(a) Orientation of A-420983, an analog of A-419259, in the Hck nucleotide-binding pocket. The overall structure of Hck is shown on the left, with the SH3 domain in red, the SH2 domain in blue, and the kinase domain in gray. The side chain of the gatekeeper residue (T338; c-Src numbering) is highlighted in magenta. The relationship of the gatekeeper residue to the pyrazolopyrimidine moiety of A-420983 is enlarged on the right. This model was produced using PyMol and the Protein Data Bank (PDB) file 2C01. (b) Chemical structures of A-419259 and A-420983.

Hck-T338M expression in K562 cells confers resistance to A-419259-induced growth arrest. K562 cells expressing wild-type Hck, the T338M mutant, as well as the vector control cells (neo) were treated with A-419259 at the indicated concentrations. Cell proliferation was monitored 24, 48 and 72 h later using the CellTiter-Blue cell viability assay as described under ‘Materials and methods’. Three replicate wells were monitored for each dose in three independent experiments and gave comparable results; a representative example is shown.

Expression of Hck-T338M in K562 cells reverses the inhibitory effects of A-419259 on Erk activation. K562 cells expressing wild-type Hck, the T338M mutant, as well as the vector control cells (neo) were treated with the indicated concentrations of A-419259 for 5 h. (a) Cell lysates were prepared and analysed for the presence of active Erk by immunoblotting with phosphospecific antibodies (pErk). Duplicate blots were probed with antibodies to Erk2 as a loading control. Representative blots are shown. (b) Phospho-Erk signal intensities from the blots of two independent experiments were normalized to the levels of Erk, and are presented as percent of control levels±s.d.

Hck-T338M protects K562 cells against the apoptotic effect of the Src family kinase (SFK) inhibitor A-419259. K562 cells expressing wild-type Hck, the T338M mutant, as well as the vector control (neo) cells were plated in the absence or presence of the indicated concentrations of A-419259 for 72 h. Apoptotic cells were detected by anti-phosphatidylserine-Alexa Fluor 488-conjugated antibody staining and flow cytometry. (a) Representative experiment with the percentage of apoptotic cells in each population shown above the bar. (b) Bar graph showing the average of three independent experiments and plotted±s.d.

Expression of Hck-T338M in K562 cells opposes the inhibitory effects of A-419259 on Stat5 activation. K562 cells expressing wild-type Hck, the T338M mutant, as well as the vector control cells (neo) were treated with the indicated concentrations of A-419259 for 5 h. (a) Stat5 tyrosine phosphorylation was assessed by immunoprecipitation of Stat5 from clarified cell lysates and immunoblotting with antiphosphotyrosine antibodies (pStat5). Duplicate membranes were blotted with an anti-Stat5 antibody to insure equal loading (Stat5). Representative blots are shown. (b) Stat5 phosphotyrosine signal intensities from the blots of two independent experiments were normalized to the levels of Stat5 protein, and the results are presented as percent of control levels±s.d.

In vitro kinase assay of recombinant wild type and T338M forms of Hck. Recombinant wild-type Hck and Hck-T338M were purified from Sf9 insect cells in their downregulated conformations and assayed for kinase activity with a peptide substrate in vitro in the presence or absence of the indicated concentrations of A-419259. A representative experiment is shown and the extent of inhibition is expressed as mean±s.d. of four assays. The entire experiment was repeated twice with comparable results. Data from two independent experiments were best fit by nonlinear regression analysis and yielded IC₅₀ values of 11.26±1.23 nM for wild-type Hck and 315.6±80.3 nM for Hck-T338M.

A-419259 induces growth arrest and apoptosis in CD34⁺ progenitor cells from chronic myelogenous leukemia (CML) patients. Purified CD34⁺ cells from three chronic phase CML patients were plated in triplicate in 96-well plates at 10⁵ cells/ml and incubated for 16 or 48 h in the presence of A-419259 or imatinib at the concentrations indicated. Cell proliferation was monitored using the CellTiter-Blue viability assay as described in ‘Materials and methods’. The average fold increase in the relative number of cells from three replicate assays is shown±s.d. (left panels). Apoptosis was measured in each well using the Apo-One Caspase-3/-7 assay as described in ‘Materials and methods’. The average fold increase in caspase-3/-7 signal intensity from three replicate wells is shown±s.d. (right panels).

