Isolation of biologically active small peptide from KSHV LANA protein sequence, which induces CHD4 degradation to promote cell differentiation and inhibits leukemic cell growth (RNA-seq)

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, in which most viral genes are silenced except a few latent proteins such as latency-associated nuclear antigen (LANA). In latent viral chromatin, viral genes are poised to be transcribed, and KSHV LANA plays a major role in maintaining such transcription status. In the poised chromatins, LANA recruits cellular CHD4 (Chromodomain Helicase DNA binding protein 4) and suppresses inducible viral gene promoters. The CHD4 is known to regulate cell differentiation by restricting enhancer-promoter interactions and its mutation or overexpression deregulates the host cell transcription program and therefore associates with tumorigenesis. Here, we identified a putative CHD4 inhibitor from the LANA amino acid sequence from the LANA-CHD4 interaction surface. The small peptide interacts with CHD4 at its PHD domain with 14 nM KD (dissociation constant). The introduction of the peptide into the primary effusion lymphomas induces caspase-mediated CHD4 cleavage and subsequently triggered cell apoptosis and autophagy. A series of MTT assays demonstrated that the peptide preferentially killed lymphoma and leukemia cell lines at low micromolar concentrations, while peripheral mononuclear cells or adhesion cell lines were found to be more resistant to the peptide treatment. A monocyte cell differentiation model demonstrated that pre-treatment with the peptide at a low dose substantially enhanced transitioning into M2 macrophage (by reducing CHD4 occupancies from gene promoters), and globally altered the repertories of phorbol myristate acetate target genes in U937 cells. Finally, the PEL xenograft mouse model inhibited tumor growth without measurable side effects, and PEL cells isolated from xenograft tumors showed reduced CHD4 and LANA expression with terminally differentiated phenotype. We propose that the peptide isolated from the KSHV LANA sequence is biologically active and may function as a CHD4 inhibitor. Overall design: The goal of this study was to define the effects of VGN73 on the regulation of phorbol-12-myristate-13-acetate (PMA)-mediated differentiation of U937 monocyte-macrophage cells. U937 cells were pretreated with VGN73 (4 or 8 µM) for 24 hours, and then stimulated with PMA (10 ng/ml final concentration) for an additional 24 hours. Controls groups included PMA stimulation (10 ng/ml, 24 hrs) without pretreatment and VGN treatment alone (4 µM, 48 hrs). All treatments were performed in triplicate. Cells were harvested at the completion of the treatments and followed by total RNA isolation and RNA-sequencing.

卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus, KSHV）可建立潜伏感染，此时多数病毒基因处于沉默状态，仅表达少量潜伏蛋白，例如潜伏相关核抗原（latency-associated nuclear antigen, LANA）。在潜伏状态的病毒染色质中，病毒基因处于转录预备状态，而KSHV LANA在维持该转录状态中发挥核心作用。在这类预备状态的染色质中，LANA可招募宿主细胞的染色质域解旋酶DNA结合蛋白4（Chromodomain Helicase DNA binding protein 4, CHD4），并抑制可诱导型病毒基因启动子的活性。已知CHD4通过限制增强子-启动子相互作用调控细胞分化，其突变或过表达会扰乱宿主细胞的转录程序，因此与肿瘤发生密切相关。本研究从LANA与CHD4的互作界面的LANA氨基酸序列中，筛选得到一种潜在的CHD4抑制剂。该小肽可与CHD4的PHD结构域结合，解离常数（dissociation constant, KD）达14 nM。将该小肽导入原发性渗出性淋巴瘤（primary effusion lymphomas, PEL）细胞后，可诱导半胱天冬酶介导的CHD4裂解，进而触发细胞凋亡与自噬。一系列MTT实验结果显示，该小肽在低微摩尔浓度下即可优先杀伤淋巴瘤与白血病细胞系，而外周血单个核细胞及黏附细胞系对该小肽处理的耐受性更强。利用单核细胞分化模型开展的实验表明，低剂量预暴露于该小肽，可通过减少CHD4在基因启动子区域的占据量，显著增强细胞向M2型巨噬细胞的转化，并全面改变U937细胞中佛波醇肉豆蔻酸乙酸酯（phorbol myristate acetate, PMA）靶基因的表达谱。最后，PEL异种移植小鼠模型实验显示，该小肽可抑制肿瘤生长且未检测到明显副作用；从异种移植瘤中分离得到的PEL细胞，其CHD4与LANA的表达水平降低，并呈现终末分化表型。本研究提出，从KSHV LANA序列中分离得到的该小肽具有生物学活性，可作为CHD4抑制剂发挥功能。总体实验设计：本研究的核心目标是阐明VGN73对佛波醇-12-肉豆蔻酸-13-乙酸酯（phorbol-12-myristate-13-acetate, PMA）介导的U937单核细胞-巨噬细胞分化的调控作用。实验将U937细胞分为三组：① 用4或8 μM的VGN73预处理24小时，随后以终浓度10 ng/ml的PMA继续刺激24小时；② 仅用10 ng/ml PMA刺激24小时（无预处理对照组）；③ 仅用4 μM VGN73处理48小时（单独处理对照组）。所有处理均设置3次生物学重复。处理结束后收集细胞，进行总RNA提取与RNA测序。