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CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer

Cell Death & Differentiation (2025)Cite this article

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Abstract

In the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired resistance remains a significant challenge. Elucidating the underlying mechanisms of resistance is crucial for developing novel strategies to overcome or delay therapeutic escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance and explore actionable targets for intervention. We investigated resistance mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The multi-omics approach revealed that CCT2 is a critical mediator of resistance to third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination and enhancing protein stability. This post-translational modification (PTM) promotes TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring resistance. Importantly, pharmacological inhibition with the compound HY-10127, identified through virtual screening, effectively restored EGFR-TKIs sensitivity in resistant cell lines and delayed the development of resistance in xenograft models. The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 represents a promising strategy to overcome resistance to third-generation EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries advance our understanding of molecular resistance mechanisms and offer potential therapeutic targets for improving lung cancer prognosis.

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Fig. 1: Genome-wide CRISPR/Cas9 screening identified CCT2 as a driver of lung cancer EGFR-TKI resistance.
Fig. 2: CCT2 promotes acquired EGFR-TKI resistance in NSCLC.
Fig. 3: CCT2 stabilizes TMX1 by removal of ubiquitination of TMX1.
Fig. 4: TRIM28 inhibits the ubiquitination of TMX1 by modulating its SUMO2 modification, thereby stabilizing TMX1.
Fig. 5: CCT2 recruits TRIM28 to SUMOylate TMX1 with SUMO2, and inhibiting its ubiquitination and degradation.
Fig. 6: TMX1 is the key player in CCT2-mediated EGFR-TKI resistance.
Fig. 7: The CCT2 inhibitor HY-10127 reverses furmonertinib resistance in combination with furmonertinib.

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Data are available on reasonable request.

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Funding

The National Natural Science Foundation of China (Grant no. 82300230). The National Natural Science Foundation of China (Grant no. 32200619). The National Natural Science Foundation of China (Grant no. 82103350). Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (Grant no. 2024KY1517). Natural Science Foundation of Zhejiang Province (Grant no. LKLY25H160008). Key Project of Zhejiang Natural Science Foundation (Grant no. LKLZ25H010001). The “Spark Program” of Cancer Treatment Clinical Research Innovation and Development Project Fund (Grant no. XH-A037). The Basic Research Project of Taizhou Clinical Medical College, Nanjing Medical University (Grant no. TZKY20230108).

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Author notes

  1. These authors contributed equally: Zihao Ke, Qi Zhang, Xingyu Chen.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China

    Zihao Ke, Xingyu Chen, Qianhua Cao, Ke Zhu, Shihui Wei, Jiajin Chen, Weina Huang, Kunlong Xiong & Chao Cao

  2. Department of Oncology, the Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou, Jiangsu, China

    Qi Zhang & Gaohua Han

  3. Department of Pathology, the Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou, Jiangsu, China

    Rongrong Jin

  4. Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China

    Qian Wang, Meiling Zhang & Kaihua Lu

  5. Institute of Engineering Medicine, the First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China

    Ying Chen

  6. Department of Thoracic Surgery, the First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China

    Kaimin Li

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Contributions

Chao Cao, Ying Chen and Kaihua Lu planned, reviewed and revised the manuscript. Zihao Ke, Qi Zhang, Xingyu Chen and Qianhua Cao conducted experiments and wrote the draft. Rongrong Jin performed the IHC assay. Gaohua Han, Ke Zhu, Shihui Wei, Jiajin Chen designed figures. Qian Wang, Meiling Zhang, Weina Huang, Kaimin Li, Kunlong Xiong modified the grammar errors and notation mistakes. All authors approved the submission of this manuscript.

Corresponding authors

Correspondence to Kaihua Lu, Ying Chen or Chao Cao.

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The authors declare no competing interests.

Ethics approval and consent to participate

All methods were performed in accordance with the relevant guidelines and regulations. All participants provided informed consent. About the use of patient data was approved by the institutional review boards of the Affiliated Taizhou People’s Hospital of Nanjing Medical University (No.KY2023-174-01). Animal experiments involved in this study were approved by the Committee on the Ethics of Animal Experiments of the Nanjing Medical University (No. IACUC-2201042).

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Ke, Z., Zhang, Q., Chen, X. et al. CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer. Cell Death Differ (2025). https://doi.org/10.1038/s41418-025-01600-w

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