CREB-binding Protein Research Articles

Inhibition of β-Catenin/CREB Binding Protein Signaling Attenuates House Dust Mite-Induced Goblet Cell Metaplasia in Mice.

Excessive mucus production is a major feature of allergic asthma. Disruption of epithelial junctions by allergens such as house dust mite (HDM) results in the activation of β-catenin signaling, which has been reported to stimulate goblet cell differentiation. β-catenin interacts with various co-activators including CREB binding protein (CBP) and p300, thereby regulating the expression of genes involved in cell proliferation and differentiation, respectively. We specifically investigated the role of the β-catenin/CBP signaling pathway in goblet cell metaplasia in a HDM-induced allergic airway disease model in mice using ICG-001, a small molecule inhibitor that blocks the binding of CBP to β-catenin. Female 6- 8-week-old BALB/c mice were sensitized to HDM/saline on days 0, 1, and 2, followed by intranasal challenge with HDM/saline with or without subcutaneous ICG-001/vehicle treatment from days 14 to 17, and samples harvested 24 h after the last challenge/treatment. Differential inflammatory cells in bronchoalveolar lavage (BAL) fluid were enumerated. Alcian blue (AB)/Periodic acid–Schiff (PAS) staining was used to identify goblet cells/mucus production, and airway hyperresponsiveness (AHR) was assessed using invasive plethysmography. Exposure to HDM induced airway inflammation, goblet cell metaplasia and increased AHR, with increased airway resistance in response to the non-specific spasmogen methacholine. Inhibition of the β-catenin/CBP pathway using treatment with ICG-001 significantly attenuated the HDM-induced goblet cell metaplasia and infiltration of macrophages, but had no effect on eosinophils, neutrophils, lymphocytes or AHR. Increased β-catenin/CBP signaling may promote HDM-induced goblet cell metaplasia in mice.

Individual functions of the histone acetyl transferases CBP and p300 in regulating the inflammatory response of synovial fibroblasts

Chromatin remodeling, and a persistent histone 3 lysine 27 acetylation (H3K27ac) in particular, are associated with a sustained inflammatory response of synovial fibroblasts (SF) in rheumatoid arthritis (RA). Here we investigated individual functions of the writers of H3K27ac marks, the homologues histone acetyl transferases (HAT) CBP and p300, in controlling the constitutive and inflammatory gene expression in RA SF. We applied a silencing strategy, followed by RNA-sequencing and pathway analysis, complemented with the treatment of SF with inhibitors targeting the HAT (C646) or bromo domains (I-CBP) of CBP and p300. We showed that CBP and p300 undertook overlapping and, in particular at gene levels, distinct regulatory functions in SF. p300 is the major HAT for H3K27ac in SF and regulated more diverse pathways than CBP. Whereas both factors regulated genes associated with extracellular matrix remodeling, adhesion and proliferation, p300 specifically controlled developmental genes associated with limb development. Silencing of CBP specifically down regulated the TNF-induced expression of interferon-signature genes. In contrast, silencing of p300 resulted in anti- and pro-inflammatory effects. Integration of data sets derived from RNA-sequencing and chromatin immunoprecipitation sequencing for H3K27ac revealed that changes in gene expression after CBP or p300 silencing could be only partially explained by changes in levels of H3K27ac. Inhibition of CBP/p300 using HAT and bromo domain inhibitors strongly mirrored effects obtained by silencing of p300, including anti- and pro-inflammatory effects, indicating that such inhibitors are not sufficient to be used as anti-inflammatory drugs.

Nrf2, the Major Regulator of the Cellular Oxidative Stress Response, is Partially Disordered

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.

ZFP36 protects lungs from intestinal I/R-induced injury and fibrosis through the CREBBP/p53/p21/Bax pathway

