RB1-I680T mutation potentiates tumor growth and chemotherapy sensitivity in non-small cell lung cancer via derepressing E2F1 transcription.

[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.