Histone Acetyltransferase Domain Research Articles

CREBBP histone acetyltransferase domain mutations predict response to mTOR inhibition in relapsed/refractory follicular lymphoma.

Despite the clinical and molecular heterogeneity of follicular lymphoma (FL), there remains a lack of biomarker-directed therapeutic approaches in routine clinical practice, with the notable exception of the EZH2 inhibitor tazemetostat in EZH2-mutant FL. Here we examined whether gene mutation status predicts response to clinical mTOR inhibitors (mTORi) in FL, by performing targeted mutational profiling of biopsies from 21 relapsed/refractory FL patients treated with mTORi everolimus or temsirolimus within clinical trials. We observed an enrichment of mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP in mTORi-responders, and describe distinct transcriptional characteristics and co-occurring mutations of FL harbouring these mutations; reinforcing the growing appreciation of CREBBPHAT mutation as a key biological determinant and its promise as a therapeutic biomarker in FL.

HyperCas12a enables highly-multiplexed epigenome editing screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We also show that the dCas12a platform can perform simultaneous activation and repression using a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, demonstrate that the dCas12a system is highly effective for high-throughput screens. We use dHyperLbCas12a-KRAB and a ~19,000-member barcoded library of crRNA arrays containing six crRNAs each to dissect the independent and combinatorial contributions of CREs to the dose-dependent control of gene expression at a glucocorticoid-responsive locus. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

Molecular Mechanism of Fusarium Fungus Inhibition by Phenazine-1-carboxamide.

Fusarium head blight caused by Fusarium graminearum is a devastating disease in wheat that seriously endangers food security and human health. Previous studies have found that the secondary metabolite phenazine-1-carboxamide produced by biocontrol bacteria inhibited F. graminearum by binding to and inhibiting the activity of histone acetyltransferase Gcn5 (FgGcn5). However, the detailed mechanism of this inhibition remains unknown. Our structural and biochemical studies revealed that phenazine-1-carboxamide (PCN) binds to the histone acetyltransferase (HAT) domain of FgGcn5 at its cosubstrate acetyl-CoA binding site, thus competitively inhibiting the histone acetylation function of the enzyme. Alanine substitution of the residues in the binding site shared by PCN and acetyl-CoA not only decreased the histone acetylation level of the enzyme but also dramatically impacted the development, mycotoxin synthesis, and virulence of the strain. Taken together, our study elucidated a competitive inhibition mechanism of Fusarium fungus by PCN and provided a structural template for designing more potent phenazine-based fungicides.

Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

Abstract 5891: An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802

Abstract Resistance to targeted therapies is a major challenge in oncology. Genetic mechanisms of resistance such as gatekeeper mutations in the primary drug target can be readily identified. However, drug targets responsible for non-genetic resistance mechanisms such as transcriptional reprogramming remain elusive. Here we describe an innovative phenotypic screening approach that led to the development of the clinical candidate TT125-802 and identification of its target CBP/p300 which we discovered as a novel regulator of transcriptional resistance to a range of targeted therapies. Targeted cancer therapies have been reported to induce profound transcriptional reprogramming leading to drug resistance. For instance, inhibition of oncogenic MAPK signaling using a BRAF/MEK inhibitor combination in BRAF-V600E mutated melanoma, triggers expression of the pluripotency transcription factor SOX2 along with broader expression of associated stemness and EMT genes. We used this system as a model to screen for small molecules that prevent transcriptional escape mechanisms to targeted cancer therapies. For this, we developed an automated high-throughput immunofluorescence staining and analysis method of SOX2 as a surrogate marker for resistance-conferring transcriptional reprogramming. A hit-like high diversity library of 16’000 small molecules was screened using an image-based multi-parameter readout with an integrated counter-screen for general cell toxicity. A battery of downstream assessments including patentability, synthetic tractability, physico-chemical properties, and dose-dependent modulation of transcriptional resistance signatures was used to select the chemical scaffold TT125 for further hit to lead optimization. Hit to lead optimization was initially target agnostic. To de-orphan the scaffold, a chemical probe of TT125 was developed in house and used in a chemoproteomics assay. The transcriptional co-activators CBP/p300 were identified as the target of TT125. Biochemical assays revealed that the TT125 scaffold binds specifically to the bromodomain of CBP/p300 but not the histone acetyl transferase domain. Moreover, TT125 was confirmed to bind highly selectively to the bromodomain of CBP/p300 but not other bromodomain-containing proteins such as BRD2 or BRD4. Structure-based drug design along with in vivo PD models using transcriptional short-term readouts were used for lead optimization that resulted in the development of the potent and highly selective clinical candidate TT125-802. Validating our phenotypic approach to drug discovery and development, TT125-802 is currently being evaluated in a phase I oncology trial. The modular setup of our phenotypic screening system has the potential to power the discovery of additional novel modulators of transcriptional drug resistance. Citation Format: Dorothea Gruber, Charles-Henry Fabritius, Thomas Bohnacker, Martin Schwill, Sara Laudato, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Stefanie Flückiger-Mangual. An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5891.