Acute lung injury induced by ischemia–reperfusion (I/R)-associated pulmonary inflammation is associated with high rates of morbidity. Despite advances in the clinical management of lung disease, molecular therapeutic options for I/R-associated lung injury are limited. Zinc finger protein 36 (ZFP36) is an AU-rich element-binding protein that is known to suppress the inflammatory response. A ZFP36 binding site occurs in the 3ʹ UTR of the cAMP‐response element-binding protein (CREB) binding protein (CREBBP) gene, which is known to interact with apoptotic proteins to promote apoptosis. In this study, we investigate the involvement of ZFP36 and CREBBP on I/R-induced lung injury in vivo and in vitro. Intestinal ischemia/reperfusion (I/R) activates inflammatory responses, resulting in injury to different organs including the lung. Lung tissues from ZFP36-knockdown mice and mouse lung epithelial (MLE)-2 cells were subjected to either Intestinal I/R or hypoxia/reperfusion, respectively, and then analyzed by Western blotting, immunohistochemistry, and real-time PCR. Silico analyses, pull down and RIP assays were used to analyze the relationship between ZFP36 and CREBBP. ZFP36 deficiency upregulated CREBBP, enhanced I/R-induced lung injury, apoptosis, and inflammation, and increased I/R-induced lung fibrosis. In silico analyses indicated that ZFP36 was a strong negative regulator of CREBBP mRNA stability. Results of pull down and RIP assays confirmed that ZFP36 direct interacted with CREBBP mRNA. Our results indicated that ZFP36 can mediate the level of inflammation-associated lung damage following I/R via interactions with the CREBBP/p53/p21/Bax pathway. The downregulation of ZFP36 increased the level of fibrosis.

Abstract 1146: Targeted degradation of the enhancer lysine acetyltransferases CBP and p300

Abstract The chromatin regulators CREB-binding protein (CBP) and p300 are factors critical for establishing and activating enhancer-mediated transcription. These large proteins are made up of at least ten distinct functional domains and are known to interact with over 400 other proteins including DNA-binding transcription factors, other chromatin regulators, and components of the general transcription apparatus. In addition to mediating protein-protein interactions on chromatin, p300/CBP exert enzymatic activity through a lysine acetyltransferase (KAT) domain. p300/CBP can dynamically acetylate ~21,000 lysines on over 5,000 proteins including more than three dozen known enhancer-associated regulators. In cancer, p300/CBP have been implicated as both oncogenes and tumor suppressors in a large number of malignant cell types and active drug development projects by a number of groups have focused on the discovery of potent and selective compounds. Small molecules that target the p300/CBP bromodomain and KAT domains have provided a unique toolset for chromatin and chemical biologists to probe various p300/CBP functions at enhancers. However, it is clear that simultaneous inhibition of multiple functional domains, or even complete protein depletion, is required to ablate the enhancer activity mediated by p300/CBP. To pursue this goal further and to provide the research community with additional tools to probe the function of this important class of chromatin regulators, we have designed and characterized the first heterobifunctional chemical degrader of p300/CBP, dCBP-1. Bifunctional, or chimeric, degraders that leverage the activity of E3 ligases such as cereblon (CRBN) through chemical-induced proximity to proteins of interest have transformed the capabilities for chemical degradation. Aided by in silico modeling of a ternary complex of ligand-bound CRBN with the KAT and bromodomains of p300/CBP, we were able to efficiently synthesize a highly potent and selective degrader, dCBP-1. Using dCBP-1 as a tool, we profile the consequences of complete p300/CBP loss in cellular models of multiple myeloma, a tumor type uniquely addicted to the activity of both p300 and CBP. Proteomics profiling reveals selective effects on p300 and CBP, with loss of oncogenic MYC expression. Epigenomic profiles of gene expression and chromatin structures with dCBP-1 treatment reveals augmented effects of oncogene expression compared to inhibitors alone or in combination, with complete ablation of histone lysine acetylation and chromatin accessibility at enhancers, including the immunoglobulin locus enhancer that can drive oncogenic MYC expression in ~20% of multiple myeloma. Taken together, dCBP-1 is the first rapid potent and selective chemical degrader of p300/CBP and enables pharmacologic disruption of p300/CBP-regulated enhancers. Citation Format: Samuel Ojeda, Raghu Vannam, Jan Sayilgan, Barbara Karakyriakou, Eileen Hu, Johannes Kreuzer, Robert Morris, Xcanda Ixchel Herrera Lopez, Sumit Rai, Wilhelm Haas, Michael Lawrence, Christopher J. Ott. Targeted degradation of the enhancer lysine acetyltransferases CBP and p300 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1146.

