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An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259

Oncogene volume 27, pages 7055–7069 (2008)Cite this article

Abstract

Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro. Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34+ CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (CA101828 and GM077629 to TES). We thank Dr David Calderwood of the Abbott Bioresearch Center for the generous gift of the Src family kinase inhibitor, A-419259 and Dr Elisabeth Buchdunger of Novartis, Inc. for providing imatinib mesylate.

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  1. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    T Pene-Dumitrescu & T E Smithgall

  2. Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI, USA

    L F Peterson & N J Donato

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  1. T Pene-Dumitrescu
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Correspondence to T E Smithgall.

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Pene-Dumitrescu, T., Peterson, L., Donato, N. et al. An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259. Oncogene 27, 7055–7069 (2008). https://doi.org/10.1038/onc.2008.330

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