PROJECT SUMMARY − PROJECT 1 In recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC), immune checkpoint blockade has changed the standard of care, with trials from HN SPORE PI Dr. Robert Ferris and co-I (Project 2) Dr. Barbara Burtness pioneering the use of anti-PD-1 in this setting. Unfortunately, only a minority of patients benefit, due to resistance to anti-PD-1 therapy, pointing to an urgent need to better understand the tumor microenvironment. With HN SPORE support, first through a Developmental Research Program award and later following elevation to Continuing Project 1, we have identified a connection between tumor metabolism, hypoxia and T cell dysfunction that may be partially driving resistance to anti-PD-1 (Zandberg, et al, ASCO 2020). To further investigate this question, we are proposing to conduct two newly designed, novel therapeutic clinical trials within Project 1, in R/M HNSCC naïve to anti-PD-1 (HCC 18-190/NCT04114136) or progressed on anti-PD-1 (HCC 18-156/NCT04326257). The former is a trial of metabolic modulators and anti- PD1, the latter a trial of anti-PD-1 combined with either anti-CTLA4 or anti-LAG3. Utilizing these trials and pre- clinical models of HNSCC, we will examine the following questions. First, what is the relationship between anti-PD-1 resistance, tumor metabolism and hypoxia in HNSCC? With the assistance of Core B, we will address this question via multiplexed tissue analysis in R/M HNSCC samples from our clinical trials as well as radiomics-based approaches of determining tumor hypoxia. Second, does hypoxia promote resistance to combinatorial immunotherapy in HNSCC? We will test how hypoxia may impede immunotherapy with nivolumab plus relatlimab (anti-LAG3), or ipilimumab (anti-CTLA4) in R/M HNSCC patients who have progressed on anti-PD-1, evaluating tissue before and after therapy. Third, can metabolically targeted therapy be combined with anti-PD-1 to overcome anti-PD1 resistance in HNSCC? We will evaluate tumor samples obtained before and after treatment with anti-PD-1 plus either metformin or rosiglitazone and determine changes in tumor metabolism and hypoxia. We will also test combinatorial immunotherapy (as in Aim 2) with metabolic modulation in pre-clinical HNSCC models rendered anti-PD1 resistant. Our trial of metabolic inhibitors combined with anti-PD1 therapy, if positive, will directly lead to larger scale clinical studies, opening up an entirely novel avenue of combinatorial immunotherapy; while the project as a whole will also provide a platform for developing future personalized immunotherapy trials by adding metabolic analysis and/or modulation as a component.

PROJECT NARRATIVE − PROJECT 1 Despite the promise of immune checkpoint inhibition in head and neck squamous cell carcinoma (HNSCC), only a minority of patients ultimately benefit. Continuing Project 1 has already identified tumor metabolism and hypoxia as potential drivers of anti-PD-1 resistance in this disease and now seek to examine the interaction between tumor hypoxia and metabolism, combinatorial immunotherapy, and response. The goal of this project will be to identify the next generation of metabolically driven immunotherapy combination for clinical trial.