Dataset Information

Phosphoproteomics landscape of lung DTP

ABSTRACT: Osimertinib is the first-line therapy for EGFR-mutated non-small cell lung cancer, but acquired resistance commonly develops, linked to a drug-tolerant persister (DTP) cell state that promotes survival and eventual resistance. Using data-independent acquisition mass spectrometry (DIA-MS), we mapped dynamic proteomic and phosphoproteomic changes in DTPs. While osimertinib initially blocks EGFR signaling, DTPs show activation in ribosome synthesis and protein translation pathways, with resistance emerging via EGFR reactivation and anti-apoptotic mechanisms, including YAP1 and mTOR-BAD hyperphosphorylation. Elevated phosphorylation of CDK1 substrates in DTPs was observed, and inhibiting CDK1-mediated SAMHD1 activation impaired DTP growth. This study highlights key DTP mechanisms, suggesting biomarkers and therapeutic targets to delay resistance.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Hsiang-En Hsu

LAB HEAD: Yu-Ju Chen

PROVIDER: PXD058009 | Pride | 2025-11-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AZ_PhosphoDIA36x_lib216x.sne	Other
	AZ_ProteoDIA36x_lib227x.sne	Other
	PhosphoDIA_120min_lysate_Acute_10min_01.raw	Raw
	PhosphoDIA_120min_lysate_Acute_10min_02.raw	Raw
	PhosphoDIA_120min_lysate_Acute_5min_01.raw	Raw

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Publications

Phosphoproteomics of osimertinib-tolerant persister cells reveals targetable kinase-substrate signatures.

Hsu Hsiang-En HE Martin Matthew J MJ Weng Shao-Hsing SH Kitata Reta Birhanu RB Nagelli Srikar S Chang Chiung-Yun CY Hess Sonja S Chen Yu-Ju YJ

Molecular systems biology 20250929 11

Osimertinib is the first-line therapy for EGFR-mutated non-small cell lung cancer, but acquired resistance emerges in most patients and remains a major barrier for complete cure. This phenomenon is most likely associated with the drug-tolerant persister (DTP) cell phenotype, a reversible state that enables survival under treatment and leads to irreversible drug resistance. To uncover the molecular mechanism driving this distinct phenotype, we applied data-independent acquisition mass spectrometr ...[more]

PMID: 41023502

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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.