American Association for Cancer Research
10780432ccr131559-sup-fig1.pdf (102.2 kB)

Supplementary Figure 1 from Tyrosine Phosphoproteomics Identifies Both Codrivers and Cotargeting Strategies for T790M-Related EGFR-TKI Resistance in Non–Small Cell Lung Cancer

Download (102.2 kB)
journal contribution
posted on 2023-03-31, 17:47 authored by Takeshi Yoshida, Guolin Zhang, Matthew A. Smith, Alex S. Lopez, Yun Bai, Jiannong Li, Bin Fang, John Koomen, Bhupendra Rawal, Kate J. Fisher, Ann Y. Chen, Michiko Kitano, Yume Morita, Haruka Yamaguchi, Kiyoko Shibata, Takafumi Okabe, Isamu Okamoto, Kazuhiko Nakagawa, Eric B. Haura

PDF file - 104KB, Supplementary Figure 1A-B: PC9GR cells acquired drug resistance to both reversible and irreversible EGFR-TKI. A: PC9 and PC9GR cells were treated for 72 h with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. B: PC9 and PC9GR cells were incubated for 1 hour in the absence or presence of erlotinib (1 �M) as indicated. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pSrc, pAkt, and pErk along with antibodies to β-actin as a loading control. C-D: Gene status in PC9 and PC9GR cells. C: PC9GR cells acquired T790M while retaining exon 19 deletion as determined by PCR-invader assay. Values of red bars above zero represent positive results with the detection probe for indicated mutation sequences of EGFR, while values of light blue bars are quality controls showing good PCR quality, as previously described (1). The value of red bars = (fluorescence of the samples with detection probe for mutation sequences) - (fluorescence of the normal control with the same probe � 2). The value of light blue bars = (fluorescence of the samples with detection probe for wild type sequences) - (fluorescence of the normal control with the same probe � 0.8). DEL1 represents E746-A750del type1 (2235-2249del GGAATTAAGAGAAGC). DEL2 represents E746-A750del type2 (2236-2250del GAATTAAGAGAAGCA). DEL3 represents L747-P753del insS. D: FISH analysis (CEP7 (green)/D7S522 (red)) was done in PC9 and PC9GR cells as previously described (2). No additional copy of D7S522 compared to CEP 7 was found in these cells, explaining that neither PC9 nor PC9GR cells has MET amplification.



Purpose: 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.Experimental Design: 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.Results: 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.Conclusions: 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. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.