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Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.

ABSTRACT: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined.We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib-treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M.

SUBMITTER: Yoshida T

PROVIDER: S-EPMC4119578 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.

Yoshida Takeshi T Zhang Guolin G Smith Matthew A MA Lopez Alex S AS Bai Yun Y Li Jiannong J Fang Bin B Koomen John J Rawal Bhupendra B Fisher Kate J KJ Chen Y Ann YA Kitano Michiko M Morita Yume Y Yamaguchi Haruka H Shibata Kiyoko K Okabe Takafumi T Okamoto Isamu I Nakagawa Kazuhiko K Haura Eric B EB

Clinical cancer research : an official journal of the American Association for Cancer Research 20140611 15

<h4>Purpose</h4>Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.<h4>Experimental design</h4> ...[more]

PMID: 24919575

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Project description:Simple Summary Treatment outcomes for non-small cell lung cancer (NSCLC) patients have significantly improved since the introduction of targeted therapy using tyrosine kinase inhibitors (TKI). Despite the initial promising results, most patients develop resistance due to on- or off-target mechanisms. On-target mutations prevent the effective binding of TKIs. In this study, we determined whether the most common on-target resistance mutation for treatment with EGFR-TKI mutation, i.e., the EGFR T790M mutation, can be detected in pre-treatment tissue samples and can predict treatment outcome. We included 33 patients who progressed with a T790M positive relapse upon treatment with EGFR-TKI between 2013 to 2019. Progression-free survival (PFS) time of the single T790M positive patient was 9 months, while the T790M negative patients had a median PFS of 10 months (range 2–27). Our results show that the presence of EGFR T790M mutations in pre-TKI samples is rare, even in patients who subsequently progressed with an EGFR T790M mutation. Abstract EGFR-mutated non-small cell lung cancer (NSCLC) patients can be effectively treated with tyrosine kinase inhibitors (TKI) but frequently present with an EGFR T790M resistance mutation at relapse. We aimed to screen for T790M in pre-treatment formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients with a confirmed T790M mutation at progression. We analyzed 33 pre-treatment DNA samples of NSCLC patients who progressed upon TKI between 2013 to 2019. To establish storage-time dependent formalin fixation-induced background levels for C>T mutations, we analyzed DNA isolated from archival (stored >1 year, n = 22) and recently generated (stored <1 month, n = 11) FFPE samples and included DNA isolated from white blood cells (WBC) (n = 24) as controls. DNA samples were analyzed by droplet digital (dd)PCR, and positivity was defined by outlier detection according to Grubb’s criterion. The T790M background allele frequency levels were 0.160% in DNA isolated from archival-FFPE, 0.100% in fresh FFPE, and 0.035% in WBC. Progression-free survival (PFS) time of the single T790M positive patient was 9 months, while T790M negative patients had a median PFS of 10 months (range 2–27). Proper storage time matched FFPE control samples are essential for reliable detection of T790M mutation at low VAF. The presence of EGFR T790M mutations in pre-TKI samples is rare, even in patients who progressed with EGFR T790M mutations.

Dataset Information

Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.

Publications

Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.

Similar Datasets

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