Discovery and Characterization of Active CBP/EP300 Degraders Targeting the HAT Domain.

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that simultaneously bind an E3 ligase and a protein of interest, inducing degradation of the latter via the ubiquitin-proteasome system. Here we present the development of degraders targeting CREB-binding protein (CBP) and E1A-associated protein (EP300)-two homologous multidomain enzymes crucial for enhancer-mediated transcription. Our PROTAC campaign focused on CPI-1612, a reported inhibitor of the histone acetyltransferase (HAT) domain of these two proteins. A novel asymmetric synthesis of this ligand was devised, while PROTAC-SAR was explored by measuring degradation, target engagement, and ternary complex formation in cellulo. Our study demonstrates that engagement of Cereblon (CRBN) and a sufficiently long linker between the E3 and CBP/EP300 binders (≥21 atoms) are required for PROTAC-mediated degradation using CPI-1612 resulting in a new active PROTAC dCE-1. Lessons learned from this campaign, particularly the importance of cell-based assays to understand the reasons underlying PROTAC performance, are likely applicable to other targets to assist the development of degraders.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of the acetyl-coenzyme A competitive p300/CBP catalytic inhibitor A-485 in biological samples.

The small molecule A-485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic-adenosine monophosphate response element-binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A-485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC-tandem mass spectrometry method has been developed and validated to quantify A-485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A-485 utilized a reversed-phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A-485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A-485, the analysis method can be seamlessly transferred to human samples.

Abstract A047: Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers

Abstract E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed histone acetyl transferases (HAT). They act by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, c-Myc, PD-L1, Estrogen receptor (ER) and Androgen receptor (AR). CBP and p300 are multidomain proteins that harbour different functional units imperative for chromatin remodelling and transcription like Bromodomain (BD), Histone acetyl transferase (HAT) domain, KIX domain etc. These two closely related epigenetic modulators are known to play oncogenic role in a variety of cancers. Conventional CBP/p300 inhibitors targeting either BD, HAT or KIX domain do not discriminate between CBP and p300 proteins due to high homology. Because of the indiscriminate inhibition of both proteins, HAT domain inhibitors showed tolerability issues preventing their clinical advancement. In contrast, bromodomain inhibitors are better tolerated because they only block coactivation function at the transcription level without impacting the scaffolding or global histone acetylation function. They show less than desirable efficacy in various cancer models within very well tolerated doses. The implication of selective inhibition of either of these proteins in various cancer has been reported in the literature due to differential dependency on either of the paralogs. A synthetic lethal relationship between these paralogs in cancer set up has also been well established recently. However, none of the reported inhibitors of the various domains described above could selectively target CBP or p300 alone due to high sequence homology. Paralog selectivity in recent time has been proved to be possible with degrader approach due to differentiated ternary complex formation. Structure-guided modeling and iterative medicinal chemistry approaches were applied to identify paralog selective and potent first in class degraders with diverse linker length and linker composition. Identified compounds showed pronounced paralog selective degradation of target proteins and cellular effects in a panel of cell lines with loss of function mutation for either of the paralog proteins but not in wild type cells. The paralog selective lead degraders are currently being further optimized to identify development candidates. Selective degraders of CBP and p300 are expected to enhance efficacy of SOC drugs or immune checkpoint blockers by governing the acetylation and transcription of oncogenic factors and co-stimulatory molecules involved in tumor progression. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Krishna Chaitanya T, Achala Apte, Naveen Kumar R, Avinash Kumar, Dabbeeru Madhu Babu, Shwetha S, Aishwarya B Kamath, Monalisha Mandal, Rituparna Panigrahi, Sivapriya Marappan, Subhendu Mukherjee, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A047.

