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. Author manuscript; available in PMC: 2023 Feb 23.
Published in final edited form as: Leukemia. 2022 Dec 1;37(2):396–407. doi: 10.1038/s41375-022-01774-z

Analysis and Therapeutic Targeting of the EP300 and CREBBP Acetyltransferases in Anaplastic Large Cell Lymphoma and Hodgkin Lymphoma

Wei Wei 1, Zhihui Song 1, Masahiro Chiba 2, Wenjun Wu 1, Subin Jeong 1, Jing-Ping Zhang 1, Marshall E Kadin 3, Masao Nakagawa 2, Yibin Yang 1,*
PMCID: PMC9949602  NIHMSID: NIHMS1870355  PMID: 36456744

Abstract

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (HL) share a similar cytological and high surface expression of CD30, and novel therapeutic strategies are needed. The EP300 and CREBBP acetyltransferases play essential roles in the pathogenesis of non-Hodgkin B cell lymphoma, but their functions in ALCL and HL are unknown. In the current study, we investigated the physiological roles of EP300 and CREBBP in both ALCL and HL, and exploited the therapeutic potential of EP300/CREBBP small molecule inhibitors that target either the HAT or bromodomain activities. Our studies demonstrated distinct roles for EP300 and CREBBP in supporting the viability of ALCL and HL, which was bolstered by the transcriptome analyses. Specifically, EP300 but not CREBBP directly modulated the expression of oncogenic MYC/IRF4 network, surface receptor CD30, immunoregulatory cytokines IL10 and LTA, and immune checkpoint protein PD-L1. Importantly, EP300/CREBBP HAT inhibitor A-485 and bromodomain inhibitor CPI-637 exhibited strong activities against ALCL and HL in vitro and in xenograft mouse models, and inhibited PD-L1 mediated tumor immune escape. Thus, our studies revealed critical insights into the physiological roles of EP300/CREBBP in these lymphomas, and provided opportunities for developing novel strategies for both targeted and immune therapies.

Keywords: EP300, CREBBP, HAT, Bromodomain, CD30, PD-L1, IRF4, MYC, ALCL, HL

Introduction

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (HL), which share a similar cytological tumor cell phenotype and high surface expression of the TNF receptor family member CD30, are common in children and young adults. ALCL comprises 10-15% of pediatric lymphomas1, 2; and Hodgkin lymphoma (HL) is the most common (6,000 to 7,000 new cases per year) cancer in young adulthood3. Although there has been significant progress in treating ALCL and HL over the last few decades, the survival rate for patients diagnosed at an advanced stage or with relapsed/refractory disease remains low. Thus, developing novel, preferably small molecule-based targeted therapies for these lymphoid malignancies are needed.

Epigenetic mechanisms are essential for regulating gene expression without altering the DNA sequence. Deregulation of the epigenetic processes is commonly observed in tumors, leading to altered gene function and contributing to tumor pathogenesis4. An essential epigenetic mediator of transcription is histone acetylation. Three classes of proteins mainly regulate this epigenetic event: histone acetyltransferases (HATs), “writers,” which install acetyl groups; histone deacetylases (HDACs), “erasers,” which catalyze the removal of acetyl functional groups; and bromodomain (BRD) proteins, “readers,” that recognize and bind to acetylated lysine residues on histone tails5. The cyclic AMP response element binding protein (CREB)-binding protein (CREBBP or CBP) and E1A interacting protein of 300 kDa (EP300) are closely related HATs that regulate a number of critical cellular events6, 7, which contain bromodomains (BRDs), in addition to their catalytic HAT domain. Both CREBBP and EP300 are often mutated in human cancers, including Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma (FL)8, 9. Interestingly, heterozygous somatic mutations in these genes are predominantly mono-allelic, suggesting that some EP300 and CREBBP activity is required for these tumors. Functional screening suggested that EP300 is a specific synthetic lethal gene in CREBBP-deficient lung cancer cells10. A recent study further showed that the synthetic lethal interaction between CREBBP and EP300 was obtained in both normal germinal center (GC) B cells and CREBBP-mutant DLBCL cells11, providing the basis for the development of small molecules targeting EP300 and CREBBP in these lymphoma cells.

Similar to what is seen in DLBCL and FL, the somatic mutations of CREBBP and/or EP300 have been recently reported in ALCL and HL12–16, though limited by the small number of cases. While these findings have provided critical insights into the potential critical roles of CREBBP/EP300 in ALCL and HL pathogenesis, these advances are mainly based on cancer genome sequencing and lack detailed mechanism studies. In our previous effort to identify unique factors involved in ALCL pathogenesis, we performed CRISPR library screenings for PD-L1 regulators in ALCL17. EP300, unexpectedly, was identified as a strong positive hit in our library screenings17, indicating its potential critical role in ALCL pathogenesis. In this study, we decided to investigate the physiological roles of these two acetyltransferases in both ALCL and HL, and exploit the therapeutic potentials of EP300/CREBBP small molecules inhibitors in these lymphoid malignancies.

