Histone acetyltransferase p300/CBP as a therapeutic target to ameliorate gvhd-induced inflammatory memory

Abstract

Abstract Background: Recently, emerging evidence indicated that inflammation-induced epigenetic remodeling in tissue stem cells enhances either degenerative or regenerative responses of epithelial cells to subsequent inflammatory stimuli, a concept recently termed “inflammatory memory” (Zhao D: Blood 2025). We have previously demonstrated that GVHD induces epigenetic remodeling in intestinal stem cells (ISCs) in an IFN-γ-dependent manner, leading to enhanced antigen presentation, particularly via MHC class II (MHC-II), and increased susceptibility to apoptosis in intestinal epithelial cells (IECs) upon subsequent inflammatory stimulation (Miyajima T: 66th ASH annual meeting). In the current study, we investigated the mechanisms by which GVHD induces inflammatory memory in intestinal stem cells (ISCs) and evaluated strategies to suppress this process, using a mouse allogeneic hematopoietic cell transplantation (HCT) model, high-dose IFN-γ administration in naive mice, and an intestinal organoid culture system. Methods: Lethally irradiated BDF1 (H-2b/d) recipient mice were transplanted from allogeneic B6 (H-2b/b) or syngeneic BDF1 donors. Intestinal organoids were generated from the small intestines on day 21 post-transplant, cultured for 6 days, and subjected to RNA sequencing (RNA-seq), ATAC sequencing (ATAC-seq), and chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR). We performed QIAGEN Ingenuity Pathway Analysis (IPA) of differentially expressed genes obtained from our RNA-seq data. Additionally, small intestines isolated on day 21 post-transplant were subjected to immunofluorescence staining for histopathological assessment. Results: IPA of RNA-seq data from intestinal organoids generated 21 days after mouse allogeneic or syngeneic HCT, focusing on epigenetic modifiers, identified histone acetyltransferase E1A-binding protein (p300) and its paralogue, CREB-binding protein (CBP), as candidate upstream regulators of GVHD-induced transcriptional modifications in IECs. Moreover, ATAC-seq of intestinal organoids revealed enrichment of ATF-3-binding motifs, a transcription factor that interacts with p300 and CBP, in chromatin regions that became significantly more accessible in GVHD compared to syngeneic controls. Consistent with the role of p300/CBP in promoting transcription through histone 3 lysine 27 acetylation (H3K27ac), immunofluorescence staining of the small intestine showed an increased number of H3K27ac+ IECs in GVHD mice. ChIP-qPCR analysis demonstrated that H3K27ac was enriched at the promoters of MHC-II genes (e.g., H2-Aa and H2-Eb1) in IECs from GVHD mice. High-dose IFN-γ also promoted H3K27ac enrichment at the promoters of IFN-γ-responsive genes in IECs, leading to enhanced antigen presentation and increased susceptibility to apoptosis upon subsequent exposure to low-dose IFN-γ. To assess whether p300/CBP is essential for inflammation-induced epigenetic modification, we knocked down (KD) Ep300 (encoding p300) and Cbp in intestinal organoids generated from naïve mice using lentiviral transduction of short hairpin RNA (shRNA). Ep300- or Cbp-KD organoids were treated with a high concentration of IFN-γ to induce epigenetic remodeling and subsequently rechallenged with a low level of IFN-γ. Compared with organoids transduced with nontargeting control shRNA, Ep300- or Cbp-KD organoids exhibited significantly attenuated enhancements of MHC-II upregulation and apoptosis induction following low-dose IFN-γ rechallenge, with Cbp-KD organoids exhibiting particularly strong suppression of inflammatory memory. This was further supported by experiments demonstrating that treatment of IFN-γ-stimulated intestinal organoids with inobrodib, a p300/CBP inhibitor, abrogated IEC hyperresponsiveness to IFN-γ rechallenge, whereas p300-specific inhibitors C646 and curcumin only partially suppressed this response. Conclusions: We found that GVHD induces epigenetic remodeling in ISCs through a p300/CBP-dependent manner, in which IFN-γ activates p300/CBP, leading to H3K27ac enrichment at target genes involved in IFN-γ responses. Given the emerging efficacy of p300/CBP inhibitors in various hematological malignancies (Nicosia L: Cancer Cell 2023, Welsh SJ: Blood Cancer Discov 2024), these agents represent promising therapeutic candidates for GVHD by suppressing inflammatory memory in ISCs, while potentially preserving GVL effects and additionally exerting direct anti-tumor activity.