KAT Activity Research Articles

The small inhibitor WM-1119 effectively targets KAT6A-rearranged AML, but not KMT2A-rearranged AML, despite shared KAT6 genetic dependency

BackgroundThe epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors’ self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs.MethodsWe investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs.ResultsWe found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML.ConclusionOur study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs.

P300/CBP KATs Are Critical for Maturation and Differentiation of Adult Neural Progenitors.

Epigenetic modifications play a pivotal role in the process of neurogenesis. Among these modifications, reversible acetylation fine-tunes gene expression for both embryonic and adult neurogenesis. The CBP/KAT3A and its paralogue p300/KAT3B are well-known lysine acetyltransferases with transcriptional coactivation ability that engage in neural plasticity and memory. The exclusive role of their KAT activity in neurogenesis and memory could not be addressed due to the absence of a p300/CBP modulator, which can cross the blood-brain barrier. Previous work from our laboratory has shown that a small molecule activator, TTK21, specific to CBP/p300, when conjugated to glucose-derived carbon nanospheres (CSP), is efficiently delivered to the mouse brain and could induce dendritic branching and extend long-term memory. However, the molecular mechanisms of p300 acetyltransferase activity-dependent enhanced dendritogenesis are yet to be understood. Here, we report that CSP-TTK21 treatment to primary neuronal culture derived from mouse embryo enhances the expression of five critical genes: Neurod1 (central nervous system development), Tubb3 (immature neural marker), Camk2a (synaptic plasticity and LTP), Snap25 (spine morphogenesis plasticity), and Scn2a (propagation of the action potential). Activation of these genes by inducing the p300/CBP KAT activity presumably promotes the maturation and differentiation of adult neuronal progenitors and thereby the formation of long and highly branched doublecortin-positive functional neurons in the subgranular zone of the dentate gyrus.

Abstract A062 Therapeutic targeting of the SAGA KAT module impairs MYCN-amplified neuroblastoma growth through reduction of the MYCN oncogenic gene expression program

Abstract Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. While direct targeting of transcription factors is difficult, one method to circumvent this problem is to target co-factors and chromatin complexes that are necessary for their oncogenic function. To evaluate protein complex dependencies enriched in MYCN-amplified neuroblastoma, a disease of dysregulated development driven by aberrant expression of an oncogenic expression program, we curated a list of protein complexes using the CORUM database and mined the Dependency Map using single sample gene set enrichment analysis. This analysis identified the KAT module of the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as a selective dependency in MYCN-amplified neuroblastoma. Loss of SAGA complex activity in neuroblastoma through both genetic knockout and induced protein degradation of the lysine acetyltransferase (KAT) module subunit TADA2B resulted in a global reduction of histone lysine acetylation and impaired neuroblastoma cell growth. Genetic knockout of TADA2B did not impair the growth of pediatric sarcoma cell lines, suggesting that SAGA loss-of-function is not broadly deleterious. Furthermore, SAGA loss-of-function reduced MYCN binding to chromatin, resulting in suppression of the MYCN-driven gene expression program and impaired cell cycle progression. Importantly, the SAGA complex is pharmacologically targetable in vitro and in vivo with GSK-699, a PROTAC that induces proteolysis of both KAT2A and KAT2B paralogs. As observed with genetic knockout studies, loss of SAGA activity through KAT2A/B degradation reduced the MYCN-driven transcriptional signature and impaired cell cycle progression. Our findings expand our understanding of the chromatin complexes that maintain the oncogenic transcriptional state in MYCN-amplified neuroblastoma and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma. Citation Format: Nathaniel W. Mabe, Clare F. Malone, Alexandra B. Forman, Gabriela Alexe, Kathleen L. Engel, Ying-Jiun C. Chen, Melinda Soeung, Silvi Salhotra, Allen Basanthakumar, Bin Liu, Sharon YR Dent, Kimberly Stegmaier. Therapeutic targeting of the SAGA KAT module impairs MYCN-amplified neuroblastoma growth through reduction of the MYCN oncogenic gene expression program [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A062.

The KAT module of the SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma.

Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.

Biological Domain Network Analysis Identifies Potential Triadic Interaction between Mitochondria, Nuclear Epigenetics, and Synaptic Viability

AbstractBackgroundThe AD endophenotypes initially described in the Common Alzheimer’s Disease Research Ontology (CADRO) were augmented and transformed into computationally implementable definitions for 19 discrete Biological Domains. These AD biological domains were employed in the development of molecular interaction networks depicting the signaling and protein‐protein interaction as a central bioinformatics resource for the TREAT‐AD consortium.Method19 AD biological domains were identified by iterative literature culling and model testing based on gene set enrichment analysis and gene ontology term identification. All 19 biological domains are defined by an exhaustive set of GO terms, allowing scientists everywhere to use the same criteria to define their large datasets in an unbiased manner. Network models were constructed from the leading‐edge genes from all AD‐risk enriched GO terms within each biological domain. The network models incorporate data from over 1700 transcriptomic brain studies and 1100 proteomic brains studies examining shifts in gene expression. These are employed as a reference tool for the formulation of specific hypotheses.ResultThe analysis of mitochondrial gene expression changes at both the transcriptomic and proteomic level identify deficits in both mitochondrial energy production and metabolism along with a decrease in acetyl‐CoA production, a necessary element for many reactions including histone acetylation. This suggests that mitochondrial metabolic tone may modify epigenetic regulation through lysine acetyltransferase (KATs) independently from ATP production. Examination of decrements in KAT activity from other model systems suggest a particularly robust impact on synaptic gene expression, implying a potential interaction between mitochondrial metabolism and synaptic gene expression independent of energy production. Intriguingly, some elements of a protein module identified by TREAT‐AD large‐scale deep proteomic analysis found to be most tightly associated with AD pathology and cognitive decline, are tightly associated with this model.ConclusionThe biological domains were constructed as a computational resource to facilitate hypothesis generation within the TREAT‐AD consortium, and here are implemented to highlight one emerging interaction between domains that may imply new mechanisms of disease regulation coupling between mitochondria and synaptic viability through shifts in epigenetic regulation.

