Abstract 265: Cotargeting EGFR, STAT3 and Src-Notch pathways: a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients
Authors: Karachaliou Niki , Chaib Imane , Pilotto Sara , Codony Jordi , Cai Xueting , Li Xuefei , Drozdowskyj Ana , Codony Carles , Cardona Andrés Felipe , López-Vivanco Guillermo , Vergnenègre Alain , Sánchez José Miguel , Provencio Mariano , de Marinis Filippo , Carcereny Enric , Reguart Noemí , García-Campelo Rosario , Marin Silvia , Teixido Cristina , Sperduti Isabella , Rodríguez Sonia , Estrada Roger , Puig de la Bellacasa Raimon , Ramírez José Luis , Molina-Vila Miguel Angel , Zhou Caicun , Cao Peng , Ma Patrick , Bivona Trever , Rosell Rafael 
Published: July 2016

Abstract
Intrinsic or acquired resistance limits the clinical effectiveness of EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations. One of the signaling mediators downstream of activated EGFR is signal transducer and activator of transcription 3 (STAT3). Not only does gefitinib not inhibit STAT3, but it also augments STAT3 tyrosine phosphorylation. EGFR blockade enriches lung cancer stem cells (CSCs) through NOTCH3-dependent signaling. A co-receptor of IL-6 (gp130) associates with Src and triggers activation of YAP and NOTCH. Our study is designed with three parallel objectives: firstly, to demonstrate that single EGFR TKI treatment cannot abrogate STAT3 and Src in EGFR mutant NSCLC cell lines; secondly, to examine whether the combination of gefitinib with compounds that target STAT3, (TPCA-1) and Src (saracatinib), suppresses the mechanisms of resistance; thirdly, to identify biomarkers in clinical tumor samples that may help us predict the outcome of EGFR TKIs and design effective combination therapies. Cell viability assay (MTT), western blotting, quantitative-real time PCR (qRT-PCR) and aldefluor assay-flow cytometry were used. We found that gefitinib increases pSTAT3 Y705 in PC-9 cells (that harbor the exon 19 deletion) in a time- and dose-dependent manner. Nine days after gefitinib treatment STAT3 mRNA level was significantly elevated. PC-9 cells showed dramatic increase in the fraction of ALDH+ cells upon treatment with gefitinib. TPCA-1 increased sensitivity to gefitinib in the PC-9 cells. Combination of gefitinib with TPCA-1 abrogated pSTAT3 Y705 but neither inhibited pPaxillin Y118 (Src induced) and pYAP S127 nor prevented the increment in the ALDH+ CSCs subpopulation. The triple combination of gefitinib, TPCA-1 and saracatinib was highly synergistic and abrogated pSTAT3 Y705, pPaxillin Y118 and pYAP S127. We performed qRT-PCR at baseline tumor samples of 64 EGFR mutant NSCLC p treated with first line EGFR TKIs and found that high expression of STAT3 and YAP were significantly correlated with shorter median progression-free survival (mPFS). mPFS was 9.6 months (m) (95% CI, 5.9 to 14.1) for p with low STAT3 and 18.4m (95% CI, 8.8 to 30.2) for p with high STAT3 mRNA expression (P&lt;0.001). mPFS was 9.6 months (95% CI, 7.7 to 15.2) for p with low YAP and 23.4 months (95% CI, 13.0 to 28.1) for p with high YAP mRNA expression (P = 0.005). A combined STAT3 and YAP risk group model was constructed since the mRNA expression of the 2 transcripts was weakly correlated (r = .0.15; P = 0.305). mPFS was 25.7 months for p with low STAT3 and YAP mRNA (95% CI, 8.5 to 60.9), 9.4 months for p with high STAT3 and YAP mRNA (95% CI, 2.8 to 15.2), and 14.1 months for others (95% CI, 8.2 to 23.4) (P = 0.004). Single EGFR TKI treatment can no longer be considered adequate for p with EGFR mutant lung cancer and a clinical trial co-targeting STAT3 and Src is warranted.
Citation Format: Niki Karachaliou, Imane Chaib, Sara Pilotto, Jordi Codony, Xueting Cai, Xuefei Li, Ana Drozdowskyj, Carles Codony, Andrés Felipe Cardona, Guillermo López-Vivanco, Alain Vergnenègre, José Miguel Sánchez, Mariano Provencio, Filippo de Marinis, Enric Carcereny, Noemí Reguart, Rosario García-Campelo, Silvia Marin, Cristina Teixido, Isabella Sperduti, Sonia Rodríguez, Roger Estrada, Raimon Puig de la Bellacasa, José Luis Ramírez, Miguel Angel Molina-Vila, Caicun Zhou, Peng Cao, Patrick Ma, Trever Bivona, Rafael Rosell. Cotargeting EGFR, STAT3 and Src-Notch pathways: a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 265.