Abstract 1437: E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, an investigational anti-cancer agent, inhibits the binding of β-catenin to its transcriptional co-activator, CREB binding protein (CBP), thereby modulating Wnt/β-catenin signaling. We previously reported that E7386 suppressed tumor growth in preclinical mouse models with aberrant activation of Wnt/β-catenin signaling. Moreover, the combination of E7386 plus lenvatinib, a multiple receptor tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor receptors and fibroblast growth factor receptors, showed stronger antitumor and antiangiogenic activities than each treatment alone. Here, we investigated the molecular mechanism of the combination activities of E7386 plus lenvatinib. Methods: We examined antitumor activity of E7386 plus lenvatinib in multiple human tumor xenograft and mouse syngeneic models. Tumor samples underwent RNA sequencing (RNA-seq) followed by weighted gene correlation network analysis. Furthermore quantitative PCR was used to assess changes in the expression of some hypoxia-related genes. The protein interaction between β-catenin and HIF1 was analyzed by immunoprecipitation-western blotting. Results: RNA-seq analysis of the effects of E7386, lenvatinib, and E7386 plus lenvatinib on the tumor microenvironment in the Hepa1-6 hepatocellular carcinoma syngeneic tumor model in mice showed that lenvatinib suppressed the expression of angiogenesis-related genes and that E7386 plus lenvatinib enhanced this suppression. Although lenvatinib increased the expression of hypoxia-related genes, E7386 plus lenvatinib attenuated this phenomenon. According to these results, we hypothesized that E7386 inhibits the formation of the β-catenin/HIF-1 complex. To confirm the effect of E7386 on the hypoxic response, we conducted an in vitro quantitative PCR assay under hypoxic conditions in Hepa1-6 cells and the human hepatocellular carcinoma cell lines, HepG2 and Hep3B. In accordance with the RNA-seq result, E7386 significantly suppressed the expression levels of hypoxia-related genes in the HepG2 and Hep3B cells. Finally, data from immunoprecipitation-western blotting demonstrated that β-catenin formed a complex with HIF-1 and that E7386 clearly inhibited this complex formation in HepG2 cells. Conclusion: These results suggest that E7386 enhances angiogenesis inhibition when used in combination with lenvatinib and that E7386 suppresses the hypoxic response induced by angiogenesis inhibition. Further analysis is required to clarify the precise mechanisms of actions of the combination of E7386 plus lenvatinib. Citation Format: Mizuki Kimura, Yusaku Hori, Megumi Kuronishi, Takayuki Kimura, Ryoga Ishida, Kenji Ichikawa, Yoichi Ozawa, Yu Kato. E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1437.

Abstract 1605: CERBBP mutation promotes antitumor immunity and as predictive biomarkers for immune checkpoint therapy in colorectal cancer

Abstract Background: The prognosis of colorectal cancer (CRC) remains unsatisfactory, only few CRC patients can benefit from immune checkpoint inhibitors (ICIs) treatment. Tumor immune microenvironment (TME), the Tumor mutation burden (TMB) and PD-L1 expression of ICIs have been confirmed to be promising predictive biomarkers of ICIs in pan-cancer. The roles of CREBBP gene in CRC have not been reported before, and the mutation correlation with ICIs predictive biomarkers and immune response remain unknown. Methods: Whole exome sequencing data of 526 CRC patients was obtained from the Cancer Genome Altas (TCGA). Next generation sequencing (NGS) was performed on FFPE tumor samples from 2198 Chinese CRC patients (Chinese cohort). NGS data and clinical data of 110 advanced CRC patients treated with ICIs were obtained from the MSK-IMPACT Clinical Sequencing cohort (MSKCC). The tumor-related immune cells infiltration level difference between mutant and wild-type tumors were inferred using TIMER2.0. Tumor mutation burden (TMB) examined by 387/733 gene panel (3D-Med) sequencing. PD-L1 expression was determined by immunohistochemistry (IHC) staining assay using SP263. Survival analysis was determined by Kaplan-Meier (KM) analysis. Results: In total, 8.62% (43/526) of CRC patients in TCGA harboring CREBBP mutation and 2.00% (44/2198) in Chinese cohort, the alternation frequency of CREBBP in Chinese cohorts was lower. In TCGA cohort, the level of infiltration of tumor-related immune cells, including CD8+ T cell, CD4+ T cell, T cell regulatory (Tregs) and NK cell, were all significantly increased in mutant tumors compared with wild-type tumors (Wilcoxon test, p < 0.05). The mutant tumors with significantly higher inferred M1 macrophages than wild-type (p=0.0029), but not in M2 macrophages. In the Chinese cohort, the TMB level was higher in mutant group compared to wild-type group (median TMB, mut vs. wt = 82.26 vs. 7.26 Mut/Mb, p < 0.001). There was an elevated PD-L1 expression in CREBBP mutant tumors (29.63%, n=44) compared to wild-type tumors (15.73%, n=2154, p = 0.062). The survival analysis was performed on patients in MSKCC CRC patients treated with ICIs. The overall survival (OS) of CREBBP mutation group (n=16) were significantly longer than wild-type group (n=94) (median OS, mutation vs. wild-type = 34 vs. 13 months; HR 0.23[95% CI 0.07-0.75]; P = 0.008). Conclusion: The CREBBP gene mutation was associated with higher tumor-related immune cell infiltration and TMB level. CRC patients harboring CREBBP mutation might be a survival benefit from ICIs. Citation Format: Youen Lin, Jiewei Zheng, Tingting Chen, Wenzhuan Xie, Mengli Huang. CERBBP mutation promotes antitumor immunity and as predictive biomarkers for immune checkpoint therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1605.