Materials and Methods

(See Supplemental Experimental Procedures)

Results

EP300 plays an essential role in supporting the viability and surface CD30 expression of ALCL and HL cells

To understand the role of EP300 and CREBBP (CBP) acetyltransferases in supporting the viability of ALCL and HL, we designed sgRNAs to deplete EP300 or CREBBP (two for each gene) (Supplementary Fig. 1A). In ALCL, two variants have been described, including anaplastic lymphoma kinase (ALK)-positive (ALK+) and ALK-negative (ALK−)18, 19. Therefore, we used a large panel of lymphoma cell lines covering ALK+ ALCL, ALK− ALCL and HL; and we measured EP300 or CREBBP sgRNAs toxicities in these cells (Fig. 1A). Depletion of EP300 expression was generally toxic to all ALCL and HL cell lines we examined (Fig. 1A). Interestingly, although CREBBP and EP300 share 60% amino acid identity and parallel domain organization, depletion of CREBBP expression had little effect on ALCL or HL viabilities (Fig. 1A), indicating the distinct function of these two proteins in ALCL and HL pathogenesis.

Figure 1: EP300 supports the viability and surface CD30 expression of ALCL and HL cells.

Figure 1:

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(A) ALCL and HL cell lines were transduced with EP300, CREBBP, or Ctrl sgRNAs along with GFP. The fraction of viable, GFP+/sgRNA+ cells relative to the live cell fraction is plotted at the indicated times following sgRNA induction, normalized to day 0 values. Error bars denote the SD of triplicates. (B) Indicated ALCL and HL lines were treated with EP300/CREBBP HAT inhibitor A-485 (upper) or bromodomain inhibitor CPI-637 (lower) at the indicated concentrations for four days. Viability was measured by an MTS assay and normalized to DMSO-treated cells. Error bars denote SEM of triplicates. (C) Indicated ALCL and HL lines were treated with A-485 (10μM) or CPI-637 (10μM) for 24 hours. Surface CD30 expression was measured by flow cytometry. The relative CD30 MFI was normalized to the DMSO-treated cells (right). Error bars denote SEM of triplicates.

In DLBCL, EP300 and CREBBP mutation status affect sgRNA toxicities11. We therefore sequenced all the available ALCL and HL cell line models (n=20) (Supplementary Fig. 1B–D). In our analysis, mutations were more frequently identified in CREBBP than in EP300 (5/20 vs. 3/20, Supplementary Fig. 1B), which is in agreement with the studies in DLBCL8. Notably, most of these mutations are missense mutations, and many of them are located in the HAT domain. Two heterozygous CREBBP frame-shift or nonsense mutations were also identified. However, no correlations of these mutations with EP300/CREBBP sgRNA toxicities were observed. Notably, CRISPR ablation of CREBBP in ALCL and HL lines made these cells significantly more vulnerable to EP300 deletion (Supplementary Fig. 2A), indicating the CREBBP protein expression level might determine the synthetic lethality of EP300 depletion in these cells.

The EP300 sgRNA results (Fig. 1A) led us to test the cytotoxicity of EP300 inhibitors in ALCL and HL cells. Although there are no specific EP300 inhibitors available, we took advantage of two newly developed small molecule EP300/CREBBP inhibitors: A-485, a pre-clinical HAT catalytic inhibitor20; and CPI-637, a selective bromodomain (BRD) inhibitor21. To investigate the therapeutic potential of EP300/CREBBP inhibition in ALCL and HL, we determined the viability of lymphoma lines after treatment with A-485 or CPI-637 (Fig. 1B). In all ALCL and HL lines we tested, both A-485 and CPI-637 were toxic in a dose-dependent manner (Fig. 1B). Compared with ALK− counterpart, the ALK+ lines appeared to be the more resistant to these inhibitors, which would occur due to the NPM-ALK oncogenic protein expression in these cells. The NPM-ALK chimeric protein in ALK+ ALCL is constitutively activated22, 23, and mediates the activation of multiple oncogenic pathways, including the MEK/MAPK signaling that is generally involved in drug resistance24, 25. We next examined the effects of these EP300/CREBBP inhibitors in ALCL and HL pathogenesis, by measuring the surface expression of the CD30 tumor maker, which is characteristically expressed in ALCL and Reed-Sternberg cells26, 27. Interestingly, treatment with A-485 or CPI-637 significantly impaired CD30 surface expression in all ALCL and HL lines we tested (Fig. 1C). In line with the sgRNA toxicity results (Fig. 1A), EP300 deletions strongly attenuated the expression of CD30 in ALCL and HL lines, while CREBBP depletion has no effect (Supplementary Fig. 2B).

