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Concurrent single cell RNA and targeted DNA sequencing on a microfluidics device allows high-throughput identification of transcriptome and genome alterations during the development of tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer cells

ABSTRACT: Within the clonal heterogeneity of a tumor, the co-measurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information, for instance a somatic mutation, is correlated with a transcriptomic and cancer-related phenotype. However, there is currently no microfluidics-based high-throughput assay available that allows this analysis. To resolve this, we developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on a microfluidic high-throughput platform. Its operation is simple, at moderate reagent cost, with excellent data quality and good accessibility for a broad scientific community. We implemented this new method to dissect the cellular consequences of mutations that result in resistance to targeted therapy. Using different tyrosine kinase inhibitor (TKI) resistant PC9 lung cancer cell lines as our study model, we detected signatures of TKI resistant genes as well as expansion of T790M mutation and EGFR amplification in the resistant cells. Interestingly, we identify transcriptomic characteristics for resistance that are not dependent on the presence of T790M, the most common resistance mutation in EGFR, indicating that phenotypically similar resistance scenarios can emerge without T790M mutation and other genomic alterations or epigenetic modifications are required for the manifestation of the resistance phenotype. Cumulatively, the application of this method allows accurate evaluation of the genomic and transcriptomic changes in cancer during development of drug resistance at single cell level in uncovering potential new biomarker and therapeutic targets in resistant cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112274 | GEO | 2019/03/23

REPOSITORIES: GEO

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Project description:Lung cancer continues to be the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are two 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance to it. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative proteomic and phosphoproteomic dataset to uncover alterations in signaling pathways, and to reveal a proteomic signature of EMT and kinases / phosphatases with altered protein expression and phosphorylation in the TKI resistant cells. We validated the significant role of SHP2 in the activation of RAS/MAPK and PI3K/AKT signaling pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified that dactolisib, a PI3K/mTOR inhibitor, in combination with osimertinib, may overcome osimertinib resistance both in vitro and in vivo. Introduction Lung cancer continues to be the leading cause of cancer mortality in the world (1). Many lung adenocarcinoma patients with activating epidermal growth factor receptor (EGFR) mutations initially respond dramaticlly to the first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). However, they eventually develop resistance. The most common mechanism of acquired resistance is the EGFR T790M gatekeeper site residue mutation (2). Osimertinib, a third generation irreversible EGFR TKI has been approved by the FDA to treat patients harboring the EGFR T790M mutation who have developed resistance to first- and second- generation EGFR TKIs (3). Recently, osimertinib was also approved for the front-line treatment of patients harboring EGFR mutations (4). Rociletinib is another irreversible inhibitor targeting the EGFR T790M mutation, which has minimal activity against wild-type EGFR. Both drugs have therapeutic benefits and have demonstrated activity in tumors with T790M-mediated resistance to other EGFR tyrosine kinase inhibitors (5, 6). Further development of rociletinib was ceased in 2016 due to less than expected efficacy, poor brain penetration leading to tumor progression in brain tissues and off-target effects on IGFR activation leading to hyperglycemia (7, 8). Although 3rd-generation TKIs provide clinical benefit to most patients with EGFR mutations, some patients, demonstrating primary resistance, still do not respond to these inhibitors. Complete responses are rare, and all patients eventually develop resistance, suggesting primary and acquired resistance mechanisms decrease the efficacy of the drugs (9, 10). Genomic approaches have been used primarily to interrogate osimertinib resistance mechanisms (9, 11-15). Several mechanisms of osimertinib resistance have been identified (16), including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events (17). However, the resistance mechanism is complex and still not fully understood. Previously, we have used SILAC-based quantitative phosphoproteomics to identify the global dynamic modification which occur upon treatment of TKI-sensitive and -resistant lung adenocarcinoma cells with the 1st and 2nd generation EGFR TKIs, erlotinib and afatinib. Utilizing this strategy, we identified the targets of mutant EGFR signaling pathways responsible for TKI resistance, and possible off-target effects of the drugs (18, 19). In this study, we employed SILAC-based quantitative mass spectrometry to characterize alterations in the proteome and phosphoproteome which occur upon acquired resistance and sought to identify novel mechanisms of resistance to the third generation EGFR TKIs, osimertinib and rociletinib. To our knowledge, this is the most comprehensive 3rd generation EGFR TKI resistant proteome and phosphoproteome analysis resource available to date.

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Concurrent single cell RNA and targeted DNA sequencing on a microfluidics device allows high-throughput identification of transcriptome and genome alterations during the development of tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer cells

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