Abstract 3083: NT219 overcomes immune evasion-resistant mechanisms in head and neck squamous cell carcinoma (HNSCC)

Abstract Background: NT219 is a small molecule that promotes insulin receptor substrate 1/2 (IRS) degradation and suppresses STAT3 phosphorylation, two major complementary signaling pathways that play a key role in cancer and drug resistance. We present NT219 suppressive effect on HNSCC tumor and tumor microenvironment (TME) and on cancer stem cells (CSC) which contribute to drug resistance and disease recurrence Method: Tumor microarrays (TMA) of 54/57 HNSCC and 9 normal tissue cases were analyzed for pSTAT3(Y705) and IRS2 by IHC NT219 and cetuximab effect on CSC was assessed using 3D spheroid assay of HPV- HNSCC lines, evaluating size (microscopic measurements), cell viability (CellTiterGlo) and stemness marker expression (FACS analysis). Ex vivo patient-derived tumor explant (PDE), treated with NT219 and/or nivolumab was analyzed for efficacy and cytokine secretion. In vivo efficacy was tested in a stem cell-enriched (NutriStem®) patient-derived xenograft (PDX). Results: NT219 with cetuximab suppressed established spheroids of SCC-9 HNSCC, in terms of size (100% disintegration) and CSC viability (94% reduction), while cetuximab had no effect (P<0.001). NT219 alone showed a significant effect on CSC viability (82% reduction, P<0.001). HNSCC FaDu spheroids showed the same trend. Spheroids showed enrichment in OCT3/4 or NANOG positive CSC (4-fold vs 2D growth), completely reversed by NT219 and cetuximab. Inhibitory effect was also demonstrated in established FaDu spheroids including major stemness markers; OCT3/4, NANOG, SOX2, ALDH, CD166, CD44. In a stem cell-enriched PDX HNSCC model, NT219 synergized with cetuximab to suppress tumor growth. Accordingly, delayed tumor recurrence demonstrated. A significant upregulation of STAT3(Y705) phosphorylation and IRS1/2 were detected in tumor and/or TME fibroblasts and TILs vs normal tissue. PDX models demonstrated that blockage of both STAT3 and IGF1R/IRS pathways is required to overcome resistance to EGFR therapy in terms of survival and tumor size. In a HNSCC PDE resistant to nivolumab, NT219 and nivolumab combination induced synergistic tumor cell death (66%) accompanied by immuno-cytotoxic activation (10 and 1.5-fold increase of IL2 and granzyme B). Nivolumab-induced immunosuppressive effects (8-fold increase of IL-10, 2-fold decrease of TNFα) were completely blocked by the combination. In a phase 1 study of HNSCC patients treated with NT219 50-100 mg/kg and cetuximab standard dose weekly, significant differences in patient’s biopsy pIGF1R and pSTAT3 were observed in 2 responders (PR) compared to the 4 progressed disease pts. Conclusions: NT219 mitigates several immune evasion mechanisms including CSC-mediated resistance and tumor recurrence, and sensitizes resistant HNSCC tumors to PD1 and EGFR therapies. Upregulation of pIGF1R and pSTAT3 were correlated with patient response and suggested as potential biomarkers for NT219 treatment. Citation Format: Hadas Reuveni, Hava Ben David, Katy Sigalov, Daphna Miron. NT219 overcomes immune evasion-resistant mechanisms in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3083.