Inhibition of p300/CREBBP catalytic activity drives context-dependent transcriptional activation in AML. [Perturb-Seq]

The lysine acetyltransferase (KAT) activity of p300/CREBBP has traditionally been linked to transcriptional activation. This has been attributed largely to acetylation of histone residues such as H3K27ac, a defining hallmark of active regulatory elements. Here we show that, in acute myeloid leukemia (AML), inhibition of p300/CREBBP catalysis can paradoxically increase transcription. We combined time-resolved dynamics of nascent and total transcription with chromatin binding dynamics of p300/CREBBP and their associated TFs/co-regulators (inferred from chromatin pull-down proteomics, acetyl-proteomics and motif enrichment) to uncover mechanisms of transcriptional rewiring after p300/CREBBP catalytic inhibition. In parallel, we dissected the functional contribution of individual p300/CREBBP acetyl-interactome members to KAT inhibition using genome-wide CRISPR-Cas9 dropout and focused Perturb-seq screens. Together, these approaches revealed that KAT inhibition paradoxically retains p300/CREBBP and promotes cooperative TF assembly and increased H3K27 acetylation at a subset of regulatory elements. The effect was most pronounced at IRF motif-enriched loci, including interferon-stimulated genes (ISGs), where KAT inhibition triggered p300/CREBBP accumulation and enhanced combinatorial TF binding, enabling recruitment of the ISG activator STAT1. Consequently, ISG loci were converted into transcriptionally active states that induced cell-cycle arrest, differentiation and apoptosis. Therapeutically, combining KAT inhibition with interferon-alpha augmented ISG expression, synergistically drove AML cell death in vitro and significantly extended survival in both AML xenografts and murine models. These findings refine our understanding of p300/CREBBP KAT activity, demonstrating that cooperative TF assembly can reconfigure p300/CREBBP-containing complexes under catalytic inhibition to induce transcription, with translational implications for reprogramming interferon-driven programs through catalytic inhibition in AML and beyond. Overall design: Cells were transduced with a targeted sgRNA library (186 sgRNAs). After selection cells were treated with either A-485 or DMSO. Cell Fixation performed directly 24 hours after treatment. Subsequent barcoding performed with Kits from ParseBiosciences (Mini/Mega v3) plus CRISPR Detect.

p300/CREBBP的赖氨酸乙酰转移酶（KAT）活性传统上被认为与转录激活相关，这一功能主要归因于其对组蛋白残基的乙酰化修饰，例如作为活性调控元件标志性特征的H3K27ac。本研究发现，在急性髓系白血病（AML）中，抑制p300/CREBBP的催化活性反而会增强转录。我们结合了新生转录本与总转录本的时间分辨动力学数据、p300/CREBBP及其关联转录因子/共调控因子的染色质结合动力学数据（从染色质拉下蛋白质组学、乙酰化蛋白质组学与基序富集分析中推导得到），以揭示p300/CREBBP催化抑制后发生的转录重编程机制。同时，我们通过全基因组CRISPR-Cas9敲除筛选与靶向Perturb-seq筛选，解析了单个p300/CREBBP乙酰化相互作用组成员在KAT抑制过程中的功能贡献。

上述研究手段共同揭示：KAT抑制反而会保留p300/CREBBP的结合，并促进协同转录因子组装，在部分调控元件处增强H3K27乙酰化修饰。这一效应在IRF基序富集位点最为显著，包括干扰素刺激基因（ISGs）位点：KAT抑制在此处触发p300/CREBBP的聚集，并增强组合式转录因子结合，进而招募干扰素刺激基因的激活因子STAT1。最终，干扰素刺激基因位点被转化为转录激活状态，诱导细胞周期阻滞、细胞分化与细胞凋亡。

在治疗层面，将KAT抑制与α干扰素联合使用可增强干扰素刺激基因的表达，在体外实验中协同诱导AML细胞死亡，并显著延长AML异种移植模型与小鼠模型的存活期。本研究更新了我们对p300/CREBBP KAT活性的认知，证明在催化抑制条件下，协同转录因子组装可重构包含p300/CREBBP的复合物以诱导转录，这为通过催化抑制在AML及其他疾病中重编程干扰素驱动的基因程序提供了转化应用前景。

实验整体设计：将靶向单向导RNA文库（共186条sgRNA）转导至细胞中，经过筛选后，用A-485或二甲基亚砜（DMSO）处理细胞。处理24小时后直接进行细胞固定。后续条形码标记使用ParseBiosciences公司的Mini/Mega v3试剂盒搭配CRISPR Detect完成。