Hck-T338M is resistant to inhibition by A-419259 in K562 cells. (a) Wild-type and Hck-T338M proteins were expressed in K562 cells using recombinant retroviruses. K562-Hck wild-type and K562-Hck-T338M cells were treated with A-419259 at the indicated concentrations for 5 h. Hck activity was assessed by immunoprecipitation of Hck from clarified cell lysates and immunoblotting with phosphospecific antibodies against the Hck activation loop phosphotyrosine residue (pHck). Duplicate blots of the immunoprecipitates were blotted with the anti-Hck antibody to insure equal loading. Representative blots are shown at the top. Phosphotyrosine signal intensities from the blots of two independent experiments were normalized to the levels of Hck. Results are presented as percent of control levels±s.d. in the bar chart. (b) A-419259 does not affect Bcr-Abl activation loop autophosphorylation. Lysates from A-419259-treated cells in part A were immunoblotted with a phosphospecific antibody to the Abl activation loop phosphotyrosine residue (pY412; upper panels). Control blots were performed with an antibody to the Abl protein (Bcr-Abl; lower panels). (c) Inhibition of Bcr-Abl activation loop autophosphorylation by imatinib. K-562 cell populations from part A were treated with imatinib at the concentrations shown. Cell lysates were then immunoblotted with a phosphospecific antibody to the Abl activation loop phosphotyrosine residue (pY412; upper panels) as well as an antibody to the Abl protein (Bcr-Abl; lower panels).

Hck-T338M maintains its biological activity and A-419259 resistance in fibroblasts. Rat-2 fibroblasts were infected with recombinant Hck-YF and Hck-T338M-YF retroviruses. Cells infected with a virus carrying only the selection marker (neo) or wild-type Hck served as negative controls. Upon G418 selection, cells were plated in soft agar in the presence or absence of the indicated concentrations of A-419259 for 10–14 days. Transformed colonies were visualized using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. (a) Colony numbers for each cell line were determined using scanned images of the plates and BioRad QuantityOne colony-counting software. Results from a representative experiment are shown as the mean number of colonies±s.d. The entire experiment was performed twice and yielded comparable results. (b) Lysates from each of the cell lines shown in panel a were probed with phosphospecific antibodies against the Hck activation loop phosphotyrosine residue (pHck). Replicate membranes were probed with a general antiphosphotyrosine antibody to determine phosphorylation of the endogenous Hck substrate pp40, with an anti-Hck antibody to determine Hck expression levels, and with anti-actin as a loading control.

Expression of Hck-T338M protects TF-1/Bcr-Abl cells against the apoptotic effects of A-419259. (a) Transformation of TF-1 cells to cytokine independence with Bcr-Abl. TF-1 cells were first transduced with Bcr-Abl retroviruses or the corresponding vector control and selected with G-418. The resulting populations were then transduced with wild-type Hck, Hck-T338M or vector control retroviruses and selected with puromycin. The six resulting cell populations were then tested for growth in the absence of GM-CSF using the CellTiter Blue assay (see Materials and methods). The average fold increase in the relative number of cells from three replicate assays is shown±s.d. Two separate experiments from independently derived cell populations gave comparable results; a representative example is shown. (b) TF-1/Bcr-Abl cells expressing the wild-type and T338M Hck proteins along with vector control cells were plated in the absence or presence of the indicated concentrations of A-419259 for 72 h. Apoptotic cells were detected by antiphosphatidylserine-Alexa Fluor 488-conjugated antibody staining and flow cytometry as described in ‘Materials and methods’. The bargraph shows the average of two independent experiments plotted±s.d. (c) TF-1/Bcr-Abl cells expressing wild-type and T338M-Hck along with the vector control were treated with the indicated concentrations of A-419259 for 5 h. Hck and Stat5 were immunoprecipitated from clarified cell lysates and immunoblotted with an anti-Hck phosphospecific pY418 antibody (pHck) or an antiphosphotyrosine antibody (pStat5), respectively. Duplicate membranes were blotted with an anti-Hck or anti-Stat5 antibody to insure equal loading. Representative blots are shown.