Novel Tip60 HAT activators as neuroepigenetic therapeutic modulators for Alzheimer’s Disease

AbstractBackgroundAlzheimer’s Disease (AD) etiology involves a complex interplay of genetics, age, and environmental factors that influence neuroepigenetic mediated neural gene control. Reduced histone acetylation in the AD brain is a widespread hallmark underlying neural gene repression and cognitive impairment in AD etiology. As histone acetylation is dynamically regulated by antagonistic histone deacetylases (HDACs) and histone acetyltransferases (HATs), pharmacological treatments aiming to restore histone acetylation via HDAC inhibition are a research hotspot for developing AD cognitive enhancing drugs. Although promising, HDAC inhibitor treatments in the AD animal models are rarely translated into clinical trials due to non‐specific global hyperacetylation. Alternatively, enhancing activity of specific HATs with non‐redundant functions serves as an exciting new therapeutic strategy that remains unexplored. To this end, our lab has identified and published a neuroprotective role for HAT Tip60 in multiple neural circuits impaired in AD pathology. Human and Drosophila AD brains exhibit reduced Tip60 and enhanced repressor HDAC2 that triggers histone deacetylation with concomitant repression of cognition genes. All these deficits are prevented by genetically increasing Tip60 in the Drosophila AD brain. Here, we test that pharmacologically enhancing Tip60’s HAT function replicates this AD neuroprotective function.MethodWe used in silico structural modeling and a novel pharmacophore based virtual screening approach to design and synthesize novel small molecule compounds strongly predicted to target and enhance Tip60’s HAT activity.ResultOur screen produced 19,000 hits that we individually docked on Tip60’s HAT domain using GOLD docking software. 13 compounds were identified that exhibited significantly high Tip60 docking scores, and optimal ease of synthesis, drug like properties and blood brain barrier peneteration. These compounds were custom synthesized. We show that several of these compounds fully rescue locomotor neural based deficits and cognition gene expression alterations in both Tip60 knockdown and AD Drosophila in vivo models with no observable side effects, underscoring their therapeutic effectiveness. These compounds will ultimately be assessed for further specificity and for their therapeutic effectiveness in AD animal models in vivo.ConclusionWe propose our first‐in‐class small molecule Tip60 HAT activators will serve as powerful novel chemical entities for development of specific acetylation‐based cognition enhancing drugs for AD.

Genetic Alterations of Follicular Lymphoma Can Predict Response to Very Low Dose Radiotherapy

