Abstract IA15: Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition
Authors: Karam Sana 
Published: September 2020

Abstract
Radiation is commonly used in the definitive management of for head and neck cancer. Yet, many patients still fail treatment and develop resistance, especially non-virally driven tumors. Head and neck tumors are highly enriched in regulatory T cells that dampen the response to radiotherapy by creating an immune-inhibitory microenvironment. A key way by which CD4 T cells convert to FOXP3 positive regulatory T cells (Tregs) is through activation of STAT3.
We explored mechanisms of Treg signaling and infiltration and assessed their modulation by RT in murine models of head and neck squamous cell carcinoma (HNSCC). Mechanisms of Treg infiltration were investigated in orthotopic murine HNSCC tumors using flow cytometry, multiplex cytokine arrays and targeted therapeutics. Mice were treated with STAT3 ASO, anti-CTLA-4, anti-CD25, and/or anti-PD-L1 alone and in combination with RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Multiplex ELISA was performed for assessment of cytokines.
Treatment with anti-CD25 concurrently with RT led to tumor eradication, enhanced T-cell cytotoxicity, induced an antitumor antigen-specific memory response, decreased Tregs, and improved survival in orthotopic HNSCC tumor-bearing mice. Treg depletion induced an influx of CD8 and CD4 T cells when combined with RT. In addition, Treg depletion in combination with RT transformed myeloid populations, decreasing M2 macrophages and myeloid-derived suppressor cells (MDSCs) and increasing M1 macrophages. In contrast, RT alone or when combined with anti-CTLA4 and/or anti-PD-L1 did not lead to durable tumor control. To assess whether STAT3 inhibition can target Tregs we employed the STAT3 anti-sense oligonucleotide, AZD5149. STAT3 inhibition in combination with RT, but not as single agent, led to significant tumor growth delay and resulted in significant downregulation of immunosuppressive cytokines including TGF-β1 and IL10 and upregulation of IL2. Analysis of immune populations showed that STAT3 inhibition when combined with RT significantly decreases intratumoral Tregs and enhanced effector T-cell function. Experiments in nude mice demonstrated complete abolishment of the benefit of STAT3 ASO and RT, demonstrating the dependency on T cells for achieving tumor control.
These data reveal a critical role for Tregs in mediating resistance to RT. Targeted depletion of Tregs represents an important mechanism of sensitizing tumors to RT. We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving RT.
Citation Format: Sana Karam. Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA15.