RNA decay defines the therapeutic response to transcriptional perturbation in leukemia [SLAMseq THP1 RNAPII targeting]. RNA decay defines the therapeutic response to transcriptional perturbation in leukemia [SLAMseq THP1 RNAPII targeting]

Therapeutic targeting of dysregulated transcriptional programs has arisen as a promising strategy for the treatment of leukemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression networks driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. Using technologies such as thiol (SH)-linked alkylation for the metabolic sequencing of RNA sequencing (SLAM-seq) to profile messenger RNA (mRNA) decay and production rates, we demonstrate that gene intrinsic properties largely govern the selectivity associated with transcriptional inhibition, where total mRNA response signatures are dominated with genes that have short transcript half-lives, including those regulated by SEs and oncogenic TFs. Further highlighting that gene sensitivities only occur in the context of short transcript half-lives, stabilisation of the c-MYC transcript through changes in the 3’ UTR rendered it insensitive to transcriptional targeting. However, this was not sufficient to rescue c-MYC target gene transcription and anti-leukemia effects following transcriptional inhibition. Importantly, long-lived mRNAs encoding essential genes that evade transcriptional targeting can be rendered sensitive via modulation of mRNA decay kinetics through inhibition of the RNA Binding Protein (RBP), ELAV Like RNA binding protein 1 (ELAVL1)/ Human Antigen R (HuR). Taken together, these data demonstrate that mRNA decay shapes the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukemia. Overall design: THP-1 cells (1 x 10^6 per treatment) were first pre-treated with small molecule inhibitors for a total time of 2 hours to pre-establish protein-target inhibition. Newly synthesized RNA in THP-1 cells was then labelled using 1mM 4-sU in the final 10 minutes of treatment at 37°C and 5% carbon dioxide.

靶向失调转录程序的治疗策略，已成为白血病治疗领域极具前景的手段。针对含溴结构域蛋白（Bromodomain-Containing Proteins, BRD，如BRD2、BRD4）、P300/环腺苷酸应答元件结合蛋白（P300/cAMP-response element binding protein, CBP）以及细胞周期蛋白依赖性激酶（Cyclin Dependent Kinases, CDKs）的小分子抑制剂，其治疗应答通常被认为是通过选择性破坏由增强子、超级增强子（super-enhancers, SEs）以及谱系特异性转录因子（lineage-specific transcription factors, TFs，包括c-MYC癌基因）驱动的致癌基因表达网络而实现的。本研究利用巯基（SH）连接烷基化代谢RNA测序技术（SLAM-seq）等手段，对信使RNA（messenger RNA, mRNA）的降解与生成速率进行表征，结果表明基因固有特性在很大程度上决定了转录抑制相关的选择性：总mRNA应答特征主要由转录本半衰期较短的基因主导，其中包括受SEs与致癌性TFs调控的基因。进一步研究显示，基因敏感性仅存在于转录本半衰期较短的情境中：通过改变3'非翻译区（3’ UTR）稳定c-MYC转录本后，该基因对转录靶向治疗不再敏感，但这不足以挽救转录抑制后c-MYC靶基因的转录过程与抗白血病效应。值得注意的是，编码逃避转录靶向的必需基因的长寿命mRNA，可通过抑制RNA结合蛋白（RNA Binding Protein, RBP）ELAV样RNA结合蛋白1（ELAV Like RNA binding protein 1, ELAVL1）/人抗原R（Human Antigen R, HuR），调节mRNA降解动力学，从而使其获得治疗敏感性。综上，本研究数据表明，mRNA降解塑造了细胞对转录扰动的治疗应答，且可通过白血病治疗中的转录靶向药物调节该过程，以实现新型治疗效果。实验整体设计：将THP-1细胞（每处理组接种1×10^6个细胞）首先用小分子抑制剂预处理总计2小时，以预先建立蛋白靶点抑制效果。随后在37℃、5%二氧化碳培养条件下，于处理的最后10分钟使用1mM 4-sU对THP-1细胞中新合成的RNA进行标记。