EP300/CREBBP HAT and bromodomain activities control partially overlapped gene expression programs in ALCL

Although the EP300/CREBBP HAT catalytic inhibitor A-485 and the bromodomain inhibitor CPI-637 efficiently diminished ALCL cell proliferation (Fig. 1B), their detailed mechanisms of action are still unclear. We therefore profiled gene expression changes upon A-485 and CPI-637 treatment in two ALK+ ALCL lines (DEL and Karpas299) and one ALK− ALCL line (MAC1), using RNA-seq analysis, which allowed us to define a set of genes that were consistently regulated by A-485 and CPI-637 treatment in all cell lines (Fig. 2A). Although each ALCL line we tested responded slightly differently to these inhibitors, we focused on the common genes that changed in at least two of the three ALCL lines. In our analysis, A-485 treatment decreased 455 genes expression, containing immunoregulatory cytokine IL-10, surface receptors (TNFRSF8 (CD30), CD274 (PD-L1), IL1R1, IL1R2, IL21R, IL7R, CCR7, and IL6R), oncogenic genes (MYC, IRF4, and BATF3), as well as genes coding immunoglobulin heavy chains (Fig. 2A). In parallel, CPI-637 treatment decreased a similar set of genes (n=509), including IL10, TNFRSF8, CCR7, IL1R1, IL1R2, IL21R, MYC, and BATF (Fig. 2A). Interestingly, a set of human leukocyte antigen (HLA) genes involved in antigen presentation and processing were preferentially reduced upon CPI-637 treatment but not with A-485 treatment, indicating that the HAT and bromodomain domains of EP300/CREBBP might also have non-overlapped actives. In our analyses, 170 genes showed significantly reduced expression, and 261 genes with increased expression were shared between these two treatments (Supplementary Fig. 3A).

Figure 2: The transcriptional programs regulated by EP300/CREBBP inhibitors in ALCL.

Figure 2:

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(A) Heatmap of mRNA levels changed upon the treatment of A-485 or CPI-637 at the indicated concentration (10μM) for 24 hours relative to DMSO-treated cells, obtained from RNA-seq analysis in DEL, Karpas299, and MAC1 ALCL lines. Genes decreased or increased by at least 1.87-fold (at least in two of three lines) in A-485 or CPI-637 treatment conditions are shown. Green represents decreased expression and red represents increased expression. Only representative transcripts are highlighted. Top significantly enriched (p < 0.05) biological programs/signaling pathways identified among the list of genes with reduced or increased expression upon the treatment were shown (right), as determined by GSEA. (B) ALCL lines were treated with A-485 (left) or CPI-637 (right) at 10μM. Indicated gene expressions were measured by real-time PCR after 24 hours of treatment. Error bars denote SEM of triplicates. (C) Indicated ALCL lines were treated with A-485 (upper) or CPI-637 (lower) at 10μM for 24 hours. Lysates were analyzed by immunoblotting for the indicated proteins.

To better understand the pathways impacted by EP300/CREBBP HAT and bromodomain inhibitions, we carried out Gene Set Enrichment Analysis (GSEA)28, and the significantly enriched signatures (p<0.05) were shown in Fig. 2A, right. Notably, gene signatures negatively correlated with A-485 or CPI-637 treatments were dominated by MYC-dependent transcriptional pathways (Fig. 2A and Supplementary Fig. 3B), indicating MYC is the major target of EP300/CREBBP in ALCL. In addition to MYC, we noted that a subset of genes downregulated by A-485 or CPI-637 overlapped with IRF4 target genes in ALCL29. Indeed, IRF4/BATF axis has been shown to play essential roles in ALCL17, 29–32, arguing that the IRF4/BATF transcriptional axis may be selectively targeted by EP300/CREBBP in ALCL.

