Data from Nrf2 Hyperactivation as a Driver of Radiotherapy Resistance and Suppressed Antitumor Immunity in Head and Neck Squamous Cell Carcinoma

<div>AbstractPurpose:<p>Alterations in the <i>KEAP1</i>/<i>NFE2L2</i> (NRF2)/<i>CUL3</i> pathway occur in ∼20% of human head and neck squamous cell carcinomas (HNSCC) and are associated with resistance to standard-of-care therapy. However, this pathway’s role in radiotherapy resistance in HNSCC has not been well studied.</p>Experimental Design:<p>We generated genetically engineered mouse models and developed primary murine cancer cell lines harboring mutations commonly observed in human HNSCC, including inducible activation of <i>PIK3CA</i> and deletion of <i>Trp53</i>, with or without <i>Keap1</i> loss. Primary tumors were initiated via 4-hydroxytamoxifen injection ± the tobacco carcinogen benzo[a]pyrene (BAP) into the oral buccal mucosa. Tumors were analyzed by Western blotting, IHC, and RNA sequencing and subjected to fractionated radiotherapy to investigate the role of the KEAP1/NRF2 pathway in radioresistance and modulation of the tumor-immune microenvironment.</p>Results:<p>BAP exposure accelerated primary tumor formation within 1 month, with histologic analysis confirming invasive squamous cell carcinoma, validated by cytokeratin and differentiation marker expression. Primary cell lines derived from <i>Keap1</i>-haploinsufficient tumors exhibited upregulation of NRF2 target genes and a radioresistant phenotype, which was reversed after Nrf2 knockdown <i>in vitro</i>. Bulk RNA sequencing revealed that <i>Keap1</i> haploinsufficiency correlated with NRF2 pathway activation, increased myeloid infiltration, and enhanced angiogenic signatures. <i>In vivo</i>, <i>Keap1</i> haploinsufficiency promoted accelerated tumor growth and decreased survival. Finally, using fractionated radiotherapy, we showed that <i>Keap1</i>-haploinsufficient primary tumors were significantly more radioresistant than <i>Keap1</i>-proficient tumors, regardless of BAP exposure.</p>Conclusions:<p>These data demonstrate that <i>Keap1</i> haploinsufficiency in HNSCC is linked to unfavorable tumor-immune microenvironment, aggressive growth, and a radioresistant phenotype.</p></div>