Abstract 1143: Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers

Abstract Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play oncogenic roles in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent its coactivator function at AR, thereby potentially inhibit growth of AR-dependent prostate cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized therapies. Experimental procedures and Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors have been identified by iterative medicinal chemistry and SAR based approaches. The lead compound, AU-18069 was optimized towards attaining good potency, physicochemical properties and DMPK profile. AU-18069 potently inhibited viability and proliferation of a wide range of cell lines derived from prostate cancer, CBP mutant and MYC-dependent hematological cancers and demonstrated improved PK profile in rodent models in comparison with a compound, currently in clinical trials. Excellent efficacy with significant tumor growth inhibition (TGI) was observed in MV4-11 xenograft model at well-tolerated doses along with downregulation of cMYC in a single dose PK-PD study. AU-18069 also showed modulation of different immune phenotypes including CD4+ T cell subsets. In summary, our lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in vitro and in vivo. Further evaluation of immune activation potential, efficacy studies in various xenograft models, long term toxicological evaluation in different species and other IND enabling studies are in progress. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Ramesh S Senaiar, Subhendu Mukherjee, Mahaboobi Jaleel, Sivapriya Marappan, Raghavendra N R, Girish Aggunda Renukappa, Aravind A B, Naveen Kumar R, Venkata Siva Reddy, Asha Babu, Akhila Srinivas, Prasad Yadlapalli, Suraj T Gore, Pathange Hemasankar, Mamon Dey, Samiulla D S, Chandranath D Naik, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, Mikko Myllymäki, Gerd Wohlfahrt, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1143.

Abstract 2236: A Pan-cancer Analysis of CREBBP as a potential predictor for immune checkpoint therapy

Abstract Background: EP300 (hereafter referred to as p300) and its closely related paralog CREB-binding protein (CREBBP, hereafter CBP) are ubiquitously expressed transcriptional co-activators and major lysine acetyltransferases (KATs) in metazoans. The roles of CREBBP in the pathogenesis of leukemia and malignant lymphomas have been reported before, but not been extensively studied in solid tumors. Tumor mutation burden (TMB) has been regarded as the most prevalent biomarker to predict immunotherapy, the CREBBP mutation status correlation with TMB and immune response remain unknown. Methods: Next generation sequencing (NGS) and clinical data of 1661 advanced cancer patients treated with ICI were obtained from the MSK-IMPACT Clinical Sequencing cohort (MSKCC). NGS (381/733 gene 3DMed panel) was performed on FFPE tumor samples from 17144 Chinese pan-cancer patients (Chinese cohort). The association between CREBBP mutation and TMB level in different tumor types were explored. The statistical significance of CREBBP expression level between normal tissues and tumors in pan-cancer was analyzed by the Wilcoxon test using TIMER2.0. Results: In total, 7.50% (108/1661) of pan-cancer patients in MSKCC harbored CREBBP mutation and 1.38% (237/17144) in Chinese cohort. In MSKCC cohort, the highest CREBBP mutation frequency tumor type was colorectal cancer (CRC, 14.55%, 16/110), Bladder urothelial carcinoma (BLCA, 14.42%, 31/215) and melanoma (SKCM, 7.5%, 24/320) come in second and third, respectively. While in Chinese cohort, the top three CREBBP mutation frequency tumor types were Small-cell lung cancer (SCLC, 9.86%, 14/142), Bladder Urothelial carcinoma (8.81%, 26/295) and Uterine corpus endometrial carcinoma (UCEC, 5.38%, 12/223), respectively. CREBBP mutation was also associated with higher TMB in both MSKCC cohort (median TMB, mutation vs wild-type = 22.12 vs 5.9 Muts/Mb, P < 0.0001) and Chinese pan-cancer cohort (median TMB, mutation vs wild-type = 43.40 vs 9.00 Muts/Mb, P < 0.0001). The highest median TMB level of CREBBP mutation tumor type was UCEC with 18.63 Muts/Mb (n=65). The CREBBP expression level of some tumor types were significantly up-regulated in tumors compared with normal tissues (p < 0.001), such as Cholangiocarcinoma, Head and Neck cancer and Liver hepatocellular carcinoma. The survival analysis were performed on patients in MSKCC cohort treatment with ICIs. The overall survival (OS) of CREBBP mutation group (n=106) were significantly longer than wild-type group (n=1555) (median OS, not reach vs 17 months; HR 0.54 [95% CI 0·38-0·76]; P < 0.001). Conclusions: The results indicated that CREBBP gene mutation was associated with a higher TMB level in pan-cancer, and patients harboring these genes mutations might easily benefit from ICIs treatment. Citation Format: Jiaping Li, Wenzhe Fan, Yue Zhao, Miao Xue, Tingting Chen, Wenzhuan Xie, Mengli Huang. A Pan-cancer Analysis of CREBBP as a potential predictor for immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2236.