Introduction Follicular lymphoma (FL) is typically indolent, but relapses and transformation to higher grade disease are common. FL is often radiosensitive and can show complete response (CR) even to very low dose radiotherapy (VLDRT, 4Gy). There are no known genetic markers of FL radiosensitivity. We sought to identify molecular signatures of FL radiosensitivity to aid in patient selection for VLDRT. Methods We analyzed our institutional database and identified 110 FL patients with 113 tumors treated with radiotherapy (RT) and preexisting MSK-IMPACT, a targeted exon sequencing panel. For each patient, we collected key clinicodemographic information including pre- and post-RT treatment history, prior large cell lymphoma, and Ann Arbor stage at RT. For treated tumors, we obtained tumor site, grade, diameter, maximum SUV, BCL2 translocation (trans) status through fluorescence in situ hybridization, RT dose and fractionation, and the treatment strategy ( i.e. treating all sites of disease vs a subsite of disease). Tumor site was dichotomized by pelvic tumors vs all others, as this was the most frequently altered site. The presence of mutant (mut) genes compared to wild type (WT) were associated with RT response, measured by PET scan within 6 months of RT, using logistic regression. Log-rank testing and Cox proportional hazards models were used to analyze local progression free survival (LPFS), censored at start of unplanned therapy, with 2-year (2y) survival reported. Multivariate modeling was used to adjust gene associations with outcomes, controlling for the clinicodemographic and tumor characteristics mentioned above. Results The patient and tumor characteristics are summarized in table 1. We found CREBBP to be the most frequently altered gene (66% of tumors). We identified 5 signatures of altered genes, most of which showed altered CREBBP and relatively long LPFS (e.g. one signature showed CREBBP mut, TNFRSF14 mut and IRF8/STAT6 mut with a 2y LPFS 78%); however, one signature with concurrent KMT2D mut and altered BCL2 including trans and mut had significantly shorter LPFS than all other signatures (2y LPFS 47%, p < 0.01) and lower odds of CR relative to the other signatures (p= 0.03). CREBBP mut was the only alteration associated with increased odds of CR (Odds ratio: 2.39 (95% CI: 1.06-5.37, p = 0.04), and this effect remained significant after adjusting for pelvic disease site (p=0.04). Mutations of BCL2, IRF8, and KMT2D were associated with LPFS using log-rank testing (p<0.01, p=0.02, and p=0.04, respectively). BCL2 mut or concurrent BCL2 mut and BCL2 trans (mut/trans) had shorter LPFS for the overall cohort (p<0.01 for both), while for VLDRT, only BCL2 mut/trans was associated with shorter LPFS (p=0.02). In the VLDRT cohort, we found that CREBBP histone acetyltransferase (HAT) mut was associated with improved LPFS compared to WT (n=39, 2y LPFS 74% vs 52%, HR:0.41 (95% CI: 0.18-0.93, p=0.03)), but this was not observed for tumors receiving >4Gy (n=44, HR: 1.27 (95% CI: 0.32-5.08), p=0.74)). We observed that either BCL2 mut, BCL2 trans, or BCL2 mut/trans and CREBBP HAT WT had a 2y LPFS of 13% whereas BCL2 mut, BCL2 trans or BCL2 mut/trans and CREBBP HAT mut had a 2y LPFS of 83% (p<0.01), and this relationship persisted on multivariate analysis (Figure 1). Conclusions Incorporating genetic signatures associated with radiosensitivity, and specifically alterations involving BCL2 and CREBBP, may independently improve patient selection for RT. CREBBP HAT domain mutations are potentially targetable and may have important implications for augmenting radiosensitivity to VLDRT.

Enhancer remodeling drives MLL oncogene-dependent transcriptional dysregulation in leukemia stem cells.

Acute myeloid leukemia (AML) with mixed-lineage leukemia (MLL) gene rearrangement (MLLr) comprises a cellular hierarchy in which a subpopulation of cells serves as functional leukemia stem cells (LSCs). They are maintained by a unique gene expression program and chromatin states, which are thought to reflect the actions of enhancers. Here, we delineate the active enhancer landscape and observe pervasive enhancer malfunction in LSCs. Reconstruction of regulatory networks revealed a master set of hematopoietic transcription factors. We show that EP300 is an essential transcriptional coregulator for maintaining LSC oncogenic potential because it controls essential gene expression through modulation of H3K27 acetylation and assessments of transcription factor dependencies. Moreover, the EP300 inhibitor A-485 affects LSC growth by targeting enhancer activity via histone acetyltransferase domain inhibition. Together, these data implicate a perturbed MLLr-specific enhancer accessibility landscape, suggesting the possibility for disruption of the LSC enhancer regulatory axis as a promising therapeutic strategy in AML.