Extending our observations from the RNA-seq analyses, we examined the effects of A-485 and CPI-637 treatment for critical gene expressions in a comprehensive set of ALCL cell lines, including both ALK+ (n=5) and ALK− (n=7) subtypes (Fig. 2B and Supplementary Fig. 4A). The HAT catalytic inhibitor A-485 treatment strongly impaired MYC, IL10, CD30, IRF4, and BATF3 mRNA transcription in all ALCL lines we tested. To a less extent, the bromodomain inhibitor CPI-637 treatment also impaired the expression of these genes in most cell lines. Furthermore, the ability of A-485 and CPI-637 to inhibit MYC and IRF4 protein expression was confirmed by immunoblot (Fig. 2C). A recent study in ALCL demonstrated a key transcription factor complex involving IRF4, BATF3, and JUNB, which binds to AP1-IRF composite (AICE) DNA motifs 32. In line with the RNA-seq analysis, A-485 treatment strongly impaired BATF3 and JUNB protein expression in all ALCL lines we tested, while CPI-637 had less effect in most cell lines (Supplementary Fig. 4B).

EP300 and CREBBP regulate distinct sets of gene expression programs in ALCL

Although EP300 and CREBBP are closely related, they appear to have distinct functions in ALCL (Fig. 1A). In order to elucidate the mechanistic basis for the differential effects of EP300 and CREBBP loss on ALCL pathogenies, we performed transcriptomic analyses upon sgRNAs depletion of EP300 or CREBBP in ALCL cell line DEL (Fig. 3A). EP300 depletion repressed a set of genes expression which were similar to those upon HAT or bromodomain inhibitors treatments, including IL-10, oncogenic genes (MYC, IRF4, and MYB), surface receptors (IL1R2, IL21R CCR7, and IL6R), as well as HLA genes (Fig. 3A). Surprisingly, CREBBP depletion likely has the opposite effects on gene expression programs in ALCL (Fig. 3A). In fact, only 1.9% (n = 18 out of 940) of the genes showing significantly reduced expression were shared between EP300 and CREBBP depletion, and 1.5% (n= 15 out of 985) genes with increased expression were shared between these two conditions (Supplementary Fig. 5A). Further GSEA pathway analysis revealed the distinct roles of EP300 and CREBBP in supporting ALCL pathogenies (Fig. 3A, right). In particular, MYC-dependent transcriptional pathway signatures were significantly enriched in EP300 sgRNA downregulated genes, but were enriched among CREBBP sgRNA upregulated genes (Fig. 3A and Supplementary Fig. 5B), in line with our sgRNA toxicity results (Fig. 1A).

Figure 3: EP300 and CREBBP modulate distinct functional programs in ALCL.

Figure 3:

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(A) Heatmap of mRNA levels changed upon EP300 or CREBBP depletion, compared with CTRL sgRNA, obtained from RNA-seq analysis in DEL ALCL line. Genes decreased or increased by at least 2.0-fold are shown. Green represents decreased expression and red represents increased expression. Only representative transcripts are highlighted. The top significantly enriched (p < 0.05) biological programs/signaling pathways identified among the list of genes with reduced or increased expression upon EP300 or CREBBP depletion were shown (right), as determined by GSEA. (B) Indicated ALCL lines were transduced with EP300, CREBBP, or Ctrl sgRNAs, selected and expression induced, and indicated gene expressions were measured by real-time PCR. (C) Chromatin IPs from indicated antibodies were subjected to real-time PCR analysis for negative control locus, or MYC, IRF4, CD30, and PIK3R promoter regions in ALCL line DEL. All error bars denote SEM of triplicates in this Figure.

The ability of CREBBP and EP300 to regulate distinct sets of genes was confirmed by real-time PCR (Fig. 3B). While depletion of EP300 largely impaired IL10, MYC, and IRF4 expression in all three ALCL cell lines, CREBBP depletion had little or opposite effects (Fig. 3B), consistent with our RNA-seq results. In line with these observations, our chromatin immunoprecipitation (ChIP)-coupled real-time PCR experiments demonstrated that EP300 bound avidly to DNA fragments (~300bp) containing the MYC, IRF4, and CD30 transcription start sites in ALCL line DEL (Fig. 3C), supporting that EP300 directly mediates these genes expression in ALCL. In contrast, no binding of CREBBP to these DNA fragments was observed (Fig. 3C). Both EP300 and CREBBP bound to PIK3R6 (one of the genes downregulated by both EP300 and CREBBP in our RNA-seq analysis) transcription start site, demonstrating the validity of the ChIP setup (Fig. 3C). Furthermore, depletion of EP300, but not CREBBP, reduced MYC and IRF4 protein levels in three ALCL cell lines (Supplementary Fig. 6A). Lastly, A-485 and CPI-637 treatment resulted in the reduction of H3K18 acetylation at the MYC and IRF4 promoter regions (Supplementary Fig. 6B), indicating that both the HAT and BRD activities of EP300 are required for the apposite stabilization of EP300 in the chromatin and acetylation of histone tails.