Effects of H3 and H4 histones acetylation and bindings of CREB binding protein and p300 at the promoter on hepatic expression of gamma-glutamyltransferase gene in a streptozotocin-induced moderate hypoinsulinemic rat model.

Gamma-glutamyltransferase (GGT), a marker of liver disease, has been shown to be associated with increased risk of diabetes and relative insulin secretion deficiency. However, the mechanism of hepatic Ggt regulation has not been explored fully. In this study, we made a concerted effort to understand the mechanism by investigating the effects of acetylation of histones H3 and H4, and bindings of histone acetyltransferases, CREB binding protein (CBP) and p300, at the Ggt promoter on the regulation of the expression of Ggt gene in the livers of streptozotocin (STZ)-induced moderate hypoinsulinemia rat model. The rats treated with STZ showed remarkably higher serum GGT level and hepatic Ggt/GGT expression than the untreated control rats. Furthermore, the acetylation of histones H3 and H4, and the binding of CBP not p300 at the Ggt promoter regions were significantly higher in the livers of STZ rats than those of the control rats. These results suggest that an enhanced hepatic expression of Ggt is associated with increased acetylation of histones H3 and H4 and CBP binding at the Ggt promoter in STZ-induced moderate hypoinsulinemic rats.

Limited Therapeutical Impact of Extensive Genomic Profiling in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia: Population-Based Four-Year Consecutive Cohort

