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

The lysine acetyltransferase (KAT) activity of EP300 lysine acetyltransferase (p300)/CREB-binding protein (CREBBP) has traditionally been linked to transcriptional activation. This has been attributed largely to acetylation of histone residues such as histone H3 lysine 27 acetylation (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 transcription factors (TFs)/coregulators (inferred from chromatin pulldown 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 interferon regulatory factor (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.