Global proteome and phosphoproteome alterations in third generation EGFR TKI resistance reveal drug targets to circumvent resistance

Abstract

<jats:title>Abstract</jats:title><jats:p>Lung cancer is 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 3<jats:sup>rd</jats:sup> 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. 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 3<jats:sup>rd</jats:sup> generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative mass spectrometry dataset to uncover alterations in signaling pathways, reveal a proteomic signature of epithelial mesenchymal transition (EMT) and identify kinases and phosphatases with altered expression and phosphorylation in TKI resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition resulting in inhibition of RAS/MAPK and activation of PI3K/AKT 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 dactolisib, a PI3K/mTOR inhibitor, which in combination with osimertinib overcomes resistance both <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>.</jats:p><jats:sec><jats:title>One Sentence Summary</jats:title><jats:p>Global quantitative proteome and phosphoproteome analyses to examine altered signaling pathways in isogenic 3<jats:sup>rd</jats:sup> generation EGFR TKI sensitive and resistant cells.</jats:p></jats:sec>

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells

“…To understand the molecular processes governing the cellular response to LCS3, we profiled the transcriptome and proteome by RNA microarray and by stable isotope labeling of amino acids in cell culture (SILAC) (Zhang et al, 2021), respectively. To assess temporal changes in RNA abundance induced by LCS3, two sensitive LUAD cell lines-H23 (KRAS mutant) and H1650 (EGFR mutant)-were treated with 3 mM LCS3 for 3, 6, or 12 h and transcriptionally profiled at each time point (Figures 2A and S2A).…”

“…Cells were treated in duplicate (one heavy and one light) with 3 mM LCS3 for 24 hours and then harvested by cell scraping. Samples were mixed 1:1, digested with trypsin, and prepared for mass spectrometry as previously described (Zhang et al, 2017(Zhang et al, , 2021. The SILAC proteome dataset has been uploaded to PRIDE: PXD027228.…”

GZ17-6.02 and Pemetrexed Interact to Kill Osimertinib-Resistant NSCLC Cells That Express Mutant ERBB1 Proteins

“…Osimertinib is a relatively specific inhibitor of mutant active forms of ERBB1 and is, at present, the standard of care therapeutic. As with all targeted drugs in cancer, eventually NSCLC cells become osimertinib resistant, with diverse mechanisms, including gain of additional ERBB1 mutations or activation of other receptor tyrosine kinases such as c-MET and FGFRs (15)(16)(17)(18). Overcoming osimertinib resistance remains an important area for the developmental cancer therapeutics field in NSCLC.…”

A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

“…To understand the molecular processes governing the cellular response to LCS3, we profiled the transcriptome and proteome by RNA microarray and by stable isotope labeling of amino acids in cell culture (SILAC) (15), respectively. To assess temporal changes in RNA abundance induced by LCS3, two sensitive LUAD cell lines-H23 (KRAS mutant) and H1650 (EGFR mutant)were treated with 3µM LCS3 for 3, 6 or 12 hours and profiled at each time point with Affymetrix RNA microarrays (Fig.…”

“…Cells were treated in duplicate (one heavy and one light) with 3µM LCS3 for 24 hours and then harvested by cell scraping. Samples were mixed 1:1, digested with trypsin, and prepared for mass spectrometry as previously described (15,74). The SILAC proteome dataset has been provided as a supplementary file.…”