The ETS inhibitor YK-4-279 suppresses thyroid cancer progression independent of TERT promoter mutations. The ETS inhibitor YK-4-279 suppresses thyroid cancer progression independent of TERT promoter mutations

Hotspot mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been well established to associate with aggressive clinical characteristics, radioiodine refractory, tumor recurrence and mortality in thyroid cancer. Several E-twenty-six (ETS) transcription factors were reported to selectively bound to the mutant TERT promoter and activated TERT expression. In this study we aimed to investigate whether TERT promoter mutations confer sensitivity to ETS inhibitor YK-4-279 in thyroid cancer cells and whether this inhibitor could be served as a potential therapeutic agent for thyroid cancer. In vitro assays showed that YK-4-279 treatment sharply suppressed cell viability, colony formation, migration and invasion, as well as induced cell cycle arrest and apoptosis in a panel of thyroid cancer cells. The cell viability after YK-4-279 treatment were similar between cell lines harboring mutant and wild-type TERT promoter. Furthermore, YK-4-279 treatment reduced both luciferase activity and mRNA expression of TERT independent of TERT promoter mutation status. Data from RNA-seq further revealed that YK-4-279 significantly affected biological processes including DNA replication and cell cycle. Reduced DNA helicase activity and decreased expression of several helicase genes were observed after YK-4-279 treatment. Moreover, YK-4-279 significantly inhibited tumor growth and induced apoptosis in a xenograft mice model. Thus, ETS inhibitor YK-4-279 suppressed TERT expression and conferred anti-tumor activity in a TERT promoter mutation-independent manner, and it could be a potential agent for the treatment of advanced thyroid cancers. Overall design: KHM-5M cells were treated with DMSO and YK-4-279 at different dosage (0.3 μM and 1 μM) with three repllication.

端粒酶逆转录酶（telomerase reverse transcriptase, TERT）基因核心启动子区域的热点突变已被广泛证实与甲状腺癌的侵袭性临床特征、碘难治性、肿瘤复发及病死率密切相关。已有研究表明，多种E-二十六（E-twenty-six, ETS）转录因子可选择性结合突变型TERT启动子，进而激活TERT的表达。本研究旨在探究TERT启动子突变是否会使甲状腺癌细胞对ETS抑制剂YK-4-279产生敏感性，以及该抑制剂能否作为甲状腺癌的潜在治疗药物。体外实验结果显示，YK-4-279处理可显著抑制一组甲状腺癌细胞的细胞活力、集落形成、迁移与侵袭能力，同时诱导细胞周期阻滞与细胞凋亡。经YK-4-279处理后，携带突变型与野生型TERT启动子的细胞系之间的细胞活力无显著差异。进一步研究发现，YK-4-279处理可降低TERT的荧光素酶活性与mRNA表达水平，且该效应不受TERT启动子突变状态的影响。RNA测序（RNA-seq）数据分析进一步揭示，YK-4-279可显著调控包括DNA复制与细胞周期在内的多种生物学过程。经YK-4-279处理后，细胞的DNA解旋酶活性降低，且多个解旋酶基因的表达水平下调。此外，YK-4-279可显著抑制异种移植小鼠模型中的肿瘤生长并诱导肿瘤细胞凋亡。综上，ETS抑制剂YK-4-279可通过不依赖TERT启动子突变的方式抑制TERT表达并发挥抗肿瘤活性，有望成为晚期甲状腺癌的潜在治疗药物。总体实验设计：将KHM-5M细胞用二甲基亚砜（DMSO）与不同浓度（0.3 μM及1 μM）的YK-4-279处理，设置三次生物学重复。