High throughput genomic technologies, such as high-density SNP arrays and whole exome and transcriptome sequencing (WES, RNAseq), significantly deepened our insight into the genomic background and biology of acute lymphoblastic leukemia (ALL) during the past decade. Novel genetic aberrations and genomic and gene expression patterns have been described which led to the definition of several novel ALL subtypes and risk-predictive factors and to the identification of novel druggable and/or resistance-associated lesions potentially impacting therapy tailoring. Anecdotal evidence of therapeutical interventions based on the up-to-date genetic data has resulted into a great demand for these investigations. However, it is unclear what proportion of pediatric ALL patients, especially the cases of therapy-resistant leukemia, would really benefit from extensive genomic profiling.Between V/2013 and VIII/2017, relapse of Ph-negative B-cell precursor (BCP) ALL was diagnosed in 20 children (1-18 years) within the Czech Republic. In addition to the routine genetics, 18/20 cases were profiled by high density SNP array (18/18), WES (18/18) and RNAseq (17/18). The examined cohort comprised 10 B-other, 4 hyperdiploid, 3 ETV6-RUNX1 -positive and 1 KMT2A -rearranged ALL. At primary leukemia manifestation, all children were treated according to the ALL-BFM protocols [BFM 95 (n=1), BFM 2000 (n=1) and BFM 2009 (n=16)] and suffered very early (n=1), early (n=4) or late relapses (n=13). Upon disease relapse, 5, 11 and 2 cases were treated according to ALL-REZ BFM 2002 and IntReALL 2010 SR and HR protocols, respectively.Bulk bone marrow (BM) samples or sorted leukemic blasts were analyzed in isolated BM (n=15) and combined BM+CNS (n=2) relapses, while tissue from uterus infiltrated with blasts was analyzed in a single case of isolated extramedullary relapse. In addition to leukemic blasts, remission samples were analyzed by WES in all and by SNP array in selected cases to distinguish germline genetic variants. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data using commercial softwares. WES and RNAseq data were processed and somatic single nucleotide variants, indels and fusion transcripts were called using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4 gene rearrangements (r.). Hierarchical clustering based on gene expression profiles (unsupervised clustering and clustering with gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL) was performed using RNAseq data. To facilitate this analysis, gene expression data from relapse cohort were combined with primary diagnosis data from >50 B-other ALL cases. Clinically important findings were reported to national study centre.Two B-other cases were classified as DUX4 -r. and 2 other cases as Ph-like ALL. Of note, 2 cases (both treated as standard risk in primary diagnosis) had iAMP21. Apart from 3 CRLF2 -rearrangements (1 present in Ph-like case), 6 KRAS and 2 NRAS genes mutations, no potentially druggable lesions were found. While the frequency of CRLF2 -rearrangements and RAS mutations found in relapse was comparable to that seen in primary disease, relapse cohort was enriched for lesions associated with unfavorable prognosis and therapy resistance. Three and four cases harbored deletion of TP53 gene and IKZF1 plus deletion pattern; however, in 1/2 cases with available data from primary diagnosis the IKZF1 plus pattern was identified only in relapse. Deletion of CREBBP and TBL1XR1 genes, potentially associated with resistance to glucocorticoids, were found in 2 cases and 1 case, respectively. Mutations of NT5C2 gene resulting in resistance to mercaptopurine were found in 2 cases. Three out of 18 analyzed cases did not harbor any of the above mentioned lesions. None of the genetic findings directly impacted patients' treatment.To conclude, we successfully implemented advanced genomics into centralized diagnostics of resistant ALL at the national level. Although it improved our understanding of disease relapse (yet, not in 100% of cases) the results were not translated to therapy tailoring in patients treated according to the large international protocols, thus, questioning the cost/benefit ratio of such extensive genetic diagnostics.Support: AZV 15-30626A and 16-30186A, GACR 15-06049Y, Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

Rubinstein-Taybi Syndrome: A Model of Epigenetic Disorder.

The Rubinstein-Taybi syndrome (RSTS) is a rare congenital developmental disorder characterized by a typical facial dysmorphism, distal limb abnormalities, intellectual disability, and many additional phenotypical features. It occurs at between 1/100,000 and 1/125,000 births. Two genes are currently known to cause RSTS, CREBBP and EP300, mutated in around 55% and 8% of clinically diagnosed cases, respectively. To date, 500 pathogenic variants have been reported for the CREBBP gene and 118 for EP300. These two genes encode paralogs acting as lysine acetyltransferase involved in transcriptional regulation and chromatin remodeling with a key role in neuronal plasticity and cognition. Because of the clinical heterogeneity of this syndrome ranging from the typical clinical diagnosis to features overlapping with other Mendelian disorders of the epigenetic machinery, phenotype/genotype correlations remain difficult to establish. In this context, the deciphering of the patho-physiological process underlying these diseases and the definition of a specific episignature will likely improve the diagnostic efficiency but also open novel therapeutic perspectives. This review summarizes the current clinical and molecular knowledge and highlights the epigenetic regulation of RSTS as a model of chromatinopathy.

Downregulation of HERC5 E3 ligase attenuates the ubiquitination of CtBP1 to inhibit apoptosis in colorectal cancer cells.

The homologous to E6AP C-terminus (HECT) domain and RCC1-like domain-containing (HERC) proteins can function as tumour suppressors and as oncogenes, depending on the cancer type. However, the expression patterns of HERCs in colorectal cancer (CRC) cells are unclear. Here, we show that only HERC1 and HERC5 are downregulated in CRC tumours, and we focus our study on revealing HERC5-mediating signalling because the change in downregulation is much more obvious for HERC5 than for HERC1. We demonstrate that HERC5 recruits an adaptor protein, CREB-binding protein, to ubiquitinate C-terminal binding protein 1 (CtBP1) in non-cancerous colon cells. The downregulation of HERC5 in CRC cells attenuates the ubiquitination of CtBP1, which then accumulates and assembles into a transcriptional complex with histone deacetylase 1 and a transcription factor c-MYC. This transcriptional complex binds to the promoters of three proapoptotic genes, Bcl2 associated X (BAX), Bcl2 interacting killer (BIK) and p53upregulated modulator of apoptosis (PUMA), and inhibits their expression, thereby suppressing apoptotic signalling and promoting tumourigenesis. Overexpression of HERC5, downregulation of CtBP1 or blocking of the CtBP1 function with its inhibitors (NSC95397 and 4-methylthio-2-oxobutyric acid [MTOB]) significantly prevents CRC cell proliferation in vitro and tumour growth in vivo. Combining NSC95397 (or MTOB) with chemotherapeutic drugs (oxaliplatin or capecitabine) gives a much stronger inhibition of cell proliferation and tumour growth compared with their single treatments. Collectively, our results reveal that downregulation of HERC5 E3 ligase attenuates the ubiquitination of CtBP1 to inhibit apoptosis. Therefore, CtBP1 may be a promising target in CRC chemotherapy.

Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.

Histone Deacetylase 2-Mediated Epigenetic Regulation is Involved in the Early Isoflurane Exposure-Related Increase in Susceptibility to Anxiety-Like Behaviour Evoked by Chronic Variable Stress in Mice.

Increasing studies report that prolonged or multiple anaesthetic exposures early in life are associated with detrimental effects on brain function. Although studies have evaluated the detrimental effects on neurocognitive function, few have focused on long-term neuropsychiatric effects. In the present study, C57BL/6 mice received either three neonatal isoflurane exposures or control exposure. Starting on postnatal day 45, the mice were either exposed or not to a chronic variable stress (CVS) paradigm, and CVS-related neuropsychiatric performance was evaluated using a series of behavioural tests. The expression levels of histone 3 lysine 9 acetylation (acetyl-H3K9), brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein-binding protein, and histone deacetylases 1-4 in the amygdala were measured by immunoblotting or immunohistochemistry analysis. In mice with neonatal isoflurane exposure, the effects of sodium butyrate (NaB), a commonly used HDAC inhibitor, were examined on CVS-related behavioural and molecular alterations. The results showed that repeated neonatal isoflurane exposure did not affect innate depression-like and anxiety-like behaviours under non-stress conditions but facilitated the CVS-induced anxiety-like behavioural phenotype. Increased HDAC2 expression in the amygdala was associated with an increase in the CVS-induced repression of acetyl-H3K9 and BDNF expression and an enhanced CVS-evoked anxiety-like behavioural phenotype in mice neonatal isoflurane exposure. NaB significantly decreased the CVS-induced anxiety level by elevating acetyl-H3K9 and BDNF expression. These results suggested that early anaesthesia exposure facilitated chronic stress-induced neuropsychiatric outcomes, and the HDAC2-related epigenetic dysregulation of BDNF gene expression is involved in the underlying mechanism.

CBP/p300: Critical Co-Activators for Nuclear Steroid Hormone Receptors and Emerging Therapeutic Targets in Prostate and Breast Cancers.

Simple SummaryThe CREB-binding protein (CBP) and p300 are paralogous lysine acetyltransferases that serve as critical co-activators for transcription factors involved in diverse signaling pathways in cancer. Work in the last two decades has firmly established CBP and p300 as important regulators of nuclear hormone signaling mediated by nuclear receptors, such as the androgen receptor (AR) and estrogen receptor (ER). The AR and ER promote tumor growth in hormone-dependent prostate and breast cancer, respectively. Inhibitors of androgen and estrogen signaling are the standard-of-care therapeutics for treating these cancers. However, resistance to current therapies remains a significant clinical problem. Inhibition of CBP and p300 as a means to block the transactivation activity of the AR and ER is an emerging therapeutic strategy for prostate and breast cancers. This review describes how CBP and p300 regulate androgen and estrogen signaling and discusses therapeutic potential of newly discovered potent CBP/p300 inhibitors for treating prostate and breast cancer.The CREB-binding protein (CBP) and p300 are two paralogous lysine acetyltransferases (KATs) that were discovered in the 1980s–1990s. Since their discovery, CBP/p300 have emerged as important regulatory proteins due to their ability to acetylate histone and non-histone proteins to modulate transcription. Work in the last 20 years has firmly established CBP/p300 as critical regulators for nuclear hormone signaling pathways, which drive tumor growth in several cancer types. Indeed, CBP/p300 are critical co-activators for the androgen receptor (AR) and estrogen receptor (ER) signaling in prostate and breast cancer, respectively. The AR and ER are stimulated by sex hormones and function as transcription factors to regulate genes involved in cell cycle progression, metabolism, and other cellular functions that contribute to oncogenesis. Recent structural studies of the AR/p300 and ER/p300 complexes have provided critical insights into the mechanism by which p300 interacts with and activates AR- and ER-mediated transcription. Breast and prostate cancer rank the first and forth respectively in cancer diagnoses worldwide and effective treatments are urgently needed. Recent efforts have identified specific and potent CBP/p300 inhibitors that target the acetyltransferase activity and the acetytllysine-binding bromodomain (BD) of CBP/p300. These compounds inhibit AR signaling and tumor growth in prostate cancer. CBP/p300 inhibitors may also be applicable for treating breast and other hormone-dependent cancers. Here we provide an in-depth account of the critical roles of CBP/p300 in regulating the AR and ER signaling pathways and discuss the potential of CBP/p300 inhibitors for treating prostate and breast cancer.

