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Interruption of MDM2 signaling augments MDM2-targeted T cell-based antitumor immunotherapy through antigen-presenting machinery

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Abstract

Identification of immunogenic tumor antigens, their corresponding T cell epitopes and the selection of effective adjuvants are prerequisites for developing effective cancer immunotherapies such as therapeutic vaccines. Murine double minute 2 (MDM2) is an E3 ubiquitin-protein ligase that negatively regulates tumor suppressor p53. Because MDM2 overexpression serves as a poor prognosis factor in various types of tumors, it would be beneficial to develop MDM2-targeted cancer vaccines. In this report, we identified an MDM2-derived peptide epitope (MDM232-46) that elicited antigen-specific and tumor-reactive CD4+ T cell responses. These CD4+ T cells directly killed tumor cells via granzyme B. MDM2 is expressed in head and neck cancer patients with poor prognosis, and the T cells that recognize this MDM2 peptide were present in these patients. Notably, Nutlin-3 (MDM2-p53 blocker), inhibited tumor cell proliferation, was shown to augment antitumor T cell responses by increasing MDM2 expression, HLA-class I and HLA-DR through class II transactivator (CIITA). These results suggest that the use of this MDM2 peptide as a therapeutic vaccine combined with MDM2 inhibitors could represent an effective immunologic strategy to treat cancer.

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Abbreviations

APC:

Antigen-presenting cell

CIITA:

Class II transactivator

CTL:

CD8+ cytotoxic T lymphocyte

DC:

Dendritic cell

E:

T: Effector: Target

FFPE:

Formalin fixed, paraffin embedded

HNSCC:

Head and neck squamous cell carcinoma

HTL:

CD4+ helper T lymphocyte

ICI:

Immune checkpoint inhibitor

mAb:

Monoclonal antibody

MDM2:

Murine double minute 2

MFI:

Mean fluorescence intensity

PBMC:

Peripheral blood mononuclear cell

TAA:

Tumor-associated antigen

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Acknowledgements

The authors also thank Dr. Hajime Kamada (Hokuto Social Medical Corporation) for his excellent suggestions for the manuscript

Funding

This work was supported by JSPS KAKENHI Grant Number 20K209724.

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Authors and Affiliations

  1. Department of Otolaryngology-Head & Neck Surgery, Asahikawa Medical University, Asahikawa, 078-8510, Japan

    Michihisa Kono, Takumi Kumai, Ryusuke Hayashi, Hidekiyo Yamaki, Hiroki Komatsuda, Risa Wakisaka, Kenzo Ohara, Kan Kishibe, Miki Takahara, Akihiro Katada, Tatsuya Hayashi & Yasuaki Harabuchi

  2. Department of Innovative Head & Neck Cancer Research and Treatment, Asahikawa Medical University, Asahikawa, Japan

    Takumi Kumai & Tatsuya Hayashi

  3. Department of Pathology, Asahikawa Medical University, Asahikawa, Japan

    Toshihiro Nagato, Takayuki Ohkuri, Akemi Kosaka & Hiroya Kobayashi

  4. Cancer Immunology, Inflammation and Tolerance Program, Augusta University, Georgia Cancer Center, Augusta, GA, USA

    Esteban Celis

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  1. Michihisa Kono
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  2. Takumi Kumai
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Contributions

MK, TK, RH, HY, HK, RW, TN and KO took part in acquisition, analysis and interpretation of data. KK, MT and AKa involved in statistical analysis of data. TO, AKo, HK participated in material support. TK, TH and YH involved in development of methodology. TK took part in conception, design and supervision of the study. MK and TK involved in writing of the paper. TK, YH and EC participated in review of the paper.

Corresponding author

Correspondence to Takumi Kumai.

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The authors declare no potential conflicts of interest

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All experiments were approved by the institutional ethics committee on the Asahikawa Medical University (#16217). The study was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. The patients have given their written informed consent to participate and publish their case.

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Supplementary Information

262_2021_2940_MOESM1_ESM.pptx

Supplementary Table S1: Correlations between MDM2 expression and clinical characteristics in HNSCC patients. The data were analyzed using Fisher’s exact test.

Supplementary Figure S1:MDM2 inhibitor reduced the proliferation of HNSCC cells in a dose-dependent manner. Error bars indicate standard deviations of triplicates.

Supplementary Figure S2: MDM2 inhibitor (Nutlin-3, 10μM) upregulated MDM2 expression in HNSCC cell lines.

Supplementary Figure S3: Expression levels of EGFR in HNSCC cell lines co-cultured with Nultin-3 were evaluated by flow cytometry. Left panels show representative data of flow cytometry. Black: isotype control, blue: EGFR on untreated tumor cell lines, red: EGFR on tumor cell lines treated with Nutlin-3 (10µM). Right panels show averages values of mean fluorescence intensity (MFI). (***<0.001, Student’s t test).

Supplementary Figure S4: Kaplan–Meier curves show the relationship between MDM2 expression and overall survival in HNSCC patients. Statistical difference was calculated using log-rank test. Kaplan–Meier analysis was performed with GraphPad Prism 8.

Supplementary Figure S5: Expression levels of PD-L1 in HNSCC cell lines co-cultured with Nultin-3 were evaluated by flow cytometry. Left panels show representative data of flow cytometry. Black: isotype control, blue: PD-L1 on untreated tumor cell lines, red: PD-L1 on tumor cell lines treated with Nutlin-3 (10µM). Right panels show averages values of mean fluorescence intensity (MFI). (***<0.001, Student’s t test).

Supplementary file1 (PPTX 842 kb)

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Kono, M., Kumai, T., Hayashi, R. et al. Interruption of MDM2 signaling augments MDM2-targeted T cell-based antitumor immunotherapy through antigen-presenting machinery. Cancer Immunol Immunother 70, 3421–3434 (2021). https://doi.org/10.1007/s00262-021-02940-5

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