EGF receptor (EGFR)–mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKI). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Because afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. FISH analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR^T790M were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR-TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR-mutant tumors with acquired resistance to EGFR-TKIs. Significance: Because all EGFR-mutant lung adenocarcinomas eventually develop resistance to TKI therapy, understanding mechanisms of acquired resistance may improve clinical outcomes. These results implicate HER2 as a novel protein involved in the sensitivity or resistance of EGFR-mutant lung cancer and provide a rationale to assess the status of and possibly target HER2 in such tumors. Cancer Discov; 2(10); 922–33. ©2012 AACR. Read the Commentary on this article by Blakely and Bivona, p. 872. This article is highlighted in the In This Issue feature, p. 857.

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. Significance Knowledge of oncogenic alterations that drive lung adenocarcinoma formation has enabled the development of genetically engineered mouse models that are increasingly being used to study the biology and therapeutic vulnerabilities of this disease. Given the importance of genomic alterations in these processes in human lung cancer, information on the mutational landscape of the mouse tumors is valuable for the design and interpretation of these experiments. In this study, we compared whole-exome sequencing data from lung adenocarcinomas induced by different lung adenocarcinoma-associated drivers. In contrast to their human counterparts, oncogene-driven lung adenocarcinomas in genetically engineered mouse models harbor few somatic mutations. These results have important implications for the use of these models to study tumor progression and response and resistance to therapy.

Patients with EGFR-mutant lung adenocarcinomas (LUADs) who initially respond to first-generation tyrosine kinase inhibitors (TKIs) develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. The addition of rapamycin reversed resistance in vivo. Analysis of afatinib-plus-cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling, including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib plus cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs.

Purpose: EGFR exon 19 deletion (Ex19Del) mutations account for approximately 60% of lung cancer–associated EGFR mutations and include a heterogeneous group of mutations. Although they are associated with benefit from tyrosine kinase inhibitors (TKI), the relative inhibitor sensitivity of individual Ex19Del mutations is unknown. Experimental Design: We studied the TKI sensitivity and structural features of common Ex19Del mutations and the consequences for patient outcomes on TKI treatment. Results: We found that the L747-A750>P mutation, which represents about 4% of all Ex19Del mutations, displays unique inhibitor selectivity. L747-A750>P differs from other Ex19Del mutations in not being suppressed completely by erlotinib or osimertinib, yet is completely inhibited by low doses of afatinib. The HCC4006 cell line (with the L747-A750>P mutation) exhibited increased sensitivity to afatinib over erlotinib and osimertinib, and computational modeling suggests explanations for this sensitivity pattern. Clinically, patients with EGFR L747-A750>P mutant tumors showed inferior outcomes when treated with erlotinib than patients with E746-A750 mutant tumors. Conclusions: These results highlight important differences between specific Ex19Del mutations that may be relevant for optimizing TKI choice for patients.