ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is a frequent and deadly malignancy. Despite significant advances in the understanding of the pathobiology of HNSCC, the substantial patient morbidity associated with treatment and the high frequency of tumor recurrence/metastasis result in an unacceptably low patient survival and poor quality of life. The cancer stem cell (CSC) hypothesis attempts to explain the observed heterogeneity of cancer cells within a tumor, with many malignant features of a cancer cell deriving from a shift towards stem- like features. CSCs function as drivers of tumor initiation, therapeutic evasion, and recurrence in HNSCC. Platinum-based agents such as Cisplatin have been shown to enhance the CSC fraction and self-renewal, as determined by Bmi-1 expression. Head and neck CSCs rely on cellular crosstalk within the perivascular niche, particularly on endothelial cell-secreted IL-6. Our preliminary data showed that IL-6/STAT3 inhibition prevents Cisplatin-induced CSC self-renewal. But the mechanism through which inhibition of IL-6 signaling asserts this function and its implications on therapeutic resistance and tumor recurrence remain unclear. The long-term objective of this project is to study molecular mechanisms underlying the acquisition and maintenance of the stem-like cancer cell phenotype. The overall hypothesis of this work is that therapeutic blockade of the IL- 6/STAT3 pathway suppresses the Bmi-1-mediated CSC self-renewal and inhibits HNSCC recurrence. To test this hypothesis, we propose the following specific aims: 1) to elucidate mechanisms underlying IL-6/STAT3- mediated phenotypic changes in the cancer cell population, 2) to describe the real-time effect of IL-6/STAT3 inhibition and Cisplatin therapy on CSC proliferation patterns, and 3) to determine the effect of inhibiting CSC self-renewal on resistance to conventional Cisplatin therapy and recurrence in HNSCC. To accomplish these aims, both genetic and pharmacologic approaches will be used in CSC assays in vitro and in vivo to test the hypothesis that the CSC phenotype is regulated by Bmi-1 via IL-6/STAT3 signaling. We will investigate the real- time phenotypic changes within the CSC population using a CRISPR/Cas9 reporter system to test the hypothesis that IL-6/STAT3 inhibition promotes asymmetric cell division of CSC by decreasing their self-renewal. Cisplatin- resistant cell lines and patient-derived xenograft mouse models will be used to test the hypothesis that inhibiting IL-6/STAT3 signaling will overcome evasive resistance to Cisplatin and prevent tumor recurrence. Elucidating crucial mechanisms that define the fate of head and neck cancer stem cells will inform mechanism-based therapies that have the potential to enhance the survival and quality of life of patients with head and neck cancer.