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.
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.
Pene-Dumitrescu
T
T
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Peterson
L F
LF
Donato
N J
NJ
Smithgall
T E
TE
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy*; D009154:Mutation; D051572:Proto-Oncogene Proteins c-hck / Q000235:genetics* / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D019061:src-Family Kinases / Q000378:metabolism*
C469552:A 419259; D000970:Antineoplastic Agents; D011743:Pyrimidines; D011758:Pyrroles; D016044:Fusion Proteins, bcr-abl; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases
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