EP300 mediates pro-survival transcriptional program and NF-κB activation in HL

Similar to ALCL, A-485 and CPI-637 treatment efficiently reduced HL cell proliferation (Fig. 1B). To understand the action mechanisms of these drugs in HL and compare them with the mechanisms in ALCL, we profiled gene expression changes upon A-485 and CPI-637 treatment in two HL lines (L1236 and KMH2) using RNA-seq analysis (Fig. 4A). In line with the results in ALCL, the common genes inhibited upon A-485 treatment in HL contained MYC, IRF4, BATF3, BATF, BCL2, and MYB (Fig. 4A), which appeared to be responsible for the growth inhibition phenotype observed (Fig. 1B). The expressions of many surface receptors genes (TNFRSF8 (CD30), PD-L2, IL1R1, IL1R2, IL21R, CCR4, and CCR7) were also reduced upon A-485 treatment (Fig. 4A). Interestingly, A485 significantly decreased a set of NF-κB signature genes (Fig. 4A), indicating the distinctive role of these acetyltransferases in HL cells. In parallel, the CPI-637 treatment inhibited a comparable transcriptional program with the A-485 treatment, although to a less extent (Fig. 4A). Indeed, a significant fraction of the genes showing reduced or increased expression were shared between these two treatments (Supplementary Fig. 7A).

Figure 4: Gene expression programs regulated by EP300/CREBBP inhibitors in HL.

Figure 4:

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(A) Heatmap of mRNA levels changed upon the treatment of A-485 or CPI-637 for 24 hours relative to DMSO-treated cells, obtained from RNA-seq analysis in L1236 and KMH2 HL lines. Genes decreased or increased by at least 2.0-fold (average of two lines) in A-485 or CPI-637 treatment conditions are shown. Green represents decreased expression and red represents increased expression. Only representative transcripts are highlighted. The top significantly enriched (p < 0.05) biological programs/signaling pathways identified among the list of genes showing reduced or increased expression upon the treatment were shown (right), as determined by GSEA. (B) Indicated HL lines were treated with A-485 or CPI-637 at 10μM for 24 hours. Lysates were analyzed by immunoblotting for the indicated proteins. (C) Indicated HL lines were transduced with EP300, CREBBP, or Ctrl sgRNAs, selected and expression induced, and indicated gene expressions were measured by real-time PCR. All error bars denote SEM of triplicates in this Figure.

When we compared the downregulated or upregulated genes upon treatments in ALCL and HL, the shared genes were uncommon and mainly limited to specific oncogenes and surface receptors (Supplementary Fig. 7B, Fig. 2A, and Fig. 4A). In fact, the MYC-targets signatures were among the top signatures enriched that negatively correlated with A-485 or CPI-637 treatments in HL (Fig. 4A, right and Supplementary Fig. 8), which is similar to the results in ALCL. However, besides the MYC-targets signatures, the NF-κB signaling was the top signature enriched upon A-485 or CPI-637 treatments from our GSEA analysis (Fig. 4A, right and Supplementary Fig. 8). Thus, A-485 and CPI-637 mediate MYC-dependent transcriptional program and NF-κB activation in HL.

Using a comprehensive set of HL cell lines (n=5), we examined the effects of A-485 and CPI-637 treatment on critical gene expressions. Both inhibitors effectively reduced MYC, IRF4, IL6, CD30, and BATF3 mRNA transcription in HL lines (Supplementary Fig. 9A). Correspondingly, the ability of A-485 and CPI-637 to inhibit MYC and IRF4 protein expression was confirmed by immunoblot (Fig. 4B). To further corroborate that the MYC/IRF4 suppression is due to the on-target activity of CREBBP or EP300, we used sgRNA to deplete either CREBBP or EP300 in the HL cell line. Similar to what we observed in ALCL, EP300 depletion in HL cells significantly impaired MYC and IRF4 expression, while CREBBP depletion had little effect (Fig. 4C).

