Abstract 1895: The role of the IFNγ pathway in the development of vemurafenib resistance in BRAFV600E mutant thyroid carcinoma
Authors: Limberg Jessica , Gray Katherine D. , Singh Mandeep , Ullmann Timothy M. , Wyrwas Brian , Wang Weibin , Lowenstein Zachary , Eshaky Steve El , Liang Heng , Li Wei , Zhang Tuo , Xiang Jenny , Stefanova Dessislava , Zarnegar Rasa , Fahey Thomas J. , Min Irene M. 
Published: September 2020

Abstract
BRAFV600E is the most common driver mutation in anaplastic thyroid cancer, and BRAF inhibitors are increasingly being used in the treatment of this disease. However, the therapeutic benefit of BRAFV600E inhibitors, such as vemurafenib, has been limited in thyroid cancer due to rapid development of drug resistance in clinical practice. Here, we characterize a targetable mechanism of vemurafenib resistance by studying vemurafenib-resistant clones derived from BRAFV600E mutant anaplastic thyroid cancer cell lines (8505C and FRO), and propose a drug combination to overcome resistance. Vemurafenib resistance in these established clones was verified by persistent activation of ERK1/2 after vemurafenib treatment by western blot analysis. RNA-sequencing and subsequent ingenuity pathway analysis (IPA) was used to identify differences in pathway activation after vemurafenib treatment in parental and vemurafenib-resistant cell lines. Treatment of parental cells with vemurafenib resulted in differential gene expression correlating with upstream activation of the IFNγ-STAT1-IRF1 pathway (All FDR &lt; 0.05). Furthermore, parental cells treated with vemurafenib demonstrated higher HLA-II and HLA-A2 expression than untreated cells when analyzed by flow cytometry, implying the activation of IFNγ signaling pathway. In the IPA of a vemurafenib-resistant clone (8505C Res1), IFNγ, STAT1 and IRF1 expression was upregulated compared to their respective untreated parental cell line (All FDR &lt; 0.05). Increased IRF1 expression in 8505C Res1 compared to parental cells was also demonstrated by qPCR, supporting innate IFNγ activation in the resistant clone. Inhibition of the IFNγ pathway with increasing concentrations of neutralizing anti-IFNγ antibody sensitized resistant thyroid cancer cells to vemurafenib. Pharmacologically blocking the IFNγ downstream pathway in resistant clones with AZD1480 (JAK2 inhibitor) or fludarabine (STAT1 inhibitor), resulted in decreased cell viability in combination treatment groups compared to vemurafenib treatment only (cell viability: 92% vemurafenib vs 19% fludarabine combination, p &lt; 0.05) (cell viability: 73% vemurafenib vs 37% AZD1480 combination, p &lt; 0.05). These results demonstrate that activation of the IFNγ pathway is a potential mechanism for the development of resistance of anaplastic thyroid cancer to BRAFV600E inhibitors. Our study suggests that blockade of the IFNγ pathway may potentiate the therapeutic benefit of vemurafenib treatment in this disease.
Citation Format: Jessica Limberg, Katherine D. Gray, Mandeep Singh, Timothy M. Ullmann, Brian Wyrwas, Weibin Wang, Zachary Lowenstein, Steve El Eshaky, Heng Liang, Wei Li, Tuo Zhang, Jenny Xiang, Dessislava Stefanova, Rasa Zarnegar, Thomas J. Fahey, Irene M. Min. The role of the IFNγ pathway in the development of vemurafenib resistance in BRAFV600E mutant thyroid carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1895.