Inhibition of CBP synergizes with the RNA-dependent mechanisms of azacitidine by limiting protein synthesis. Inhibition of CBP synergizes with the RNA-dependent mechanisms of azacitidine by limiting protein synthesis

The nucleotide analogue azacitidine interferes with RNA and DNA metabolism and is currently the best treatment option for a subset of patients with high-risk myelodysplastic syndromes. However, only half of treated patients respond and almost all patients that initially respond eventually relapse. We performed an optimized loss-of-function shRNA screen in combination with azacitidine treatment in an MDS-derived acute myeloid leukemia cell line to identify chromatin regulators affecting drug response. We identified CBP, as a major regulator of azacitidine sensitivity. Compounds inhibiting the enzymatic activity of CBP synergistically reduced viability of MDS-derived AML cell lines when combined with AZA. Surprisingly, this effect was specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is limited to DNA incorporation. The identification of immediate target genes suggested that the effect of CBP inhibition is mediated by downregulation of genes encoding the translational machinery, which could be confirmed in proteomic analysis of nascent proteins. Furthermore, proteins most affected by CBP inhibition include key drivers of cell cycle progression. Overall design: Nascent mRNA transcripts in the sAML cell line MOLM-13 in response to C646 treatment (2h, 10µM) identified by SLAM-seq MOLM-13 cells were either treated with 10 µM C646 or DMSO (3 replicates each) for 1 hour, labelled with 100 µM 4sU for another hour and SLAM-seq performed using the SLAMseq Anabolic Kinetics Kit (Lexogen Gmbh, Austria) followed by library preparation using the QuantSeq 3’mRNA-Seq for Illumina (FWD) and PCR Add-on Kit for Illumina (Lexogen Gmbh, Austria) according to the manufacturer’s instructions. The subsequent data analysis was performed using the software SLAM-Dunk and the R package DEseq2.

核苷酸类似物阿扎胞苷（azacitidine）可干扰RNA与DNA代谢，目前是高危骨髓增生异常综合征（myelodysplastic syndromes, MDS）亚群患者的首选治疗方案。然而，仅半数接受治疗的患者可产生应答，且几乎所有初始应答的患者最终都会复发。我们在MDS来源的急性髓系白血病（acute myeloid leukemia, AML）细胞系中，结合阿扎胞苷处理优化了功能缺失型短发夹RNA（short hairpin RNA, shRNA）筛选，以鉴定影响药物应答的染色质调控因子。我们鉴定出CBP是调控阿扎胞苷敏感性的关键因子。抑制CBP酶活性的化合物与阿扎胞苷联用时，可协同降低MDS来源AML细胞系的细胞存活率。令人意外的是，该效应仅特异性针对阿扎胞苷的RNA依赖性功能，而对仅能掺入DNA的同类化合物地西他滨（decitabine）未观察到此效果。对直接靶基因的鉴定表明，CBP抑制的效应是通过下调编码翻译机器的基因所介导，这一点可在新生蛋白质组学分析中得到验证。此外，受CBP抑制影响最显著的蛋白质包括细胞周期进程的关键调控因子。总体实验设计：通过SLAM-seq技术鉴定经C646处理（2小时，10μM）的继发性急性髓系白血病（secondary AML, sAML）细胞系MOLM-13中的新生mRNA转录本。将MOLM-13细胞分为两组，分别用10 μM C646或二甲基亚砜（DMSO）处理（每组设3个生物学重复），先处理1小时，再用100 μM 4sU标记1小时；随后使用SLAMseq Anabolic Kinetics Kit（奥地利Lexogen GmbH公司）进行SLAM-seq实验，再依照制造商说明书，使用QuantSeq 3’mRNA-Seq for Illumina（FWD）及Illumina PCR Add-on Kit（奥地利Lexogen GmbH公司）完成文库制备。后续数据分析采用SLAM-Dunk软件及R包DESeq2完成。