Abstract 3841: Development of p300-targeting PROTAC degraders with enhanced selectivity and onset of degradation

Abstract CREB-binding protein (CBP, CREBBP, KAT3A) and E1A-binding protein (EP300, p300, KAT3B) are paralogous multi-domain proteins that act as chromatin regulators and transcriptional co-activators. They contain a histone acetyltransferase (HAT) domain that catalyzes the histone H3, lysine 27 acetylation (H3K27ac) mark at regulatory elements such as enhancers and promoters. Transcription factors associate with stretches of H3K27ac marks (known as ‘super-enhancer’ elements) and result in gene transcription that ultimately establishes cell identity and fate. They are implicated in cancer pathology, and small molecule inhibition of the bromodomain (BRD) or HAT domain of CBP/p300 are considered promising therapeutic strategies for a number of cancer types. CBP and p300 are highly homologous but have distinct roles that have to date been hard to delineate, since small molecule inhibitors developed to date are unable to selectively target each protein independently. Additionally, small molecule inhibitors that target individual domains are unable to entirely abrogate the full functionality of CBP/p300. A bromodomain-recruiting dual CBP/p300 PROTAC Degrader ‘dCBP1’ was therefore recently developed to provide a chemical tool to explore the phenotypic consequences of CBP/p300 chemical knockdown. A further study demonstrated that it is possible to degrade p300 with some selectivity by converting a CBP/p300 dual HAT-domain inhibitor into a PROTAC, called ‘JQAD1’. We have used a different HAT-domain recruiting ligand to develop novel PROTACs that elicit proteasome-mediated degradation of p300 with significantly enhanced selectivity over CBP, compared with JQAD1. We additionally demonstrate a faster onset of degradation for lead PROTAC molecules and present data exploring the consequences of selective p300 degradation in CIC-DIX4 sarcoma. Citation Format: Graham P. Marsh, Sean Goggins, Darko Bosnakovski, Michael Kyba, Samuel Ojeda, Drew A. Harrison, Christopher J. Ott, Hannah J. Maple. Development of p300-targeting PROTAC degraders with enhanced selectivity and onset of degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3841.

Discovery of DS-9300: A Highly Potent, Selective, and Once-Daily Oral EP300/CBP Histone Acetyltransferase Inhibitor

Histone acetylation is a post-translational modification of histones that is catalyzed by histone acetyltransferases (HATs) and plays an essential role in cellular processes. The HAT domain of EP300/CBP has recently emerged as a potential drug target for cancer therapy. Here, we describe the identification of the novel, highly potent, and selective EP300/CBP HAT inhibitor DS-9300. Our optimization efforts using a structure-based drug design approach based on the cocrystal structures of the EP300 HAT domain in complex with compounds 2 and 3 led to the identification of compounds possessing low-nanomolar EP300 HAT inhibitory potency and the ability to inhibit cellular acetylation of histone H3K27. Optimization of the pharmacokinetic properties in this series resulted in compounds with excellent oral systemic exposure, and once-daily oral administration of 16 (DS-9300) demonstrated potent antitumor effects in a castrated VCaP xenograft mouse model without significant body weight loss.

Molecular and functional characterization of an evolutionarily conserved CREB-binding protein in the Lymnaea CNS.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.

193 (PB073) - Orally Bioavailable CBP and p300 Selective Degraders for the Treatment of AR- and ER-dependent Cancers

E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed histone acetyl transferases (HAT) that act by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, c-Myc, Estrogen receptor (ER) and Androgen receptor (AR). CBP and p300 are multidomain proteins that harbour different functional units imperative for chromatin remodelling and transcription like Bromodomain (BD), Histone acetyl transferase (HAT) domain, KIX domain etc.

Structure-activity relationship and antitumor activity of 1,4-pyrazine-containing inhibitors of histone acetyltransferases P300/CBP

Acetylation of histone lysine residues by histone acetyltransferase (HAT) p300 and its paralog CBP play important roles in gene regulation in health and diseases. The HAT domain of p300/CBP has been found to be a potential drug target for cancer. Compound screening followed by structure-activity relationship studies yielded a novel series of 1,4-pyrazine-containing inhibitors of p300/CBP HAT with their IC50s as low as 1.4 μM. Enzyme kinetics and other studies support the most potent compound 29 is a competitive inhibitor of p300 HAT against the substrate histone. It exhibited a high selectivity for p300 and CBP, with negligible activity on other classes of HATs in human. Compound 29 inhibited cellular acetylation of several histone lysine residues and showed strong activity against proliferation of a panel of solid and blood cancer cells. These results indicate it is a novel pharmacological lead for drug development targeting these cancers as well as a useful chemical probe for biological studies of p300/CBP.

