Identification of Activating Mutations in the Transmembrane and Extracellular Domains of EGFR

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer, most notably non-small-cell lung cancer and glioblastoma. While many frequently occurring EGFR mutations are known to confer constitutive EGFR activation, the situation is less clear for rarely detected variants. In fact, more than 1000 distinct EGFR mutations are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC), but for most of them, the functional consequence is unknown. To identify additional, previously unknown activating mutations in EGFR, we screened a randomly mutated EGFR library for constitutive EGFR phosphorylation using a recently developed high-throughput approach termed PhosphoFlowSeq. Enrichment of the well-known activating mutations S768I, T790M, and L858R validated the experimental approach. Importantly, we also identified the activating mutations S442I and L658Q located in the extracellular and transmembrane domains of EGFR, respectively. To the best of our knowledge, neither S442I nor L658Q has been associated with an activating phenotype before. However, both have been detected in cancer samples. Interestingly, molecular dynamics (MD) simulations suggest that the L658Q mutation located in the hydrophobic transmembrane region forms intermolecular hydrogen bonds, thereby promoting EGFR dimerization and activation. Based on these findings, we screened the COSMIC database for additional hydrophilic mutations in the EGFR transmembrane region and indeed detected moderate constitutive activation of EGFR-G652R. Together, this study demonstrates that unbiased screening for activating mutations in EGFR not only yields well-established substitutions located in the kinase domain but also activating mutations in other regions of EGFR, including the extracellular and transmembrane domains.表皮生长因子受体（epidermal growth factor receptor，EGFR）在人类癌症中频发突变，其中尤以非小细胞肺癌和胶质母细胞瘤最为显著。尽管已知多种常见EGFR突变可导致受体组成型激活，但对于罕见变异的相关功能机制仍不甚明确。事实上，癌症体细胞突变目录（Catalogue of Somatic Mutations in Cancer，COSMIC）中已收录超过1000种不同的EGFR突变，但其中绝大多数的功能效应尚未被阐明。为鉴定EGFR中此前未被发现的新增激活突变，我们采用新近开发的高通量方法——磷酸化流测序（PhosphoFlowSeq），对随机突变的EGFR文库开展组成型EGFR磷酸化筛选。已知的经典激活突变S768I、T790M与L858R的富集结果验证了本实验方法的有效性。值得注意的是，我们还分别鉴定出位于EGFR胞外域和跨膜域的激活突变S442I与L658Q。据我们所知，此前尚无研究将S442I或L658Q与激活表型相关联，但二者均已在癌症样本中被检测到。有趣的是，分子动力学（molecular dynamics，MD）模拟结果显示，位于疏水跨膜区的L658Q突变可形成分子间氢键，进而促进EGFR二聚化与激活。基于上述发现，我们在COSMIC数据库中筛选了EGFR跨膜区的其他亲水突变，确实检测到EGFR-G652R存在中等程度的组成型激活。综上，本研究表明，针对EGFR激活突变的无偏筛选不仅能够发现激酶域中已被证实的突变位点，还能鉴定出EGFR其他区域（包括胞外域与跨膜域）的激活突变。