UNDERSTANDING THE MECHANISMS BEHIND RADIOTHERAPY RESISTANCE OF IMMUNOLOGICALLY COLD HEAD AND NECK CANCER - Outcomes have stagnated for patients with locally advanced human papillomavirus (HPV)-negative head & neck squamous cell carcinoma (HNSCC) over the past few decades. Recent studies have shown that ∼15% of all HPV-negative HNSCC patients have altered KEAP1/NFE2L2(NRF2) pathway, which strongly correlates with radioresistance and poor outcomes. Now we know that many factors, such as genetic lesions, pH, oxygenation, and interactions with other cells in the tumor microenvironment, play a critical role in reprogramming cancer cell metabolism. No one has studied Keap1’s radioresistance role in HNSCC using a mouse model that closely recapitulates human disease. Consequently, we developed a novel genetically engineered mouse model (GEMM) that lacks Keap1 (or overexpress Nrf2 downstream targets) for studying HNSCC, allowing us to control the temporal and spatial induction of primary tumors. Using these in vivo models, we showed that even a partial loss of Keap1 resulted in (1) sustained hyperactivation of the Keap1/Nrf2 signaling pathway, (2) increased infiltration of myeloid cells, particularly macrophages & granulocytes, and (3) significant radioresistance of primary HNSCC tumors. Furthermore, we discovered that partial loss of Keap1 increased the radiosensitization of poly ADP ribose polymerase (PARP) inhibitors in vitro. This application aims to understand the role of the tumor immune microenvironment (TIME) in radioresistance and whether PARP inhibitors can overcome this. Aim 1 – Does NRF2 signaling reshape TIME in HNSCC tumors post-RT? We will utilize HNSCC cell lysates and supernatants at baseline and post-RT to quantify the production and secretion of inflammatory mediators, as well as their influence on various leukocyte chemotaxis. This will be achieved using Ibidi µ-slide chambers filled with cell supernatants to create a chemical gradient. Finally, we will quantify the changes in the TIME of primary tumors at baseline and following RT using a pre-validated panel of fluorescently labeled flow cytometry antibody markers for leukocyte subsets. Aim 2 – Investigate whether manipulating the DDR pathway in NRF2 overexpressing tumors could create favorable TIME. We will transiently knockdown or overexpress Nrf2 in HNSCC cell lines, treat them with RT +/- PARPi, and collect cell lysates/supernatants. We will assess (1) DNA damage, (2) DDR, (3) activation of the cGAS-STING pathway, and (4) the release of damage-associated molecular patterns (DAMPs). Finally, we will collect primary tumors following RT +/- PARPi treatment and perform RNA-seq to compute single-sample gene set enrichment analysis scores for different leukocyte subsets. The future research plans – The data and resources generated from this R03 project will provide the necessary framework for an expanded R01 grant application and ultimately lead toward improving HPV-negative HNSCC patient outcomes.
