SIRT2 is a regulator of the differentiation block driven by SMARCB1 loss in ATRT. [CUT&Run]

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive brain tumor of childhood with a poor overall survival. The salient molecular feature of ATRT is the loss of SMARCB1 which results in epigenetic dysregulation of the genome. SMARCB1 loss affects lineage commitment and differentiation by controlling gene expression. We hypothesized that additional epigenetic factors co-operate with SMARCB1 loss to control cell self-renewal and drive ATRT. We identified SIRT2 as a primary dependency in ATRT. Using a combination of RNA-seq, CUT&RUN and single RNA sequencing in model systems ATRT, we observed genome-wide reorganization of active chromatin and significantly changes in genes expression in ATRT cells with genetic (with shRNA) or chemical (with Tenovin or Thiomyristoyl (TM)) SIRT2 deactivation. Single-cell RNA transcriptome analysis of xenograft tumors revealed the elimination of tumor cells expressing stem cell genes and expansion of tumor cells expressing differentiated genes in vivo. Furthermore, SIRT2 inhibition induced apoptosis, decreased tumor growth and prolonged survival in orthotopic xenograft models. In summary we demonstrated that SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted tumors with therapeutic potential. Overall design: CUT&RUN data comparing peak height in Atypical teratoid rhabdoid tumor (ATRT) BT16 cells treated with Thiomyristoyl (TM) and DMSO. Thiomyristoyl was bought from MedChem express (HY-101278) and reconstituted in DMSO. BT16 ATRT cells were treated with 20 uM of TM for 24 hours followed by CUT&RUN assay with antibodies for H3K4me (positive control; EpiCypher #13-0041), H3K27ac (Cell signaling #8173), HeK27me3 (Cell signaling#, 9733), IgG (negative control; EpiCypher#13-0042K). For bulk RNA-seq experiment BT16 ATRT cells were treated with DMSO or 30uM TM for 72hrs. RNA-Seq data comparing gene expression in pediatric ATRT treated with TM vs DMSO. Single-cell RNA sequencing on GFP-positive BT16ATRT cells. To study the impact of TM on differentiation in vivo, we performed single-cell RNA sequencing on GFP-positive BT16 ATRT cells. Cell were growing in mice cerebellum and treatment started when tumors were established. Mice were treated with either TM (50 mg/kg IP 3/week for 4 weeks) or Vehichle control.

非典型畸胎瘤样横纹肌样瘤（Atypical teratoid rhabdoid tumor, ATRT）是一种极具侵袭性的儿童脑部肿瘤，整体预后极差。ATRT的核心分子特征为SMARCB1缺失，该突变会导致基因组表观遗传失调。SMARCB1缺失可通过调控基因表达，影响细胞谱系定型与分化过程。我们推测存在其他表观遗传因子与SMARCB1缺失协同作用，调控细胞自我更新并驱动ATRT发生发展。

我们鉴定出SIRT2是ATRT的核心依赖基因。通过在ATRT模型系统中联合运用RNA测序（RNA-seq）、CUT&RUN技术以及单细胞RNA测序，我们观察到：当通过短发夹RNA（shRNA）或化学抑制剂（替诺芬（Tenovin）或硫代肉豆蔻酰基化合物（Thiomyristoyl, TM））使SIRT2失活时，ATRT细胞的全基因组活性染色质发生重排，基因表达亦出现显著改变。

对异种移植瘤的单细胞RNA转录组分析显示，体内表达干细胞基因的肿瘤细胞被清除，而表达分化相关基因的肿瘤细胞发生扩增。此外，SIRT2抑制可诱导细胞凋亡、降低原位异种移植模型中的肿瘤生长速率并延长小鼠生存期。综上，我们证实SIRT2抑制是SMARCB1缺失肿瘤的分子脆弱位点，具备潜在治疗价值。

整体实验设计：
1. CUT&RUN实验：对比经硫代肉豆蔻酰基化合物（Thiomyristoyl, TM）与二甲基亚砜（DMSO）处理的非典型畸胎瘤样横纹肌样瘤BT16细胞的染色质峰高差异。硫代肉豆蔻酰基化合物购自MedChem Express（货号HY-101278），以DMSO进行复溶。BT16 ATRT细胞以20 μM TM处理24小时后，采用针对H3K4me（阳性对照；EpiCypher #13-0041）、H3K27ac（Cell Signaling #8173）、H3K27me3（Cell Signaling #9733）以及IgG（阴性对照；EpiCypher #13-0042K）的抗体完成CUT&RUN检测。
2. 批量RNA测序实验：BT16 ATRT细胞分别以DMSO或30 μM TM处理72小时，通过RNA-seq对比儿童ATRT细胞经TM与DMSO处理后的基因表达差异。
3. 单细胞RNA测序：针对GFP阳性的BT16 ATRT细胞开展单细胞RNA测序。为研究TM对体内细胞分化的影响，我们对植入小鼠小脑并成瘤后的GFP阳性BT16 ATRT细胞进行单细胞RNA测序。小鼠分别接受TM处理（50 mg/kg，腹腔注射，每周3次，持续4周）或赋形剂对照处理。