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Updated: Feb 2, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
RB1-I680T mutation potentiates tumor growth and chemotherapy sensitivity in non-small cell lung cancer via
Yilin Zhu1, Fengyuan Gao1, Yu Liu1,2
1Department of Thoracic Surgery, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Background:
Retinoblastoma Transcriptional Corepressor 1 (RB1) is a critical tumor suppressor restricting the malignant progression of cancer cells. Emerging evidence indicates that RB1 mutations typically promote tumorigenesis through loss of its tumor-suppressive functions. Yet, the biological significance of mutated RB1, specifically certain rare variants, in non-small cell lung cancer (NSCLC) remains elusive. Here, we first reported a rare and previously uncharacterized missense mutation in RB1, the 680th residue isoleucine replaced by threonine (RB1-I680T), in NSCLC.
Methods:
To investigate the functional and mechanistic consequences of the RB1-I680T mutation in NSCLC, we first used CRISPR-Cas9 to knock out endogenous RB1 in NSCLC cells. Then, we generated cell models harboring the RB1-I680T mutation by infecting these knockout cells with lentivirus carrying either wild-type RB1 (RB1-WT) or RB1-I680T expression constructs. The biological phenotypes mediated by RB1-I680T were investigated using in vitro and in vivo experiments. The exploration of the molecular mechanism was performed primarily through co-immunoprecipitation, immunofluorescence, dual-luciferase reporter assays, western blot analysis, and protein docking and dynamics simulation.
Results:
Our study demonstrated that the I680T mutation caused faster tumor growth and potentiated chemotherapy-induced tumor regression compared to RB1-WT control. Mechanistic studies illustrated that the I680T mutation in RB1 disrupted its inhibition of E2F1 transcriptional activity by weakening the physical interaction between RB1 and E2F1 in a manner dependent on conformational flexibility of RB1 pocket B domain, which is essential for sustaining the enhanced proliferation and chemosensitivity in NSCLC cells.
Conclusion:
Our findings elucidate that the I680T mutation-induced loss-of-function of RB1 simultaneously confers invasive proliferation and chemotherapeutic vulnerability to tumor cells, suggesting that RB1-I680T could serve as a predictive biomarker for chemotherapy response in NSCLC. Stratifying patients based on the RB1-I680T mutation status may enable personalized therapeutic strategies, particularly for tumors with E2F1 dysregulation.
Insights
A rare RB1-I680T mutation in non-small cell lung cancer (NSCLC) promotes tumor growth and enhances chemotherapy response. This RB1 mutation disrupts tumor suppression, offering a potential biomarker for personalized NSCLC treatment strategies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Retinoblastoma Transcriptional Corepressor 1 (RB1) is a key tumor suppressor gene.
- RB1 mutations often lead to cancer by losing tumor-suppressive functions.
- The role of rare RB1 variants in non-small cell lung cancer (NSCLC) is not well understood.
Purpose of the Study:
- To investigate the functional and mechanistic impact of a novel RB1 missense mutation (I680T) in NSCLC.
- To determine if RB1-I680T affects tumor growth, proliferation, and chemosensitivity.
- To elucidate the molecular mechanisms underlying the effects of the RB1-I680T mutation.
Main Methods:
- Generated NSCLC cell models with RB1 knockout and RB1-I680T or wild-type RB1 (RB1-WT) expression using CRISPR-Cas9 and lentivirus.
- Assessed biological phenotypes through in vitro and in vivo experiments.
- Explored molecular mechanisms using co-immunoprecipitation, immunofluorescence, luciferase assays, western blot, and protein simulations.
Main Results:
- The RB1-I680T mutation accelerated tumor growth compared to RB1-WT.
- RB1-I680T potentiated chemotherapy-induced tumor regression.
- The mutation impaired RB1's inhibition of E2F1 transcriptional activity by weakening RB1-E2F1 interaction, dependent on RB1 pocket B domain flexibility.
Conclusions:
- The RB1-I680T mutation leads to loss-of-function, promoting proliferation and conferring chemosensitivity in NSCLC.
- RB1-I680T may serve as a predictive biomarker for chemotherapy response in NSCLC patients.
- Stratifying patients by RB1-I680T status could enable personalized therapies, especially for E2F1-dysregulated tumors.
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