Abstract 4665: The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo in non-small cell lung cancer
Authors: Singh Ratnakar , Wang Yuehong , Wang Liguang , Nilsson Monique , Goonatilake Ruchitha , Tong Pan , Li Lerong , Giri Uma , Wang Jing , Heymach John V. , Johnson Faye M. 
Published: July 2016

Abstract
Introduction: Activation of epidermal growth factor receptor (EGFR) leads to the development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (EGFR-TKI) are effective therapy for NSCLC with activating EGFR mutations. However, acquired resistance to EGFR-TKIs is unavoidable and occurs through various molecular mechanisms including the development of T790M secondary EGFR mutations, MET amplification, epithelial-mesenchymal transition (EMT) and other mechanisms. One of potential strategy to overcome EGFR-TKIs is through inhibition of polo-like kinase 1 (PLK1). PLK1 is one of the key regulators of mitotic progression and a DNA damage recovery checkpoint. PLK1 inhibitors are at various stages of clinical development. Our previous studies showed that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibitor than epithelial ones. This study examines the efficacy of PLK1 inhibition in NSCLC cell lines with acquired resistance to the EGFR-TKI erlotinib.
Methods: Erlotinib resistant cell lines were developed in EGFR mutant, erlotinib sensitive cell lines PC9, HCC4006 and HCC827 by chronic exposure to stepwise increased concentrations of erlotinib. The effects of the PLK1 inhibitor volasertib alone or combined with erlotinib on proliferation, apoptosis, cell cycle, EGFR-dependent signaling, DNA damage and DNA damage response signaling were evaluated using CellTiter-Glo assay, TUNEL assay, BrDU incorporation assay, comet assay, western blot and immunofluorescence. Sensitivity of volasertib alone or in combination with erlotinib was also detected in the human tumor xenograft model. The combination index (CI) was calculated by Calcusyn software.
Results: Acquired erlotinib resistant NSCLC cell lines with endogenous EGFR mutations (PC9, HCC4006 and HCC827) showed diverse resistance mechanisms: T790M EGFR mutation, MET amplification and EMT. Four of the 6 ER clones that underwent EMT became more sensitive to volasertib. Volasertib and erlotinib demonstrated synergistic effects only in cells bearing T790M EGFR mutations where we observed more pronounced G2M arrest and increased apoptosis. Volasertib alone or in combination showed enhanced DNA damage, activation of CHK1/ATR and CHK2/ATM pathways and an increase in γH2AX foci but only a modest effect on canonical signaling downstream of EGFR. The combination treatment exhibited a pronounced reduction in the size of tumor compared to single agent treatment in subcutaneous xenograft model generated with T790M mutant EGFR TKI-resistant PC9 cell line.
Conclusions: These data demonstrate that PLK1 inhibition alone induces apoptosis and DNA damage in EGFR-TKI resistant cell lines with EMT. The combination of volasertib and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo.
Citation Format: Ratnakar Singh, Yuehong Wang, Liguang Wang, Monique Nilsson, Ruchitha Goonatilake, Pan Tong, Lerong Li, Uma Giri, Jing Wang, John V. Heymach, Faye M. Johnson. The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo in non-small cell lung cancer. [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 4665.