Both A-485 and CPI-637 treatment inhibited NF-κB activation, which is one of the most striking oncogenic mechanisms in HL33. Confirming these results, A-485 and CPI-637 treatment inhibited NF-κB activity in three HL lines engineered to express a luciferase reporter driven by an NF-κB response element (Fig. 5A). No expression changes of NF-κB subunits were observed upon treatments, supporting that EP300/CREBBP regulates NF-κB activity through mediating NF-κB signaling (Supplementary Fig. 9B). In our transcriptomic analyses, the immunoregulatory cytokine LTA was among the most potent hits upon EP300/CREBBP inhibition in HL cells (Fig. 4A). The autocrine LTA signaling was shown to drive NF-κB activity in HL34, suggesting EP300 might mediate NF-κB activation through LTA signaling. Indeed, A-485 or CPI-637 effectively inhibited LTA and its receptor TNFRSF14 expressions in HL cell lines (Fig. 5B). Genetically, EP300 sgRNAs effectively reduced the expression of LTA and TNFRSF14, while CREBBP sgRNA had little effect (Fig. 5C, left). Finally, our ChIP-coupled real-time PCR experiments demonstrated that EP300 bound to DNA fragments containing the MYC, IRF4, CD30, LTA, and TNFESF14 transcription start sites in HL line L428 (Fig. 5C, right), supporting that EP300 directly mediates these genes expression HL.

Figure 5: EP300 promotes NF-κB activation in HL.

Figure 5:

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(A) Relative activity of an NF-κB-dependent luciferase reporter in L1236, KMH2, and L428 HL lines after treatment of A-485 or CPI-637 at 10μM for 24 hours. (B) HL lines were treated with A-485 or CPI-637 at 10μM. LTA and TNFRSF14 expressions were measured by real-time PCR after 24 hours of treatment. (C) Indicated HL lines were transduced with EP300, CREBBP, or Ctrl sgRNAs, selected and expression induced, and indicated gene expressions were measured by real-time PCR (left). Chromatin IPs from indicated antibodies were subjected to real-time PCR analysis for negative control locus, or MYC, IRF4, CD30, LTA, and TNFRSF14 promoter regions in HL line L428 (right). All error bars denote SEM of triplicates in this Figure.

EP300 regulates PD-L1 expression and immune escape in ALCL and HL

Interestingly, the key immunosuppressive molecules on APC: PD-L1(CD274) and PD-L2 were among the genes repressed by EP300/CREBBP inhibitors in our RNA-seq analyses. While copy gains of the 9p24.1 locus and PD-L1/PD-L2 gene amplification are frequently observed in cHL35, 36, our data and others17, 37 suggest that oncogenic pathways may also contribute to the constitutive expression of PD-L1. In a panel of ALCL and HL lines, both A-485 and CPI-637 treatment inhibited PD-L1 surface expression and mRNA transcription (Fig. 6A and B), verifying the RNA-seq results. In line with these results, EP300 deletions strongly attenuated the expression of PD-L1 in ALCL and HL lines, while CREBBP depletion has no effect at all (Fig. 6C). Consistent with this observation, our ChIP-coupled real-time PCR experiments demonstrated that EP300, but not CREBBP, bound to DNA fragments containing the candidate PD-L1 enhancer region (Fig. 6D), which was identified in our previous study17.

Figure 6: EP300 mediates PD-L1 expression and immune escape in ALCL and HL.

Figure 6:

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(A) Indicated ALCL and HL lines were treated with A-485 (10μM) or CPI-637 (10μM) for 24 hours. Surface PD-L1 expression was measured by flow cytometry. The relative PD-L1 MFI was normalized to the DMSO-treated cells (right). (B) Indicated ALCL and HL lines were treated with A-485 (left) or CPI-637 (right) at 10μM. PD-L1 mRNA expression was measured by real-time PCR after 24 hours of treatment. (C) Indicated ALCL and HL lines were transduced with EP300, CREBBP, or Ctrl sgRNAs, selected and expression induced, and PD-L1 mRNA expressions were measured by real-time PCR. (D) Chromatin IPs from indicated antibodies were subjected to real-time PCR analysis for negative control locus, or PD-L1 enhancer region in the DEL line. (E) PD-1 expressed Jurkat T cells were co-cultured with DEL ALCL cells (left) or L1236 HL cells (right) for 24 hours under the indicated conditions. Relative CD69 expression was measured by flow cytometry. Tumor: T cell ratio = 4:1. (F) Same as (E), the IL-2 section was measured by ELISA and normalized to activated T cell only controls. Tumor: T cell ratio = 4:1. All error bars denote SEM of triplicates in this Figure.