Dynamics of the Histone Acetyltransferase Lysine-Rich Loop in the Catalytic Core of the CREB-Binding Protein.

The tight control of transcriptional coactivators is a fundamental aspect of gene expression in cells. The regulation of the CREB-binding protein (CBP) and p300 coactivators, two paralog multidomain proteins, involves an autoinhibitory loop (AIL) of the histone acetyltransferase (HAT) domain. There is experimental evidence for the AIL engaging with the HAT binding site, thus interrupting the acetylation of histone tails or other proteins. Both CBP and p300 contain a domain of about 110 residues (called the bromodomain) that recognizes histone tails with one or more acetylated lysine side chains. Here, we investigate by molecular dynamics simulations whether the AIL of CBP (residues 1556-1618) acetylated at the side chain of Lys1595 can bind to the bromodomain. The structural instability and fast unbinding kinetics of the AIL from the bromodomain pocket suggest that the AIL is not a ligand of the bromodomain on the same protein chain. This is further supported by the absence of strong and persistent contacts at the binding interface. Furthermore, the simulations of unbinding show an initial fast detachment of the acetylated lysine and a slower phase necessary for complete AIL dissociation. We provide further evidence for the instability of the AIL intramolecular binding by comparison with a natural ligand, the histone peptide H3K56ac, which shows higher stability in the pocket.

Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

CREBBP Histone Acetyltransferase Domain Mutations Result in Dexamethasone Resistance in B-Progenitor Acute Lymphoblastic Leukemia

Genes encoding epigenetic regulators are commonly mutated at relapse in acute lymphoblastic leukemia, but the way in which such mutations promote drug resistance, and their potential for therapeutic targeting is largely unknown. We previously identified mutations in CREBBP, encoding CREBBP-binding protein, a histone and non-histone acetyl transferase and transcriptional coregulatorin relapsed ALL and non-Hodgkin lymphoma. In view of the known role of CREBBP in mediating the transcriptional response to glucocorticoids, we hypothesized that CREBBP mutations promote glucocorticoid resistance, and sought to comprehensively define the prevalence and spectrum of mutations in relapsed ALL, and elucidate their role in resistance.In 174 children with B- and T-ALL treated on St Jude Children's Hospital Total Therapy XIII-XV protocols, and with B-ALL treated on the Children's Oncology Group AALL0232 study of high risk B-ALL experiencing relapse within 5 years of diagnosis, CREBBP mutations were observed in 37 (21.2%) of patients, representing the most common target of sequence mutation at relapse. The mutations were either preserved from diagnosis to relapse, or evolved as new mutations or from minor clones to become clonal mutations at relapse. There were hotspots of mutation at R1446 and between P1488-P1494 in the histone acetyltransferase domain, residues critical for recognition of acetylation substrates. These mutations are known or predicted to impair histone acetylation in biochemical assays and in Crebbp-/- ; Ep300-/- mouse embryonic fibroblasts reconstituted with mutant CREBBP.To examine the role of CREBBP mutations in drug resistance, we generated isogenic CREBBP wild type and mutant human ALL cell lines, and conventional and conditional Crebbp Q1501P (=human Q1500) and R1447H (=human R1446) mouse strains. Mice bearing germline Q1501P or R1447H alleles exhibited dysmorphology, reduced fertility, lymphoproliferation and reduced lifespan. This phenotype was most pronounced in Crebbp R1447 mice preventing establishment of a stable line, thus we generated a conditional Crebbp -R1447H knockin strain (CREBBP-R1447H-CKI) that expresses CREBBP R1447H following Cre-mediated recombination.We generated BCR-ABL1 pre-B ALL models by transducing E13.5 fetal liver cells with BCR-ABL1 p185 retrovirus. Consistently, leukemias derived from heterozygous or homozygous CREBBP-Q1501P or heterozygous CREBBP-R1447H-CKI mice showed profound and selective resistance to prednisolone and dexamethasone. Similarly, NALM6 (DUX4/ERG) human ALL cells harboring CREBBP mutations induced by genome editing (NALM6-C001) also showed complete resistance to glucocorticoids, without change in expression of the glucocorticoid receptor (GR). Chromatin immunoprecipitation and sequencing showed that this was accompanied by marked attenuation of recruitment of GR to chromatin, attenuation of histone 3 lysine 18 (H3K18) and H3K27 acetylation, and reduced expression of H3K18Ac-marked genes following dexamethasone treatment. Global acetylome profiling showed that CREBBP mutations resulted in perturbed acetylation of multiple histone and non-histone targets, including CREBBP itself, IKZF1 and MYC, with reciprocal increase in EP300 acetylation.To identify agents that kill CREBBP mutant leukemic cells and that may reverse steroid resistance, we screened NALM6-C001 with a collection of 11,293 chemicals (5,832 unique compounds enriched for FDA-approved drugs) dosed at 10 uM in the presence or absence of dexamethasone for 48 or 96 hrs. 223 active compounds and 220 compounds targeting epigenetic modifications were tested in a 10-point dose response screen. Seventeen compounds with IC50 <10 nM were identified, including GNE-049, a selective CREBBP/EP300 bromodomain inhibitor (IC50 1.8nM). Notably, this compound showed reduced activity in CREBBP wild-type NALM6 cells. Moreover, GNE-049 restored dexamethasone responsiveness of CREBBP-R1447H-CKI leukemic cells, which were insensitive to steroids or GNE-049 when either drug was used alone.These results demonstrate the high prevalence of CREBBP in relapsed ALL, and show that these mutations have a central role in driving glucocorticoid resistance. These data also suggest that these mutations create a therapeutic vulnerability through EP300 acetylation that may be exploited by bromodomain inhibition. DisclosuresWoessner:Boerhinger Ingelheim: Employment. Hunger:Jazz Pharmaceuticals: Honoraria; Amgen: Consultancy, Equity Ownership; Erytech Pharmaceuticals: Consultancy; Novartis: Consultancy. Gascoigne:Roche: Equity Ownership; Genentech: Employment. Mullighan:Amgen: Consultancy; Loxo Oncology: Research Funding.

