Arena Sabrina, Pisacane Alberto, Mazzone Massimiliano, Comoglio Paolo Maria, Bardelli Alberto
Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, I-10060 Candiolo, Turin, Italy.
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11412-7. doi: 10.1073/pnas.0703205104. Epub 2007 Jun 26.
The development of kinase inhibitors is revolutionizing cancer treatment. Assessing the oncogenic potential of individual kinase activities and ensuring that a drug of interest acts by direct inhibition of its putative target kinase are clear priorities. We developed a genetic strategy to selectively inactivate the catalytic activity of kinases. This approach generates isogenic cells in which a given kinase gene is expressed but is devoid of enzymatic activity. As a model to test this approach, we chose the MET receptor, which is involved in multiple cancers and is the focus of several therapeutic efforts. The exon encoding the ATP-binding site of MET was deleted from the genome of colorectal, bladder, and endometrial cancer cells. The derivative isogenic cells expressed a kinase-inactive Met (MET-KD) and were completely unresponsive to its ligand hepatocyte growth factor (HGF), indicating the exclusivity of this ligand-receptor axis. The in vivo tumorigenic potential of MET-KD cells was reduced but could be partially restored by HGF, suggesting that concomitant targeting of the receptor and its ligand should be therapeutically exploited. A reportedly selective Met-kinase inhibitor (SU-11274) markedly affected the growth of MET-KD cancer cells, indicating this compound exerts its effects not only through the intended target. The genetic strategy presented here is not limited to kinase genes but could be broadly applicable to any drug/protein combination in which the target enzymatic domain is known.
激酶抑制剂的发展正在彻底改变癌症治疗。评估个体激酶活性的致癌潜力，并确保所关注的药物通过直接抑制其假定的靶激酶发挥作用，这显然是首要任务。我们开发了一种遗传策略来选择性地使激酶的催化活性失活。这种方法产生了同基因细胞，其中给定的激酶基因表达但缺乏酶活性。作为测试该方法的模型，我们选择了MET受体，它与多种癌症有关，并且是多项治疗研究的重点。从结肠直肠癌、膀胱癌和子宫内膜癌细胞的基因组中删除了编码MET的ATP结合位点的外显子。衍生的同基因细胞表达激酶失活的Met（MET-KD），并且对其配体肝细胞生长因子（HGF）完全无反应，表明该配体-受体轴的专一性。MET-KD细胞的体内致瘤潜力降低，但可通过HGF部分恢复，这表明受体及其配体的联合靶向在治疗上应加以利用。一种据报道具有选择性的Met激酶抑制剂（SU-11274）显著影响MET-KD癌细胞的生长，表明该化合物不仅通过预期靶点发挥作用。这里介绍的遗传策略不仅限于激酶基因，还可广泛应用于任何已知靶酶结构域的药物/蛋白质组合。
