Abstract IA18: Interrogating anti-PD1 immunotherapy resistance mechanisms
Authors: Uppaluri Ravindra 
Published: September 2020

Background: The response rate of 15-20% with anti-PD1 targeting in head and neck squamous cell carcinoma (HNSCC) highlights the need for strategies to overcome resistance. We have focused on delineating mechanistic aspects of immune checkpoint response and resistance using novel clinical trial approaches coupled with analyses in high-fidelity immunocompetent murine oral carcinoma (MOC) model to study HNSCC immunobiology.

Methods: Using the MOC HNSCC model, we generated anti-PD1 resistant models. Whole-exome sequencing and RNA-Seq were used to compare mutational burden and expression signatures. Time of flight mass cytometry (CyTOF) was used to analyze tumor microenvironment (TME) remodeling in response to checkpoint inhibition. 2-D coculture system was used to assess tumor recognition by MHC class I restricted cytotoxic T cells. Tumor cells were loaded with SIINFEKL peptide and OT1 T cells were used as effectors in T-cell cytotoxicity assays. The anti-PD1 resistant MOC model overexpressing CAS9 with high editing efficiency was generated for a genome-scale CRISPR screen. The Broad Institute Genomic Perturbation Platform Brie lentiviral library, including 78,637 gRNAs targeting 19,674 with 1000 controls, was transduced into this line to define modifiers of CD8+ T cell mediated killing. Ezh2 inhibitors and knockout lines were used in characterizing the role of EZH2 in antigen presentation and antitumor immunity.

Results: To examine immunotherapy resistance we generated 2 independent anti-PD1 adaptive resistant cell line models, MOC1-esc1 and MOC1-esc2, from their isogenic MOC1 parental line. RNA-Seq showed that MOC1-esc1 upregulated Myc and E2F targets and lost inflammatory signatures whereas -esc2 tumors have upregulated DNA damage and unfolded protein response. When MOC1-esc tumors were retransplanted into naive mice, they exhibited resistance to anti-PD1, while remaining responsive to anti-CTLA4 treatment. To gain comprehensive insights into the tumor microenvironment (TME) as a contributor to adaptive resistance, we analyzed tumor-infiltrating lymphocytes (TIL) in naive MOC1 and MOC1-esc1 tumors using CyTOF with a panel of 38 markers. MOC1-esc1 tumors were highly infiltrated with regulatory T cells (Tregs) and M2-like tumor-associated macrophages (TAMs), while MOC1 tumors have more M1-like TAMs and neutrophils. Furthermore, we observed that both anti-PD1 and anti-CTLA4 treatment dramatically expanded CD8+ T cells and decreased neutrophils in MOC1-esc1 tumors. In responding MOC1-esc1 tumors, anti-CTLA4 treatment resulted in Treg depletion, decreased M2-like TAMs and neutrophils, as well as a striking increase in M1-like TAMs compared with isotype control treated tumors. In contrast, anti-PD1 treated MOC1-esc1 tumors showed decreased M1-like TAMs, while M2-like TAMs were increased compared with control. Depletion of Tregs, neutrophils, or repolarization of TAM using monoclonal antibodies confirmed their contribution in immunotherapy resistance. As the MOC1-esc1 line showed in vitro resistance to T-cell cytotoxicity compared to MOC1 and to define tumor cell intrinsic modulators of T-cell cytotoxicity, we then completed a genome-scale CRISPR screen in MOC1-esc1. This screen identified 355 candidate genes whose loss of function regulated T-cell cytotoxicity. Chromatin modifiers emerged as a major class of genes regulating T-cell recognition of tumor cells. We validated Ezh2 as a therapeutic target and showed that inhibition of this pathway enhanced Class I expression and sensitized MOC1-esc1 cells to T-cell killing in vitro. Analysis of human HNSCC cell line models and TCGA data revealed conservation of Ezh2 impact on Class I expression. Combination therapy with Ezh2 inhibitors and anti-PD1 blockade resulted in enhanced therapeutic efficacy compared to either agent alone in the anti-PD1 resistant MOC1-esc1 model.

Conclusions: In summary, this study identified (1) immune modulators within TME involved in adaptive immunotherapy resistance of HNSCC and remodeling with checkpoint therapy and (2) tumor cell intrinsic candidate pathways regulating T-cell recognition. Findings from these studies will advance our understanding of HNSCC immunotherapy resistance and will accelerate the discovery of new combination therapeutic targets and biomarkers in adaptive resistance.

Citation Format: Ravindra Uppaluri. Interrogating anti-PD1 immunotherapy resistance mechanisms [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 IA18.