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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 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.

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is the standard of care as 1st-line treatment for NSCLC patients with activating EGFR mutations (exon 19 deletion or L858R mutation). However, EGFR-TKI therapy eventually leads to acquired drug resistance approximately 1-2 years from the beginning of treatment. Multiple acquired resistance mechanisms including on-target EGFR mutations, phenotype transformation, and activation of bypass signaling molecules have been identified, but over 35% still remains unknown. CAGE, a cancer/testis antigen, was originally isolated from the sera of patients with gastric cancer and is known to regulate the autophagic flux in acquired resistance to EGFR TKI in NSCLC. Herein, the expression of CAGE was found in more than 50% of NSCLC patients using immunohistochemistry (IHC). Interestingly, the expression level was increased especially for the patients acquiring resistance to EGFR-TKIs. In addition, both PC-9/ER (Erlotinib-resistance cells) and H1975/OR (Osimertinib-resistant cells) cells demonstrated increased CAGE expression and autophagic flux, when compared with their parental cells. Thus, we developed the peptide drugs targeting the DEAD box domain of CAGE. This novel peptide effectively inhibited tumor cell proliferation in EGFR-TKI resistant cell line. Moreover, in Erlotinib-resistant patient-derived xenograft (PDX) mice, the peptide drug retarded tumor growth and enhanced anti-tumor efficacy. Collectively, our data suggest that CAGE plays a crucial role in the tumor progression and acquired resistance of EGFR TKI by the modulation of autophagic flux. Furthermore, we provided evidence that the inhibition of autophagy through CAGE- derived peptide could overcome the acquired resistance to EGFR TKIs in advanced/metastatic NSCLC patients. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Jaemoon Koh, Dooil Jeoung, Jin Hyoung Kang. Inhibition of autophagy by CAGE targeting peptide retard tumor growth in non-small cell lung cancer of EGFR TKI resistance [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 1582.