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Cytoplasmic pattern recognition receptors (PRRs) for double-stranded RNA (RIG-I/MDA5) are key mediators of anti-viral responses. PRR agonists, such as dsRNA oncolytic Reovirus type 3 Dearing (Rt3D), potently activate RNA sensors. We used an unbiased cytotoxicity screen to reveal synergistic drug-virotherapy combinations and found potent effects of Rt3D combined with the CDK4/6 inhibitor, palbociclib. The combination augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and the expression and activation/signaling of RNA sensors. Combined Rt3D-palbociclib treatment potently increased interferon production and signaling, and knockdown studies implicated key UPR proteins and the RNA sensor, RIG-I, as essential to the phenotype observed. Further experiments, using canonical RIG-I agonists and an ER stress inducer, thapsigargin, confirmed cross-talk between RNA sensing and ER stress pathways that augmented cancer cell death and interferon production. Combined Rt3D-palbociclib also increased innate immune activation within tumour cells and IFN-induced HLA expression. Analysis of the immunopeptidome revealed changes to HLA-captured peptides with Rt3D-palbociclib, including altered expression of peptides from cancer/testis antigens (CTA) and endogenous retroviral elements (ERVs). Our findings highlight cross-talk between UPR signaling and RNA-mediated PRR activation as a means of enhancing anti-cancer efficacy with potential pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, and broadens the therapeutic remit of CDK4/6 inhibitors to include roles as both ER stress and dsRNA PRR sensitizers.

BackgroundDespite therapeutic gains from immune checkpoint inhibitors (ICI) in many tumor types, new strategies are needed to extend treatment benefits, especially in patients failing to mount effective antitumor T-cell responses. Radiation and drug therapies can profoundly affect the tumor immune microenvironment. Here, we aimed to identify immunotherapies to increase the antitumor response conferred by combined ataxia telangiectasia and Rad3-related kinase inhibition and radiotherapy.MethodsUsing the human papillomavirus (HPV)-negative murine oral squamous cell carcinoma model, MOC2, we assessed the nature of the antitumor response following ataxia telangiectasia and Rad3-related inhibitor (ATRi)/radiotherapy (RT) by performing RNA sequencing and detailed flow cytometry analyses in tumors. The benefit of immunotherapies based on T cell immunoreceptor with Ig and ITIM domains (TIGIT) and Programmed cell death protein 1 (PD-1) immune checkpoint blockade following ATRi/RT treatment was assessed in the MOC2 model and confirmed in another HPV-negative murine oral squamous cell carcinoma model called SCC7. Finally, immune profiling was performed by flow cytometry on blood samples in patients with head and neck squamous cell carcinoma enrolled in the PATRIOT clinical trial of combined ATRi/RT.ResultsATRi enhances radiotherapy-induced inflammation in the tumor microenvironment, with natural killer (NK) cells playing a central role in maximizing treatment efficacy. We demonstrated that antitumor activity of NK cells can be further boosted with ICI targeting TIGIT and PD-1. Analyses of clinical samples from patients receiving ATRi (ceralasertib) confirm the translational potential of our preclinical studies.ConclusionThis work delineates a previously unrecognized role for NK cells in the antitumor immune response to radiotherapy that can be augmented by small-molecule DNA damage–response inhibitors and immune checkpoint blockade.