RNA-seq on DMSO or MAK683 treated cancer cells and xenograft tumor samples. RNA-seq on DMSO or MAK683 treated cancer cells and xenograft tumor samples

The methyltransferase Polycomb Repressive Complex 2 (PRC2), composed of EZH2, SUZ12, and EED subunits, is associated with transcriptional repression via tri-methylation of histone H3 on lysine 27 residue (H3K27me3). PRC2 is a validated drug target, as the EZH2 gain-of-function mutations identified in patient samples drive tumorigenesis. PRC2 inhibitors have been discovered and demonstrated anti-cancer efficacy in clinic. However, their pharmacological mechanisms are poorly understood. MAK683 is a potent EED inhibitor in clinical development. The overall goal of our study is to understand the molecular events leading to tumor regression after PRC2 inhibition. Our study revealed that BMP-ACVR1 signaling pathway as a critical component for the anti-lymphoma efficacy of PRC2 inhibitor. Overall design: We performed RNA-seq, H3K27me3 and H3K4me3 ChIP-seq with spike-in on MAK683-sensitive cancer cells WSU-DLCL2 treated with DMSO or PRC2 inhibitor MAK683 (3 μM for ChIP-seq, 5 μM for RNA-seq). To reveal the ACVR1-independent transcriptome change, we also constructed ACVR1 KO WSU-DLCL2 cell clone, NT and ACVR1 knock out cells that treated with DMSO or MAK683 (5 μM) were also included in the cellular RNA-seq study. Finally, we build WSU-DLCL2 and ACVR1 KO cell-derived xenograft models in mice, and obtained the expression profiles of WSU-DLCL2-derived xenograft tissues (NT or ACVR1 knock out) after treatment with vehicle or 100 mg/kg MAK683 for 28 days.

多梳蛋白抑制复合物2（Polycomb Repressive Complex 2, PRC2）是一类甲基转移酶，由EZH2、SUZ12及EED亚基组成，通过对组蛋白H3的赖氨酸27位点进行三甲基化修饰（H3K27me3）介导转录抑制。PRC2是经验证的药物靶点：患者样本中检出的EZH2功能获得性突变可驱动肿瘤发生。目前已发现PRC2抑制剂，并在临床中证实其具备抗癌疗效，但其药理机制仍不甚明确。MAK683是一种处于临床开发阶段的强效EED抑制剂。本研究的整体目标是阐明PRC2抑制后介导肿瘤消退的分子事件。本研究揭示，BMP-ACVR1信号通路是PRC2抑制剂发挥抗淋巴瘤疗效的关键组分。

实验总体设计：我们对MAK683敏感性癌细胞WSU-DLCL2分别经二甲基亚砜（DMSO）或PRC2抑制剂MAK683处理后，开展了带spike-in内参的RNA测序（RNA-seq）、H3K27me3及H3K4me3染色质免疫沉淀测序（ChIP-seq）实验（其中ChIP-seq的MAK683处理浓度为3 μM，RNA-seq为5 μM）。为揭示ACVR1非依赖的转录组变化，我们还构建了ACVR1基因敲除的WSU-DLCL2细胞克隆，并将未处理（NT）及ACVR1敲除细胞分别经DMSO或5 μM MAK683处理后，纳入本次RNA-seq研究。最后，我们构建了WSU-DLCL2及ACVR1敲除细胞的小鼠细胞源性异种移植模型，获取了经赋形剂或100 mg/kg MAK683连续处理28天后的WSU-DLCL2来源异种移植组织（未处理或ACVR1敲除）的表达谱数据。