title: 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.
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.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: T
authors.initials: T
authors.lastname: Pene-Dumitrescu
authors.forename: L F
authors.initials: LF
authors.lastname: Peterson
authors.forename: N J
authors.initials: NJ
authors.lastname: Donato
authors.forename: T E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: 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*
chemical_list: 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
references.citation: N Engl J Med. 2003 Mar 27;348(13):1201-14
references.pmid: 12660384
references.citation: Proc Natl Acad Sci U S A. 1990 Sep;87(17):6649-53
references.pmid: 2204061
references.citation: Science. 1986 Jul 11;233(4760):212-4
references.pmid: 3460176
references.citation: N Engl J Med. 2002 Feb 28;346(9):645-52
references.pmid: 11870241
references.citation: Leukemia. 2001 Jun;15(6):989-90
references.pmid: 11417489
references.citation: Leukemia. 2005 Jun;19(6):1034-41
references.pmid: 15815728
references.citation: N Engl J Med. 2005 Feb 24;352(8):786-92
references.pmid: 15728811
references.citation: Oncogene. 2002 Dec 9;21(56):8629-42
references.pmid: 12476309
references.citation: EMBO J. 1997 Oct 15;16(20):6151-61
references.pmid: 9321394
references.citation: N Engl J Med. 1999 Apr 29;340(17):1330-40
references.pmid: 10219069
references.citation: J Biol Chem. 2000 Jan 28;275(4):2721-6
references.pmid: 10644735
references.citation: Blood. 2004 Oct 1;104(7):2204-5
references.pmid: 15377577
references.citation: Mol Cell. 2003 Jul;12(1):27-37
references.pmid: 12887890
references.citation: Science. 2001 Aug 3;293(5531):876-80
references.pmid: 11423618
references.citation: Exp Cell Res. 2000 Jan 10;254(1):1-13
references.pmid: 10623460
references.citation: Blood. 2003 Jun 15;101(12):4701-7
references.pmid: 12576334
references.citation: Proc Natl Acad Sci U S A. 1987 Sep;84(18):6558-62
references.pmid: 3498165
references.citation: Cell. 1995 Sep 22;82(6):981-8
references.pmid: 7553858
references.citation: Blood. 2006 Jun 1;107(11):4532-9
references.pmid: 16469872
references.citation: J Biol Chem. 2006 Oct 13;281(41):30907-16
references.pmid: 16912036
references.citation: J Exp Med. 1996 Mar 1;183(3):811-20
references.pmid: 8642285
references.citation: J Med Chem. 2006 Nov 16;49(23):6819-32
references.pmid: 17154512
references.citation: Nature. 1973 Jun 1;243(5405):290-3
references.pmid: 4126434
references.citation: Mol Cell Biol Res Commun. 2000 May;3(5):299-305
references.pmid: 10964754
references.citation: Cancer Res. 1996 Aug 1;56(15):3589-96
references.pmid: 8758931
references.citation: J Biol Chem. 2000 Jun 16;275(24):18581-5
references.pmid: 10849448
references.citation: Science. 1990 Feb 16;247(4944):824-30
references.pmid: 2406902
references.citation: Oncogene. 2004 Oct 18;23(48):7918-27
references.pmid: 15489910
references.citation: J Biol Chem. 2003 Apr 25;278(17):15435-40
references.pmid: 12594213
references.citation: Chem Biol. 1999 Sep;6(9):671-8
references.pmid: 10467133
references.citation: Oncogene. 2002 Nov 21;21(53):8075-88
references.pmid: 12444544
references.citation: Clin Cancer Res. 2007 Feb 15;13(4):1089-97
references.pmid: 17317816
references.citation: Cell. 1993 Oct 8;75(1):175-85
references.pmid: 8402896
references.citation: J Biol Chem. 1997 Jul 18;272(29):17899-902
references.pmid: 9218412
references.citation: Mol Cell. 1999 May;3(5):629-38
references.pmid: 10360179
references.citation: Mol Cell. 1999 May;3(5):639-48
references.pmid: 10360180
references.citation: Nat Struct Biol. 2002 May;9(5):365-9
references.pmid: 11976726
references.citation: Ann Oncol. 2007 Jun;18 Suppl 6:vi42-6
references.pmid: 17591830
references.citation: Cancer Res. 2004 Jan 15;64(2):672-7
references.pmid: 14744784
references.citation: EMBO J. 2002 Nov 1;21(21):5766-74
references.pmid: 12411494
references.citation: Oncogene. 1995 Apr 6;10(7):1385-91
references.pmid: 7537361
references.citation: J Biol Chem. 2006 Sep 15;281(37):27029-38
references.pmid: 16849330
references.citation: J Biol Chem. 2005 Dec 9;280(49):40832-7
references.pmid: 16210316
references.citation: Nature. 2000 Sep 21;407(6802):395-401
references.pmid: 11014197
references.citation: J Biol Chem. 2002 Nov 22;277(47):45680-7
references.pmid: 12244095
references.citation: Blood. 2000 Sep 15;96(6):2269-76
references.pmid: 10979976
references.citation: J Biol Chem. 1999 Sep 10;274(37):26579-83
references.pmid: 10473622
references.citation: Mol Cell Biol. 2001 Feb;21(3):840-53
references.pmid: 11154271
references.citation: Chem Biol. 2004 May;11(5):691-701
references.pmid: 15157880
references.citation: Mol Cell Biol. 2000 Feb;20(4):1179-86
references.pmid: 10648603
references.citation: Bioorg Med Chem. 1998 Aug;6(8):1219-26
references.pmid: 9784863
references.citation: Oncogene. 2001 Sep 13;20(41):5826-35
references.pmid: 11593388
references.citation: Gastroenterology. 2004 Jul;127(1):294-9
references.pmid: 15236194
references.citation: J Natl Cancer Inst. 2008 Jul 2;100(13):926-39
references.pmid: 18577747
references.citation: Leukemia. 2005 Nov;19(11):1872-9
references.pmid: 16179913
references.citation: J Cell Physiol. 1989 Aug;140(2):323-34
references.pmid: 2663885
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3153-8
references.pmid: 16492761
references.citation: Oncogene. 1996 Jul 18;13(2):247-54
references.pmid: 8710363
references.citation: J Biol Chem. 1997 Dec 26;272(52):33260-70
references.pmid: 9407116
references.citation: Mol Cell Biol. 1989 Mar;9(3):1263-70
references.pmid: 2725497
references.citation: Cell. 1993 Mar 12;72(5):767-78
references.pmid: 7680959
references.citation: Cancer Cell. 2002 Jun;1(5):479-92
references.pmid: 12124177
references.citation: J Biol Chem. 2004 Aug 13;279(33):34227-39
references.pmid: 15175350
references.citation: Nat Med. 2004 Nov;10(11):1187-9
references.pmid: 15502840
references.citation: Leuk Res. 2004 May;28 Suppl 1:S71-3
references.pmid: 15036945
references.citation: Nat Genet. 2004 May;36(5):453-61
references.pmid: 15098032
references.citation: Chem Biol. 1998 Jun;5(6):321-8
references.pmid: 9653550
references.citation: J Biol Chem. 1996 Mar 1;271(9):4640-5
references.pmid: 8617726
references.citation: Blood. 2000 Jul 15;96(2):664-70
references.pmid: 10887132
references.citation: Oncogene. 1996 Feb 15;12(4):839-46
references.pmid: 8632906