PI3Kinase based degradation of NRF2 signaling in chemo-radiation resistant HNSCC

<title>Abstract</title> <p> Background. Hyperactivation of NRF2 is an established factor contributing to chemoradiation resistance in head and neck squamous cell carcinoma (HNSCC). In tumors with <italic>KEAP1</italic> loss or mutations in the oncogenic <italic>PIK3CA</italic> pathway, NRF2 accumulates, enhancing antioxidant defenses that mitigate the cytotoxic effects of cisplatin and ionizing radiation. An alternative approach to directly targeting NRF2 exploits the PI3K/AKT/GSK3 axis, which regulates a KEAP1-independent pathway for NRF2 proteasomal degradation. Methods. We evaluated the effects of the dual PI3K/mTOR inhibitor gedatolisib on NRF2-signaling and phenotype in various human and murine HNSCC cell lines, as well as genetically engineered mouse models (GEMMs) with conditional <italic>Keap1</italic> deletion and orthotopic xenografts of the cisplatin-resistant HN30R8 line. Proliferative responses were tracked using real-time IncuCyte imaging and label-free holotomographic microscopy. NRF2 protein regulation was assessed via western blot, nuclear/cytoplasmic fractionation, and RT-qPCR. The role of GSK3 and the proteasome in the gedatolisib-induced reduction of NRF2 was confirmed through pharmacological rescue with GSK3 and proteasome inhibitors and further validated by siRNA knockdown of GSK3-α, GSK3-β, β-TrCP, and SPOP. Results. Gedatolisib inhibited AKT phosphorylation and decreased NRF2 protein levels in a dose- and time-dependent manner across all tested cell lines, including those derived from GEMMs with Keap1 deletion. This reduction was post-translational, as <italic>NFE2L2</italic> mRNA was upregulated via FoxO transcription factor activation, while NRF2 protein was directed towards GSK3-dependent, β-TrCP/SPOP-mediated proteasomal degradation. Holotomographic live-cell imaging revealed that cells treated with gedatolisib primarily underwent necrotic cell death, which was accompanied by mitochondrial accumulation and growth arrest. Gedatolisib effectively suppressed tumor growth in both <italic>Keap1</italic> wild-type and <italic>Keap1</italic> -deleted GEMMs. In orthotopic HN30R8 cisplatin-resistant xenografts, gedatolisib was the primary driver of antitumor activity. Additionally, the combination of gedatolisib and radiation showed synergistic tumor suppression according to the multiplicative model in a replicated experiment. Conclusions. Gedatolisib effectively overcomes NRF2-driven resistance in HNSCC by engaging a degradation pathway that does not rely on KEAP1. The drug simultaneously activated NRF2 transcription through FoxO and accelerated the degradation of NRF2 protein via the GSK3/β-TrCP pathway, resulting in decreased NRF2 protein levels. These findings support the potential of gedatolisib as a chemoradiosensitizer in HNSCC clinical trials. </p>