Abstract 619: TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma
Authors: Shi Yewen , Ren Xiaoyong , Cao Shaolong , Xie Tongxin , Chen Xi , Michikawa Chieko , Osman Abdullah A. , Sikora Andrew G. , Myers Jeffrey N. , Rangel Roberto 
Published: April 2023

Abstract
Background: TP53 is the most common mutated gene in head and neck cancer, including oral squamous cell carcinoma (OSCC), a lethal disease with limited therapeutic options. In OSCC, for example, TP53 R175H is a hot-spot, high-risk, functional mutation that inhibits the function of WT p53, however these correlations and functional roles of high-risk p53 mutations had not been experimentally addressed using syngeneic oral tumor models. To understand the functional role of TP53 mutations in modulating the tumor immune microenvironment (TIME) and response to immunotherapy, we used a syngeneic oral tumor model (ROC3 cell) derived from a genetically engineered mouse model expressing the p53 R172H (human, R175H).
Methods: Orthotopic tongue injections were performed to (i) characterize tumor growth rate (ii) the TIME landscape by flow cytometry and opal multiplex immunofluorescence (mIF); and (iii) immunotherapy drug studies. We inactivated mutant p53 expression by shRNA and CRISPR Cas9, profiled gene expression changes by HTG EdgeSeq array, and validated these findings by qPCR, Western blot and immunochemistry. Tumor response was achieved by anti-PD1 and STING agonist therapies. Patients’ gene expression analyzed by CIBERSORT and TIME landscape performed by mIF. The spatial distribution of TIME was evaluated by Visiopharm software and UMAP.
Results: We demonstrated that disruption of mutant p53R172H in ROC3 cells altered cytokine and chemokine expression levels and consequently affected the maturation and infiltration of different immune subsets, including cytotoxic CD8+ T cells, helper CD4+ T cells, Tregs, G-MDSCs, tumor-associated macrophages and neutrophils in ROC3 tumors. In addition, our spatial TIME analysis revealed that p53R172H mutation promoted the infiltration of Tregs, M2 macrophages, and exhausted CD8+ T cells, and excluded active CD8+ and CD11+ cells in both the tumor cores and on the tumor edges. Interestingly, it seems that p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Moreover, we found that p53R172H mutation not only disrupted the phosphorylation of STING pathway which supported the recently reported finding, but also decreased the absolute amount of STING protein. Furthermore, we detected that p53-mutated tumor-infiltrating CD8+ and CD4+ T cells expressed programmed cell death protein 1 (PD-1), and that these tumors responded to anti-PD-1 and STING agonist therapy. Furthermore, Human OSCC samples also revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our ROC3 tumor model.
Conclusions: Our findings collectively demonstrate that the mutant TP53 GOF mutation modulates the tumor immune landscape and is associated with reduced overall survival in patients with OSCC.
Citation Format: Yewen Shi, Xiaoyong Ren, Shaolong Cao, Tongxin Xie, Xi Chen, Chieko Michikawa, Abdullah A. Osman, Andrew G. Sikora, Jeffrey N. Myers, Roberto Rangel. TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 619.