Abstract LB-112: Tyrosine kinase and ERAD inhibitors synergize to promote apoptosis through ATF4 induction in medullary thyroid cancer
Authors: Bagheri-Yarmand Rozita , Sinha Krishna , Lu Yue , Gagel Robert 
Published: July 2017

Abstract
The activating transcription factor 4 (ATF4) plays a central role in activation of the integrated stress response pathway. Targeted deletion of Atf4 in mouse thyroid gland causes C-cell hyperplasia, a precancerous lesion for medullary thyroid cancer (MTC). Activating mutations of the tyrosine kinase RET are associated with oncogenic function in MTC progression. We have reported that RET is a serine-threonine dual specificity kinase, phosphorylates ATF4, and inhibits expression of the ATF4 target proapoptotic genes. Forced ATF4 expression in MTC cells decreased their survival through ubiquitin-mediated degradation of RET protein levels. We hypothesize that inactivation of ATF4 by RET is a central impediment to induction of apoptosis in response to TKI therapy. Further upregulation of ATF4 would render tumor cells exquisitely sensitive to stress induced apoptosis. Here, we show that the treatment of MTC cells with tyrosine kinase inhibitors upregulates ATF4 expression and induces Endoplasmic reticulum (ER) stress. Eeyarestatin is an ER-associated protein degradation (ERAD) inhibitor that elicit an integrated stress response program at the ER, which results in activation of ATF4 to stimulate expression of the apoptotic genes, PMAIP1 and BBC3, resulting into apoptotic cell death. We found that treatment of MTC cell lines, TT and MZCRC1 cells, with eeyarestatin not only induces ATF4 but also downregulates RET protein levels. The combinations of TKIs (vandetanib, sunitinib, cabozantinib) with eeyarestatin promote apoptotic cell death in a synergistic manner shown by cleaved PARP/ cleaved casapase3 staining using flow cytometry. Western blot analysis showed that combination therapy increased ATF4 levels and its targets, NOXA and PUMA levels as compared to each drug treatment alone. We applied genome-wide Chromatin Immunoprecipation Sequencing (ChIP-seq) to identify genes directly regulated by ATF4 in eeyarestatin-treated MZCRC1 cells. ChIP-seq analysis identified many previously known and unknown ATF4 target genes. We have identified the stress response, proapoptotic genes, inhibitors of MAPK, mTOR and βcatenin-wnt pathways with tumor suppressor function, including MKNK2, TCF3, APC2, KLF9, LZTFL1, SIAH1. Gene expression analysis was performed to further validate the activation of those genes in ATF4-overexpressing and eeyarestatin-treated cells. We found that combination of TKI and eeyarestatin synergistically upregulated expression of ATF4, and its targets APC2, LZTFL1, BBC3, MKNK2, and SIAH1. These results suggest that induction of ATF4 in combination with TKIs could be a promising therapeutic strategy to treat patients with medullary thyroid cancer.
Citation Format: Rozita Bagheri-Yarmand, Krishna Sinha, Yue Lu, Robert Gagel. Tyrosine kinase and ERAD inhibitors synergize to promote apoptosis through ATF4 induction in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2017-LB-112