Abstract 1991: Notch pathway regulates proliferation of osimertinib drug-tolerant cells in epidermal growth factor receptor mutated non-small cell lung cancer
Authors: Takahashi Hirofumi , Sakakibara-Konishi Jun , Ito Shotaro , Tsuji Kosuke , Kikuchi Eiki , Kikuchi Junko , Shinagawa Naofumi , Konno Satoshi 
Published: July 2021

Background: Osimertinib is a third generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), which has shown remarkable antitumor activity in EGFR mutated patients with or without T790M mutation associated with previous-generation EGFR TKIs. But these effects are transient and the majority of patients develop resistance. A recently identified reversibly drug tolerant (DT) cells are defined as a small subpopulation of cells with remarkably reduced sensitivity to targeted drugs and noticed as non-genetic acquired resistance to EGFR-TKI. Notch is a transmembrane receptor that plays an important role in tumorigenesis. Notch pathway is associated with the resistance to previous-generation EGFR TKIs, however, the role of Notch is not fully understood in osimertinib treatment. In this study, we evaluated whether Notch was involved in resistance mechanism to osimertinib, using osimertinib DT cells and osimertinib resistant cells.

Methods: We isolated osimertinib resistant cells by stepwise exposure of human non-small cell lung cancer (NSCLC) cells with EGFR mutations (PC-9 cells and H1975 cells) to osimertinib (0.5-5 μmol/L). The osimertinib resistant cells were examined for EGFR mutations by direct sequencing, each time the concentration of osimertinib was increased. In resistant cells, original EGFR mutations were continuously observed without other new EGFR mutations. We isolated DT cells by exposure of human NSCLC cells with EGFR mutations (PC-9 cells and H1975 cells) to osimertinib (3 μmol/L) or combination of osimertinib and γ-secretase inhibitor (GSI) (1 μmol/L) for 9 days. The proteins of EGFR, ERK1/2, AKT, Notch1, HES1 and HEY were examined using western blot. The cytotoxic effect of osimertinib and GSI was evaluated using MTT assay and clonogenic assay in vitro.

Results: The phosphorylation of EGFR was decreased and Notch1 expression was increased in resistant cells compared to the parental cells. Combination of osimertinib and GSI inhibited the proliferation of parental cells compared to osimertinib or GSI single treatment, but did not reduce the viability of resistant cells. DT cells were highly insensitive to osimertinib compared with their parental cells. Continuous administration of osimertinib and GSI delayed the proliferation and colony formation of DT cells compared to osimertinib alone. The phosphorylation of EGFR, ERK1/2, and AKT was decreased and Notch1 expression was increased in DT cells compared to the parental cells. Although up-regulated Notch1 in DT cells was inhibited when GSI was administered together with osimertinib, phosphorylation of EGFR, ERK1/2 and AKT was unchanged, suggesting that other bypass pathways might be suppressed by GSI.

Conclusions: Combination of osimertinib and GSI delays proliferation of DT cells in EGFR mutated NSCLC by modulating Notch pathway.

Citation Format: Hirofumi Takahashi, Jun Sakakibara-Konishi, Shotaro Ito, Kosuke Tsuji, Eiki Kikuchi, Junko Kikuchi, Naofumi Shinagawa, Satoshi Konno. Notch pathway regulates proliferation of osimertinib drug-tolerant cells in epidermal growth factor receptor mutated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1991.