Profiling of 520 Candidate Genes in 50 Surgically Treated Chinese Small Cell Lung Cancer Patients.

Small cell lung cancer (SCLC) is one of the severe malignancies with high mortality. Surgically resected tumor tissues from 50 Chinese SCLC patients were collected for next-generation sequencing to detect 520 cancer‐related genes. The most frequently altered genes were TP53 (94.0%), RB1 (86.0%), LRP1B (44.0%), SPTA1 (26.0%) and KMT2D (24.0%). We detected that NOTCH2, JAK2 and CDK12 (P<0.05) had a significantly higher mutation frequency in Chinese SCLC compared to the Cologne and MSKCC. The single nucleotide variation (SNV) is dominated by C>A (34.1%). We found a significant association between TMB-H (≥10.3muts/Mb) and ATM (P=0.023), CREBBP (P=0.010), KMT2D(P=0.050) and LRP1B (P=0.005) gene mutations in Chinese SCLC patients. Immunostaining was performed using the following antibodies: TTF-1, CgA, CD56, Syn, and Ki-67. Correlation analysis between the expression of 6 markers and mutations in signaling pathways showed that Syn and CgA expression were associated with 4 (cGMP-PKG, Chemokine, TGF-β and Phospholipase D) and 2 (cGMP-PKG and Phosphatidylinositol) signaling pathway mutations. Kaplan-Meier curve showed that age<55 years, mutant ARID2 and high TMB (≥7muts/Mb) were associated with a better prognosis, while the prognosis of patients with mutations in the Ras pathway was significantly improved. High TMB is an important prognostic factor for SCLC patients showed by multivariate analysis. In the combined cohort composed of current and two previous studies, survival analysis showed that SCLC patients with mutant LRP1B demonstrated better OS (P=0.0017). Patients with a high TMB (≥7muts/Mb) have a better prognosis (P=0.0053), consistent with our results in the Chinese cohort. We characterized the genomic alterations profile of Chinese SCLC patients and analyzed the correlation between genomic changes and immunohistochemical phenotypes at the signaling pathway level. Our data might provide useful information in the diagnosis and treatment for Chinese SCLC patients.

A quinazoline-based bromodomain inhibitor, CN210, ameliorates indomethacin-induced ileitis in mice by inhibiting inflammatory cytokine expression.

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

Histone Deacetylase Inhibitors Ameliorate Morphological Defects and Hypoexcitability of iPSC-Neurons from Rubinstein-Taybi Patients.

Rubinstein-Taybi syndrome (RSTS) is a rare neurodevelopmental disorder caused by mutations in CREBBP or EP300 genes encoding CBP/p300 lysine acetyltransferases. We investigated the efficacy of the histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) in ameliorating morphological abnormalities of iPSC-derived young neurons from P149 and P34 CREBBP-mutated patients and hypoexcitability of mature neurons from P149. Neural progenitors from both patients’ iPSC lines were cultured one week with TSA 20 nM and, only P149, for 6 weeks with TSA 0.2 nM, in parallel to neural progenitors from controls. Immunofluorescence of MAP2/TUJ1 positive cells using the Skeletonize Image J plugin evidenced that TSA partially rescued reduced nuclear area, and decreased branch length and abnormal end points number of both 45 days patients’ neurons, but did not influence the diminished percentage of their neurons with respect to controls. Patch clamp recordings of TSA-treated post-mitotic P149 neurons showed complete/partial rescue of sodium/potassium currents and significant enhancement of neuron excitability compared to untreated replicas. Correction of abnormalities of P149 young neurons was also affected by valproic acid 1 mM for 72 h, with some variation, with respect to TSA, on the morphological parameter. These findings hold promise for development of an epigenetic therapy to attenuate RSTS patients cognitive impairment.