Abstract 2232: The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations
Authors: Kim Jeong-Oh , Shin Jung-Young , Kim Min-Young , Lee Mi-Ran , Kim Seoree , Lee Hankyu , Jeon Doyong , Baek Seung Ryel , Il Kim Joo , Lee Kyoungjune , Lee Youn Soo , Kang Jin-Hyoung 
Published: April 2023

Abstract
Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (mEGFR) well respond to EGFR TKIs, but the majority of patients eventually acquire drug resistance, leading to treatment failure. Recently, intracellular potential autophagy in addition to primary genetic alterations implicates the acquired drug resistance to targeted drugs in various carcinomas such as melanoma, pancreas, and breast cancer. We aimed to identify autophagy flux-related signal molecules which may be closely linked to acquired resistance in mEGFR NSCLC.
Methods: We constructed the TMA composed of 579 surgically resected tumor tissues from lung adenocarcinoma patients and evaluated protein expression of autophagy flux-related signal molecules including CAGE, p-Beclin1, LC3B, p-AMPK, ULK1, p62, and ATG5 using immunohistochemistry (IHC). We established H1975OSI (166-folds) and PC9ERL (3,650-folds) resistant sub cell lines through the prolonged exposure of Osimertinib and Erlotinib to H1975 (L858R/T790M) and PC9 (Del19) cells, respectively. We also explored genes that induce autophagy flux activity using mRNA seq and qRT-PCR.
Results: H-score of CAGE ( 95.6±7.9, 70±5.5), p-AMPK (73.4±7.8, 32±5.6) and p-Beclin1 (44.4±16.1, 19.7±5.5), ULK1( 57.4±4.9, 41.9±4.9) were significantly higher in mEGFR, compared to those of wild type EGFR NSCLC (P &lt;0.001). According to EGFR mutation subtype, the expressions of CAGE (98±11.2, 70±5.5) and ULK1(60.7±5.0, 41.9±4.9) were significantly higher in Del19 (P&lt;0.001), while the expressions of CAGE(93.2±5.6, 70±5.5,), p-AMPK(82.9±5.7, 32±5.6), and p-Beclin1(45.6±16.7, 19.7±5.5) were significantly higher in L858R (P&lt;0.001). In advanced/metastatic mEGFR NSCLC patients treated with EGFR TKIs, p-Beclin1(13.0±10.9, 59±7.2) expression was significantly increased, when comparing before treatment and at treatment failure (P&lt;0.018). As results of mRNA seq and qRT-PCR in H1975 parental cells and H1975OSI cells, we identified autophagy flux-related signal molecules including BECLIN1, GABAR1, NBR1, PINK1, ULK1 which were significantly increased more than 10 times.
Conclusion: Taken together, our results strongly support the evidence that autophagy flux-related signal molecules as well as known genetic alterations are closely connected to the acquired resistance during period of EGFR TKI treatment in mEGFR NSCLC patients. Now, we are undergoing additional RNA profiling and function study of identified novel autophagy-related genes to examine a possibility as potential target to overcome EGFR TKI resistance.
Citation Format: Jeong-Oh Kim, Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Seoree Kim, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Kyoungjune Lee, Youn Soo Lee, Jin-Hyoung Kang. The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2232.