Synergistic Action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells. Synergistic Action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells

Rhabdoid tumors (RT) are rare and aggressive pediatric tumors that are driven by the loss the tumor suppressor SNF5 (SMARCB1). Here we examine how RT cells respond to small molecule-mediated inhibitors of the “WIN” site of WDR5, a chromatin-associated protein that regulates a specific set of genes linked to protein synthesis. We characterize WDR5 binding in RT cell lines via ChIP-Seq and show that WIN site inhibitor rapidly and comprehensively displaces WDR5 from chromatin in these cells. Using Precision Run On-sequencing (PRO-Seq) we show that WDR5-bound protein synthesis genes are early and direct transcriptional targets of WIN site inhibitor. We also use RNA-Seq to characterize the persistent transcriptional changes in RT lines treated with WIN site inhibitor, and compare these transcriptional effects with those of the HDM2 inhibitor nutlin-3a. Overall design: G401 scr shRNA, G401 p53 shRNA, or WT TTC549, cells were treated with DMSO or WIN site inhibitor C16. ChIP-Seq was used to track interaction of WDR5 with chromatin and RNA-Seq was used to monitor long-term transcriptional changes.

横纹肌样瘤（Rhabdoid tumors, RT）是一类罕见且高侵袭性的儿科肿瘤，其发病机制源于肿瘤抑制因子SNF5（SMARCB1）的缺失。
本研究探讨横纹肌样瘤细胞对靶向WDR5“WIN”位点的小分子抑制剂的应答反应——WDR5是一类染色质相关蛋白，可调控与蛋白质合成相关的特定基因集。本研究通过染色质免疫沉淀测序（ChIP-Seq）对横纹肌样瘤细胞系中的WDR5结合情况进行表征，结果显示“WIN”位点抑制剂可快速且全面地将WDR5从这些细胞的染色质上解离下来。借助精准延伸测序（Precision Run-On Sequencing, PRO-Seq），本研究证实结合有WDR5的蛋白质合成相关基因为“WIN”位点抑制剂的早期直接转录靶标。此外，本研究通过转录组测序（RNA-Seq）表征了经“WIN”位点抑制剂处理的横纹肌样瘤细胞系中的持续性转录变化，并将该转录效应与人双微体蛋白2（HDM2）抑制剂nutlin-3a的转录效应进行了对比。

整体实验设计：将G401阴性对照短发卡RNA（scr shRNA）转染细胞、G401 p53短发卡RNA（p53 shRNA）转染细胞，或野生型TTC549细胞分别用二甲基亚砜（DMSO）或“WIN”位点抑制剂C16进行处理。通过染色质免疫沉淀测序（ChIP-Seq）检测WDR5与染色质的相互作用，通过转录组测序（RNA-Seq）监测长期转录变化。