The success of anti PD-1 or PD-L1 checkpoint therapies relies on their ability to promote anti-tumor T cell immunity38. Therefore, the EP300/CREBBP inhibitors are likely to inhibit immunosuppression of ALCL and HL, and improve the efficacy of immunotherapy. To examine this notion, we established a Jurkat T cell line constitutively expressing the PD-L1 receptor PD-1 on the surface17, and used co-culture experiments with lymphoma cells to evaluate the effects of EP300/CREBBP HAT or bromodomain inhibitors on T cell activation and proliferation. Co-culture of PD-1-expressing Jurkat T cells with ALCL line DEL inhibited induction of the T cell activation marker CD69, as expected. When we deleted EP300 expression using sgRNA in the tumor cells, T cell activation was restored (Fig. 6E and Supplementary Fig. 10). Likewise, when we pre-treated the culture with the clinically employed anti-PD1 antibody nivolumab, A-485, or CPI-637, T cell activation was restored. Notably, the tumor cells pre-treated with either A-485 or CPI-637 failed to inhibit T cell activation to a similar degree as in presence of nivolumab. Similar effects were observed in HL line L1236 (Fig. 6E and Supplementary Fig. 10). Consistent with these results, DEL or L1236 depleted of EP300, or pre-treated with either A-485 or CPI-637, failed to inhibit IL-2 secretion by PD1-expressing Jurkat cells (Fig. 6F).

Targeting EP300 for the treatment of ALCL and HL in xenograft mouse models

Our studies strongly support the notion that EP300 is an attractive target for novel therapeutic strategies in ALCL and HL. Therefore, we established both ALCL (Karpas299) and HL (KMH2) xenograft mouse models, and sought to evaluate the efficacy of the EP300 inhibitors in mouse xenograft models. Since the HAT inhibitor A-485 and the bromodomain inhibitor CPI-637 acted almost equally in our studies, we chose CPI-637 because of its accessibility.

In both ALCL and HL xenograft mouse models, oral treatment with CPI-637 (50 mg/kg/day) significantly slowed tumor growth of established tumors compared to vehicle treatment (p < 0.01 at Day 4, 7, 10, 13 of treatment in ALCL, and p < 0.01 at Day 6, 9, 12, 15, 18 of treatment in HL) (Fig. 7A). Furthermore, the effectiveness of CPI-637 for established tumors was confirmed by tumor weight and size in CPI-637 and vehicle treatment groups at the treatment endpoint, for both ALCL and HL models (Fig. 7B and Supplementary Fig. 11A). Accordingly, the reduction of MYC, IRF4, BATF3, and IL10 expression was observed in the CPI-637 treatment ALCL xenografts at the treatment endpoint (Fig. 7C, upper), indicating the on-target suppression in vivo. Similarly, CPI-637 treatment downregulated MYC, IRF4, BATF3, and LTA expression in HL models at the treatment endpoint (Fig. 7C, lower). In line with these results, CPI-637 treatment downregulated MYC, IRF4, and BATF3 protein expression in both xenograft models (Supplementary Fig. 11B). At the doses used, CPI-637 was well tolerated by mice, with no change in body weight observed in both ALCL and HL models (Supplementary Fig. 11C).

Figure 7: Targeting EP300 in ALCL and HL xenograft mouse models.

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(A) NSG mice bearing Karpas299 (left) or KMH2 (right) xenografts were treated with CPI-637 (50mg/kg daily, n=5) or vehicle controls (n=5). Tumor growth was measured as a function of tumor volume. Error bars denote SEM. P was calculated comparing vehicle and CPI-637 treatment groups. * indicates p < 0.05; ** indicates p < 0.01. (B) Tumor weight in CPI-637 and vehicle treatment groups at the treatment endpoint. ** indicates p < 0.01. (C) Tumors in A at the treatment endpoint were harvested. The mRNA expression of indicated genes was measured by real-time PCR. P was calculated comparing vehicle and CPI-637 treatment groups. * indicates p < 0.05.

Discussion

In the current study, we investigated the physiological roles of EP300 and CREBBP acetyltransferases in both ALCL and HL, and exploited the therapeutic potentials of the small molecule inhibitors that target either the HAT or bromodomain activities of these histone acetyltransferases. EP300, but not CREBBP, directly regulates the oncogenic transcriptional programs, as well as the expression of immunoregulatory cytokines, surface receptors, and immunosuppressive molecules that shape the tumor microenvironment in these tumors. As a consequence of these biological activities, EP300/CREBBP HAT or bromodomain small molecules inhibitors exhibited strong activities against ALCL and HL in vitro and in xenograft mouse models. Together, our findings provide for the first time a broad understanding of the physiological roles of EP300/CREBBP in ALCL and HL, and deliver opportunities for the development of novel strategies for both targeted and immune therapies to treat these lymphoid cance

Our gene expression analyses and functional studies revealed that EP300 and CREBBP regulate distinct transcriptional programs in ALCL and HL, despite their strong similarity. These results are consistent with recent studies on germinal center (GC) B cells and B cell lymphoma11. In our analyses, EP300 but not CREBBP depletion affects a number of critical cellular processes indicating a central role of EP300 in the molecular pathogenesis of these tumors.

