Abstract 2766: Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti-PD-L1 refractory head and neck cancer
Authors: Oweida Ayman J. , Hararah Mohammad , Phan Andy , Bhatia Shilpa , Lennon Shelby , Binder David , Raben David , Heasley Lynn , Clambey Eric , Nemenoff Raphael , Karam Sana D. 
Published: August 2018

Background: Clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that a majority of head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. Strategies aimed at sensitizing tumors to immunotherapy can provide durable response compared to conventional therapies. We previously reported that local radiation to the tumor can transform the immune landscape and render poorly immunogenic murine orthotopic HNSCC tumors sensitive to PD-L1 inhibition. However, the response to combined radiotherapy (RT) and PD-L1 inhibition was transient. In this work, we characterize the immune landscape of HNSCC tumors during RT and PD-L1 treatment and interrogate mechanisms of resistance.

Methods: We employed time-of-flight mass cytometry (CyTOF) for characterizing the tumor immune microenvironment at early and late stages of response to RT and anti-PD-L1. For studying the effect of dual checkpoint blockade and RT, poorly immunogenic murine squamous cell carcinoma cells (LY2 and MOC2) were injected into the right buccal mucosa. Mice were randomized to IgG, anti-PD-L1, anti-TIM-3, RT or combinations of RT, PD-L1 and TIM-3. Mechanistic experiments were performed on tumors harvested 72 hours after treatment. Tumors were assessed for levels of activated T cells (CD44+IFNg+) and regulatory T cells (FoxP3+CD4+) by flow cytometry and T-cell infiltration by immunohistochemistry.

Results: Tumors treated with RT and PD-L1 blockade significantly upregulated TIM-3 expression on CD4 and CD8 T cells. Furthermore, the proportion of regulatory T cells expressing TIM-3 was significantly increased during the tumor regrowth phase with RT and anti-PD-L1 treatment. Targeting TIM-3 concurrently with PD-L1 and RT led to significant tumor growth retardation, enhanced T-cell cytotoxicity and improved survival. In addition, dual targeting of PD-L1 and TIM-3 in combination with RT significantly decreased the proportion of regulatory T cells (4.1-fold decrease relative to IgG control and 2.8-fold decrease relative to RT+anti-PD-L1). However, the response to dual checkpoint blockade and RT was not durable and all tumors eventually relapsed. Analysis of relapsed tumors revealed decreased T-cell infiltration, re-emergence of regulatory T cells and decreased proportion of cytotoxic and helper T cells.

Conclusion: Our study shows upregulation of TIM-3 in response to RT and PD-L1 inhibition. However, dual targeting of TIM-3 and PD-L1 with RT did not provide a durable response in two genetically distinct HNSSCC tumor models. In light of the increased number of clinical trials employing dual immune checkpoint blockade, our findings reveal the complexity of tumor immune evasion mechanisms and underscore the need for multimodality targeting of HNSCCs.

Citation Format: Ayman J. Oweida, Mohammad Hararah, Andy Phan, Shilpa Bhatia, Shelby Lennon, David Binder, David Raben, Lynn Heasley, Eric Clambey, Raphael Nemenoff, Sana D. Karam. Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti-PD-L1 refractory head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2766.