12% NuPAGE gel followed by colloidal Coomassie staining. Bands 1–4 were excised, digested with trypsin, and analyzed by LC-MSMS (see SI Table 1). Main proteins identified in bands 1–4 are indicated. (B) Total cell extracts from U937, K562, Namalwa, Jurkat, and RAW cells and proteins that were bound by immobilized dasatinib were immunoblotted for Abl and the Tec kinases Btk, Tec, and Itk, as well as for actin as a loading and specificity control. Fifty micrograms of total cell extract and 5% of the fractions that were bound by immobilized dasatinib were loaded.

Fig. 2.

Dasatinib is a very potent inhibitor of Btk. (A) Full-length recombinant c-Abl and Btk were assayed in parallel for catalytic activity by in vitro kinase assays at the indicated concentrations of dasatinib and an optimal Abl substrate peptide as substrate. The graph shows the mean of one representative experiment done in triplicate and relative inhibition, in which kinase activity in the absence of dasatinib is set to 1.0. (B) Vitamin D3-differentiated U937 cells were either left untreated or stimulated with 1 μg/ml LPS for 6 h and treated with the indicated concentrations of dasatinib or imatinib for 1 h. Total cell extracts were prepared, and equal amounts of total protein were immunblotted with the indicated antibodies.

Fig. 3.

The gatekeeper residue is a major determinant of dasatinib sensitivity in Btk. (A) Structural superposition of the Btk kinase domain [Protein Data Bank (PDB) ID code 1K2P] and the Abl kinase domain in complex with dasatinib (PDB ID code 2GQG) identifies Thr-474 in Btk as structural homologous residue to the gatekeeper residue Thr-315 in Abl. (B) TAP-tagged Btk WT and Btk T474I were purified by IgG Sepharose precipitation, and catalytic activity was determined at the indicated concentrations of dasatinib and an optimal Abl substrate peptide as substrate. The graph shows the mean of two experiments done in duplicate and relative inhibition, in which kinase activity in the absence of dasatinib is set to 1.0. (C) Vitamin D3-differentiated U937 cells were retrovirally transduced with either Btk WT (Upper) or Btk T474I (Lower) and either left untreated or stimulated with 1 μg/ml LPS for 6 h and treated with the indicated concentrations of dasatinib, imatinib, or nilotinib for 1 h. Total cell extracts were prepared, and equal amounts of total protein were immunoblotted with the indicated antibodies.

Fig. 4.

Mutation of the gatekeeper residue in Itk confers sensitivity to dasatinib. (A) Structural superposition of the Itk kinase domain (PDB ID code 1K2P) and the Abl kinase domain in complex with dasatinib (PDB ID code 2GQG) identifies Phe-435 in Itk as structural homologous residue to the gatekeeper residue Thr-315 in Abl. (B) Closeup view of dasatinib bound to the Abl kinase domain and the gatekeeper residues Thr-315 in Abl and Phe-435 in Itk showing hydrogen bonding of the Cβ-OH group of Thr-315 in Abl with dasatinib (gray dashed line) and sterical clash of Phe-435 in Itk with dasatinib. All other residues are omitted for graphical clarity. (C) TAP-tagged Tec WT, Tec T442I, Itk WT, and Itk F435T were purified by IgG Sepharose precipitation, and catalytic activity was determined at the indicated concentrations of dasatinib and an optimal Abl substrate peptide as substrate. The graph shows the mean of two experiments done in duplicate and relative inhibition, in which kinase activity in the absence of dasatinib is set to 1.0.

Fig. 5.

Dasatinib inhibits the secretion of immunomodulators. (A) U937 cells were differentiated with vitamin D3, pretreated with the indicated concentrations of drugs for 1 h and stimulated with LPS for 6 h. TNF-α secretion was measured by ELISA. The graph shows the mean of two experiments done in duplicate. (B) IgE primed WT mast cells were activated with TNP for 24 h in the presence of 1 μM dasatinib. IL-6 secreted into the culture medium was determined by ELISA. (C) Basophils from a healthy donor were preincubated in control medium or 1 μM dasatinib (Bristol-Myers Squibb, Princeton, NJ) for 30 min. Cells were exposed to various concentrations of an anti-IgE antibody (37°C, 30 min) as indicated. Histamine was measured in cell lysates (total histamine) and cell-free supernatants. Histamine release is expressed as percent of total histamine and represents the mean of triplicate determinations in one donor. (D) BMMCs from WT, Btk−/−, and Tec−/− mice were preincubated in medium with or without 1 μM dasatinib for 30 min. Then, cells were washed and exposed to 1 μg/ml TNP (37°C, 30 min). Histamine release was measured as in C.