Carnosol Is a Novel Inhibitor of p300 Acetyltransferase in Breast Cancer.

Carnosol, a natural polyphenol abundant in edible plants such as sage, rosemary, and oregano, has shown promising anticancer activity against various types of cancers. Nonetheless, very little is known about its molecular mechanism of action or its downstream target(s). We have previously shown that carnosol inhibits cellular proliferation, migration, invasion, and metastasis as well as triggers autophagy and apoptosis in the highly invasive MDA-MB-231 breast cancer cells. Here, we report that carnosol induces histone hypoacetylation in MDA-MB-231 and Hs578T breast cancer cells. We show that, while carnosol does not affect HDACs, it promotes a ROS-dependent proteasome degradation of p300 and PCAF histone acetyl transferases (HATs) without affecting other HATs such as GCN5 and hMOF. Carnosol-induced histone hypoacetylation remains persistent even when p300 and PCAF protein levels were rescued from degradation by (i) the inhibition of the proteasome activity by the proteasome inhibitors MG-132 and bortezomib, and (ii) the inhibition of ROS accumulation by the ROS scavenger, N-acetylcysteine. In addition, we report that, in a cell-free system, carnosol efficiently inhibits histone acetyltransferase activity of recombinant p300 but not that of PCAF or GCN5. Molecular docking studies reveal that carnosol inhibits p300 HAT activity by blocking the entry of the acetyl-CoA binding pocket of the catalytic domain. The superimposition of the docked conformation of the p300 HAT domain in complex with carnosol shows a similar orientation as the p300 structure with acetyl-CoA. Carnosol occupies the region where the pantetheine arm of the acetyl-CoA is bound. This study further confirms carnosol as a promising anti-breast cancer therapeutic compound and identifies it as a novel natural p300 inhibitor that could be added to the existing panel of inhibitors.