The predominant EP300 targets in ALCL that emerged from our analyses are the oncogenic transcription factors MYC and IRF4. MYC and IRF4 are aberrantly expressed and play essential roles in various lymphoma subtypes, including ALCL29. EP300 directly binds to MYC and IRF4 promoters, which likely contribute to the elevated expression levels of these proteins. Like ALCL, EP300 also mediates MYC, IRF4, and CD30 expression in HL. However, the most enriched cellular pathway associated with EP300 depletion or HAT/bromodomain inhibition in HL is related to the NF-κB signature, in line with the central role of NF-κB activation in HL. Notably, there is a clear difference in EP300/CREBBP regulated gene sets between cHL cells in our study and germinal center (GC) B cells in the previous study11, which could be a consequence of transformation during the pathogenesis in cHL39. Unique in B cell lymphoma, cHL cells show a defective B-cell differentiation program, however escape from BCR-mediated apoptosis and immune elimination, and highly depend on the constitutive activation of numerous signaling pathways, such as NF-κB that is usually driven by the signaling input from the tumor microenvironment. Interestingly, our study demonstrated that EP300 modulates the expression of a set of immunoregulatory cytokines and surface receptors, i.e., the LTA and its receptor TNFRSF14, which would be responsible for the oncogenic NF-κB activation. However, the functional cooperation of EP300, NF-κB, and LTA signaling remains to be established.

In addition to the survival pathway, our study disclosed the role of EP300 in immunosuppressive molecule PD-L1 regulation. In prior studies, BET-bromodomain inhibitor has been reported to mediate PD-L1 expression in cancer cell lines40, 41, and EP300 was found to bind at the PD-L1 promoter region42. Unlike previous work, our study demonstrated that EP300 binds to the AP1-IRF composite element (AICE) motif, located in the transcriptional enhancer region of the PD-L1 gene17. Interestingly, IRF4 and BATF3, which are required for PD-L1 transcription, also bind to the similar enhancer region in these cells17. Given that EP300 also upregulates IRF4 and BATF3 expression in ALCL and HL, it seems conceivable that EP300 regulates PD-L1 expression through multiple mechanisms. One possible model is that IRF4 only binds weakly to DNA by itself and needs transcriptional co-factors for optimal PD-L1 transcription. However, additional analyses in the future are required to decipher the specific assemblies among these TFs.

Finally, our findings provide a comprehensive mechanistic basis for clinical trials in ALCL and HL by targeting EP300/CREBBP activity. The development of small-molecule inhibitors targeting the HAT activity and bromodomain of EP300/CREBBP hold promise for anti-cancer treatment, and many of them have been well investigated, including CCS147711, I-CBP11243, 44, SGC-CBP30/CBP3044, 45, and GNE-27246. Indeed, the EP300/CREBBP bromodomain inhibitor CCS1477 is currently undergoing clinical evaluation (NCT04068597, NCT03568656). Notably, the dual inhibitor of BET and CBP/EP300, NEO2734, has been shown to have preferential activity in hematologic cancers47. In our studies, the HAT inhibitor A-485 or the bromodomain inhibitor CPI-637 demonstrated convincing activity in blocking the oncogenic transcriptional programs, preventing the growth of ALCL and HL in xenograft mouse models, and inhibiting PD-L1 mediated tumor immune escape, supporting clinical evaluation of this treatment regimen. From this perspective, targeting EP300/CREBBP would be an attractive strategy to improve targeted and immune therapies in ALCL and HL.

Supplementary Material

Supplementary information

Acknowledgements.

This research was supported by NIH R01 CA259188 (Y.Y.), R01 CA251674 (Y.Y.), Scholar Award from Leukemia & Lymphoma Society (Y.Y.), and Cancer Research Conventional Grant from Gabrielle’s Angel Foundation and Mark Foundation (Y.Y.). Z.S. was partially supported by the Greenwald Postdoctoral Fellowship for Research, FCCC.

We thank Dr. Alan L. Epstein (USC Keck School of Medicine) for the TLBR1 and TLBR2 cell lines, and Dr. Annarosa Del Mistro (The Veneto Institute of Oncology) for the FEPD cell line.

Footnotes

Disclosure of Conflicts of Interest.

The authors declare no competing financial interests.

Data Availability.

All data generated or analyzed during this study are included in this published article and its supplementary information files. The high-throughput RNA sequencing data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession number: SUB11100974.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary information
NIHMS1870355-supplement-Supplementary_information.pdf (3.8MB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. The high-throughput RNA sequencing data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession number: SUB11100974.

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