EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.
In cancer, genetically activated proto-oncogenes often induce ''upstream'' dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce ''downstream'' dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.cancer genomics ͉ combination therapy ͉ high-throughput cell line screening ͉ oncogene dependency
BackgroundThe EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.Methodology/Principal FindingsTo study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFRT790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFRL858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFRT790M-expressing animals develop tumors with longer latency than EGFRL858R+T790M-bearing mice and in the absence of additional kinase domain mutations.Conclusions/SignificanceThese new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFRT790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.
Locus control regions (LCRs) are complex high-order chromatin structures harbouring several regulatory elements, including enhancers and boundaries. We have analysed the mouse tyrosinase LCR functions, in vitro, in cell lines and, in vivo, in transgenic mice and flies. The LCR-core (2.1 kb), located at -15 kb and carrying a previously described tissue-specific DNase I hypersensitive site, operates as a transcriptional enhancer that efficiently transactivates heterologous promoters in a cell-specific orientation-independent manner. Furthermore, we have investigated the boundary activity of these sequences in transgenic animals and cells. In mice, the LCR fragment (3.7 kb) rescued a weakly expressed reference construct that displays position effects. In Drosophila, the LCR fragment and its core insulated the expression of a white minigene reporter construct from chromosomal position effects. In cells, sequences located 5' from the LCR-core displayed putative boundary activities. We have obtained genomic sequences surrounding the LCR fragment and found a LINE1 repeated element at 5'. In B16 melanoma and L929 fibroblast mouse cells, this element was found heavily methylated, supporting the existence of putative boundary elements that could prevent the spreading of condensed chromatin from the LINE1 sequences into the LCR fragment, experimentally shown to be in an open chromatin structure.
Summary Lung cancer in never-smokers is an important disease often characterized by mutations in EGFR, yet risk reduction measures and effective chemopreventive strategies have not been established. We identify mTOR as a new and potentially valuable target for EGFR mutant lung cancer, as mTOR was activated in human lung cancers with EGFR mutations, which increased with acquisition of T790M mutation. In a mouse model of EGFR mutant lung cancer, activation of mTOR was an early event. As a single agent, the mTOR inhibitor rapamycin, prevented tumor development, prolonged overall survival, and improved outcomes after treatment with an irreversible EGFR TKI. These studies support clinical testing of mTOR inhibitors to prevent the development and progression of EGFR mutant lung cancers.
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