Background Although immune checkpoint inhibition has moved into the frontline setting for advanced and metastatic head and neck cancer (HNC), only a minority of patients experience durable treatment responses.1 Therapeutic resistance is associated with elevated hypoxia and STAT3 signaling and infiltration by myeloid derived suppressor cells (MDSC).2 3 In this study, we tested the hypothesis that resistant phenotypes are driven by tumor cell autonomous innate immune signaling.
Methods Gene Set Enrichment Analysis was used to assess transcriptional signatures of immunity in the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA). Western blotting was used to assess HIF1α, HIF2α and pSTAT3 expression. ELISA was used to test the expression of cytokines including IL-6, IL-8 and VEGF-A. Inhibitors and blocking antibodies were used to disrupt TLR2, TLR4, RAGE, IL-1R1 and HIF1α signaling in human cell lines. Transplantable murine models of immunotherapy-sensitive (MOC1) and -resistant (MOC2) oral squamous cell carcinoma were used to test the immune effects of inhibiting HIF1α and IL-1α signaling. Flow cytometry was used to assess infiltration of immune populations in murine tumors.
Results We identified patterns of constitutive STAT3 and hypoxia signaling in HNC cell lines from CCLE and confirmed that these signatures were enriched in human HNC tumors (figure 1A-C). We confirmed enrichment of cell autonomous pSTAT3 activation and HIF1a accumulation in HNC cell lines compared with non-HNC controls (figure 2A,B). Cell autonomous inflammatory signaling was associated with secretion of IL-6 and IL-8 (figure 2C). We dissected the mechanism of spontaneous inflammatory signaling and demonstrated that it depends entirely on IL-1α signaling and partly on HIF1α and TLR signaling, which are associated with increased expression of innate pattern recognition receptors (figure 2D,E).
We next confirmed the presence of cell autonomous inflammatory signaling in the murine MOC2 model and its absence in MOC1 (figure 3A,B). Echinomycin, a HIF1α inhibitor, significantly impaired the growth of MOC2 tumors (figure 3C,D). Echinomycin-treated MOC2 tumors demonstrated enhanced immune infiltration, T and NK cell infiltration and decreased MDSC compared with placebo controls (figure 3E-G).
Conclusions This study establishes a basis for self-propagating inflammatory signaling in HNC tumors driven by HIF1α, IL-1α and STAT3. Disruption of this loop by targeting the IL-1α or HIF1A signaling pathways represent promising strategies to overcome therapeutic resistance and induce anti-tumor immunity.
Acknowledgements The Yale SPORE in Head and Neck Cancer provided funding critical to the generation of this study.
Burtness B, et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet 2019;394:1915–1928.
Geiger JL, Grandis JR, Bauman JE. The STAT3 pathway as a therapeutic target in head and neck cancer: Barriers and innovations. Oral Oncol. 2016;56:84–92.
Swartz JE. et al. Poor prognosis in human papillomavirus-positive oropharyngeal squamous cell carcinomas that overexpress hypoxia inducible factor-1α. Head Neck 2016;38:1338–1346.
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