Targeting MYC Transcription with Small Peptide Derived from KSHV Transactivator (SLAM-Seq)

Herpesviruses rely on host cell transcription and translation machineries for replication. Viral proteins thus function to redirect multiple cellular proteins for viral replication. In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here we show that an evolutionally-shaped viral protein sequence is a great starting material for unique drug development to modulate cellular transcription. Cellular c-Myc protein (MYC) is overexpressed in over 70% of all types of cancer cells and therefore a very attractive target to control cancer cell growth. We identified a small functional peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta), which strongly attenuates MYC expression, reduces cell proliferation, and selectively kills cancer cells in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide blocks promoter-enhancer interactions by preventing coactivator complex consisting of Nuclear receptor coactivator 2, p300, and SWI/SNF proteins from engaging the MYC promoter in leukemia cells. Target gene profiling with SLAM-seq suggests that the viral peptide attenuates MYC expression through a mechanism likely different from that of BET bromodomain inhibitors. Furthermore, fusing the 13 amino acids peptide with humanized anti-CD22 single chain armed the antibody drug with cell killing ability, and inhibited cell growth in soft agar. Our studies thus demonstrate the utility of the peptide sequence as a therapeutics module, which may be used to modulate MYC activity in a cell type-specific manner. Overall design: The goal of these studies was to utilize thiol(SH )-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) in order to determine the dynamics of gene expression in response to treatment with a novel small functional peptide therapeutic derived from the Kaposi's sarcoma-associated herpesvirus (KSHV)-transactivator (K-Rta), which strongly attenuates MYC expression. This was performed in the context of the BCBL-1 and BC-1 cell line models, which are derived from KSHV-infected human primary effusion lymphomas (PEL) and contain latent KSHV genomes. For this study, replicate cultures from each cell line were treated with either vehicle control, wild-type peptide (24 uM), or mutant peptide (24 uM) for 30 minutes, and then for an additional 1 hour in the presence of 4-Thiouridine (s4U; 300 uM) in order to label nascent transcripts. Following treatment, the cells were then harvested for isolation of total RNA (containing s4Uracil-labeled transcripts) and followed by library preparation and next-generation sequencing (NGS).

疱疹病毒（Herpesviruses）依赖宿主细胞的转录与翻译机器完成复制。因此，病毒蛋白的功能在于重定向多种宿主蛋白以服务于病毒复制。在疱疹病毒感染的复制细胞中，宿主基因的转录常被下调，因为重要的转录装置已被病毒转录因子抢占。

本研究发现，经进化塑造的病毒蛋白序列可作为调控细胞转录的独特药物开发优质起始材料。细胞c-Myc蛋白（MYC）在超过70%的各类癌细胞中过表达，因此是调控癌细胞生长的极具吸引力的靶点。我们从卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus, KSHV）的反式激活因子（K-Rta）中筛选得到一段小型功能肽，该肽可强力减弱MYC的表达、抑制细胞增殖，并在细胞培养与异种移植瘤小鼠模型中选择性杀伤癌细胞。

机制上，该肽通过阻断由核受体共激活因子2、p300与SWI/SNF蛋白组成的共激活因子复合物结合白血病细胞中的MYC启动子，从而破坏启动子-增强子相互作用。通过SLAM-seq（基于巯基（SH）连接烷基化的RNA代谢测序技术）进行的靶基因谱分析显示，该病毒肽的MYC表达调控机制与BET溴结构域抑制剂存在显著差异。此外，将该13氨基酸肽与抗CD22人源化单链抗体融合后，可赋予该抗体药物细胞杀伤能力，并抑制软琼脂中的细胞生长。

综上，本研究证实该肽序列可作为治疗模块，实现细胞类型特异性的MYC活性调控。

总体实验设计：本研究旨在利用基于巯基（SH）连接烷基化的RNA代谢测序技术（SLAM-seq），解析经卡波西肉瘤相关疱疹病毒反式激活因子K-Rta衍生的新型小型功能肽治疗后，细胞基因表达的动态变化。实验采用BCBL-1与BC-1细胞系模型，二者均源自KSHV感染的人原发性渗出性淋巴瘤（primary effusion lymphomas, PEL），且携带潜伏态KSHV基因组。

本研究中，每种细胞系的重复培养物分别用溶剂对照、野生型肽（24 μM）或突变型肽（24 μM）处理30分钟，随后在4-硫尿苷（s4U；300 μM）存在下继续培养1小时以标记新生转录本。处理完成后，收集细胞并分离总RNA（包含s4U标记的转录本），随后进行文库制备与下一代测序（next-generation sequencing, NGS）。