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NOTCH3 attenuates cytotoxicity via RBPJ-dependent PVR upregulation to influence immunotherapy outcomes in colorectal
Qi Ma1, Shuning Xu2, Ning Ma3
1Department of General Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, Henan, China.
Introduction:
This study aimed to elucidate the function of NOTCH3 in pan-cancer and CRC progression, its impact on the tumor immune microenvironment, and its value as a therapeutic target and predictive biomarker.
Methods:
We performed a multi-omics analysis of NOTCH3 alterations (expression, mutation, copy number variation, methylation) using data from The Cancer Genome Atlas (TCGA). Immune cell infiltration was assessed using multiple algorithms and single-cell RNA sequencing (scRNA-seq) data from CRC patients. In vitro functional experiments, including co-immunoprecipitation, chromatin immunoprecipitation (ChIP), luciferase reporter assays, and CD8+ T cell cytotoxicity co-cultures, were conducted in CRC cell line. An immune-competent mouse xenograft model was used to evaluate the efficacy of anti-NOTCH3 in combination with anti-PD-L1 therapy. Clinical validation was performed using independent immunotherapy-treated cohorts from the MSKCC database and our institutional cohort (102 patients) via immunohistochemistry and survival analysis.
Results:
NOTCH3 is frequently altered across multiple cancers. In CRC, high NOTCH3 expression correlated with poor survival and fostered an immunosuppressive microenvironment. Mechanistically, NOTCH3 transcriptionally upregulates the immune checkpoint molecule PVR by binding to the transcription factor RBPJ; this process is abrogated by NOTCH3 mutations (e.g., R1669H). NOTCH3-mediated PVR upregulation suppressed CD8+ T cell cytotoxicity. scRNA-seq analysis revealed enhanced PVR-TIGIT interactions between cancer and immune cells in NOTCH3-high tumors. In vivo, NOTCH3 depletion synergized with anti-PD-L1 therapy to inhibit tumor growth and increase CD8+ T cell infiltration. Clinically, NOTCH3 mutation or low expression independently predicted improved survival in immunotherapy-treated CRC and pan-cancer cohorts.
Conclusion:
NOTCH3 is a pivotal regulator of immune evasion in CRC via the RBPJ-PVR axis.
Insights
NOTCH3 drives immune evasion in colorectal cancer (CRC) by upregulating PVR, suppressing T cell activity. Targeting NOTCH3 combined with anti-PD-L1 therapy shows promise, while NOTCH3 mutations predict better immunotherapy response.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- NOTCH3 alterations are frequent across cancers.
- NOTCH3's role in colorectal cancer (CRC) progression and its impact on the tumor immune microenvironment remain incompletely understood.
- Identifying NOTCH3's therapeutic potential and predictive biomarker value is crucial.
Purpose of the Study:
- To investigate the function of NOTCH3 in pan-cancer and CRC.
- To analyze NOTCH3's impact on the tumor immune microenvironment.
- To evaluate NOTCH3 as a therapeutic target and predictive biomarker.
Main Methods:
- Multi-omics analysis of NOTCH3 alterations in TCGA data.
- Assessment of immune cell infiltration using scRNA-seq and other algorithms.
- In vitro functional assays and in vivo xenograft models with anti-NOTCH3 and anti-PD-L1 therapy.
- Clinical validation using independent immunotherapy-treated cohorts.
Main Results:
- High NOTCH3 expression in CRC correlates with poor survival and an immunosuppressive microenvironment.
- NOTCH3 upregulates PVR via RBPJ, suppressing CD8+ T cell cytotoxicity and enhancing PVR-TIGIT interactions.
- NOTCH3 depletion combined with anti-PD-L1 therapy inhibited tumor growth and increased CD8+ T cell infiltration in vivo.
- NOTCH3 mutation or low expression predicted improved survival in immunotherapy-treated patients.
Conclusions:
- NOTCH3 is a key regulator of immune evasion in CRC through the RBPJ-PVR axis.
- NOTCH3 alterations have significant implications for tumor progression and immunotherapy response.
- Targeting NOTCH3 offers a potential therapeutic strategy for CRC.
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