Mulloy Roseann, Ferrand Audrey, Kim Youngjoo, Sordella Raffaella, Bell Daphne W, Haber Daniel A, Anderson Karen S, Settleman Jeffrey
Center for Molecular Therapeutics, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129, USA.
Cancer Res. 2007 Mar 1;67(5):2325-30. doi: 10.1158/0008-5472.CAN-06-4293.
Somatic mutations within the epidermal growth factor receptor (EGFR) kinase domain are detected in 10% to 30% of human non-small cell lung cancers and are correlated with striking clinical responses in a subset of patients treated with EGFR kinase inhibitors, such as gefitinib and erlotinib. Cell-based studies suggest that these mutant EGFRs promote increased autophosphorylating activity on a subset of EGFR COOH-terminal tyrosines and the consequent engagement of a subset of downstream effectors. Because EGFR function is regulated at multiple levels in vivo, and it is therefore difficult to assess the direct consequences of these mutations on EGFR enzyme function, we measured EGFR catalytic activity in in vitro kinase assays using purified recombinant proteins corresponding to the cytoplasmic domain of wild-type and two frequently detected EGFR mutants (DelL747-P753insS and L858R). Both mutants exhibit substantially increased autophosphorylating activity relative to wild-type EGFR, and they exhibit distinct reaction kinetics. In addition, the mutant kinases are more sensitive to kinase inhibition by gefitinib, which seems to reflect their increased drug affinity. These findings suggest that the altered signaling properties and drug sensitivity of these EGFR mutants that have been observed in vivo largely result from differences in the catalytic properties of the kinase. In addition, we find that the T790M secondary "drug resistance mutation" of EGFR, which frequently arises in relapsed patients that initially responded to treatment, confers enhanced kinase activity to primary activating EGFR alleles and may, therefore, be oncogenic in some contexts.
在10%至30%的人类非小细胞肺癌中可检测到表皮生长因子受体（EGFR）激酶结构域内的体细胞突变，并且这些突变与接受EGFR激酶抑制剂（如吉非替尼和厄洛替尼）治疗的部分患者的显著临床反应相关。基于细胞的研究表明，这些突变型EGFR可促进EGFR羧基末端酪氨酸亚群上的自身磷酸化活性增加，进而导致部分下游效应器的参与。由于EGFR功能在体内受到多个水平的调节，因此难以评估这些突变对EGFR酶功能的直接影响，我们使用对应于野生型和两种常见的EGFR突变体（DelL747 - P753insS和L858R）胞质结构域的纯化重组蛋白，在体外激酶测定中测量了EGFR催化活性。相对于野生型EGFR，这两种突变体均表现出显著增加的自身磷酸化活性，并且它们表现出不同的反应动力学。此外，突变型激酶对吉非替尼的激酶抑制更敏感，这似乎反映了它们增加的药物亲和力。这些发现表明，在体内观察到的这些EGFR突变体信号特性和药物敏感性的改变很大程度上源于激酶催化特性的差异。此外，我们发现EGFR的T790M继发性“耐药突变”，其经常出现在最初对治疗有反应的复发患者中，赋予原发性激活EGFR等位基因增强的激酶活性，因此在某些情况下可能具有致癌性。