Biological Domain Network Analysis Identifies Potential Triadic Interaction between Mitochondria, Nuclear Epigenetics, and Synaptic Viability

AbstractBackgroundThe AD endophenotypes initially described in the Common Alzheimer’s Disease Research Ontology (CADRO) were augmented and transformed into computationally implementable definitions for 19 discrete Biological Domains. These AD biological domains were employed in the development of molecular interaction networks depicting the signaling and protein‐protein interaction as a central bioinformatics resource for the TREAT‐AD consortium.Method19 AD biological domains were identified by iterative literature culling and model testing based on gene set enrichment analysis and gene ontology term identification. All 19 biological domains are defined by an exhaustive set of GO terms, allowing scientists everywhere to use the same criteria to define their large datasets in an unbiased manner. Network models were constructed from the leading‐edge genes from all AD‐risk enriched GO terms within each biological domain. The network models incorporate data from over 1700 transcriptomic brain studies and 1100 proteomic brains studies examining shifts in gene expression. These are employed as a reference tool for the formulation of specific hypotheses.ResultThe analysis of mitochondrial gene expression changes at both the transcriptomic and proteomic level identify deficits in both mitochondrial energy production and metabolism along with a decrease in acetyl‐CoA production, a necessary element for many reactions including histone acetylation. This suggests that mitochondrial metabolic tone may modify epigenetic regulation through lysine acetyltransferase (KATs) independently from ATP production. Examination of decrements in KAT activity from other model systems suggest a particularly robust impact on synaptic gene expression, implying a potential interaction between mitochondrial metabolism and synaptic gene expression independent of energy production. Intriguingly, some elements of a protein module identified by TREAT‐AD large‐scale deep proteomic analysis found to be most tightly associated with AD pathology and cognitive decline, are tightly associated with this model.ConclusionThe biological domains were constructed as a computational resource to facilitate hypothesis generation within the TREAT‐AD consortium, and here are implemented to highlight one emerging interaction between domains that may imply new mechanisms of disease regulation coupling between mitochondria and synaptic viability through shifts in epigenetic regulation.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

P300/CBP Regulates HIF-1-Dependent Sympathetic Activation and Hypertension by Intermittent Hypoxia.

Obstructive sleep apnea (OSA), a widespread breathing disorder, leads to intermittent hypoxia (IH). Patients with OSA and IH-treated rodents exhibit heightened sympathetic nerve activity and hypertension. Previous studies reported transcriptional activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) by HIF-1 (hypoxia-inducible factor-1) contribute to autonomic dysfunction in IH-treated rodents. Lysine acetylation, regulated by KATs (lysine acetyltransferases) and KDACs (lysine deacetylases), activates gene transcription and plays an important role in several physiological and pathological processes. This study tested the hypothesis that acetylation of HIF-1α by p300/CBP (CREB-binding protein) (KAT) activates Nox transcription, leading to sympathetic activation and hypertension. Experiments were performed on pheochromocytoma-12 cells and rats treated with IH. IH increased KAT activity, p300/CBP protein, HIF-1α lysine acetylation, HIF-1 transcription, and HIF-1 binding to the Nox4 gene promoter in pheochromocytoma-12 cells, and these responses were blocked by CTK7A, a selective p300/CBP inhibitor. Plasma norepinephrine (index of sympathetic activation) and blood pressures were elevated in IH-treated rats. These responses were associated with elevated p300/CBP protein, HIF-1α stabilization, transcriptional activation of Nox2 and Nox4 genes, and reactive oxygen species, and all these responses were absent in CTK7A-treated IH rats. These findings suggest lysine acetylation of HIF-1α by p300/CBP is an important contributor to sympathetic excitation and hypertension by IH.

MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins

Changes in the transcriptional machinery cause aberrant self-renewal of non-stem hematopoietic progenitors. AF10 fusions, such as CALM-AF10, are generated via chromosomal translocations, causing malignant leukemia. In this study, we demonstrate that AF10 fusion proteins cause aberrant self-renewal via ENL, which binds to MOZ/MORF lysine acetyltransferases (KATs). The interaction of ENL with MOZ, via its YEATS domain, is critical for CALM-AF10-mediated leukemic transformation. The MOZ/ENL complex recruits DOT1L/AF10 fusion complexes and maintains their chromatin retention via KAT activity. Therefore, inhibitors of MOZ/MORF KATs directly suppress the functions of AF10 fusion proteins, thereby exhibiting strong antitumor effects on AF10 translocation-induced leukemia. Combinatorial inhibition of MOZ/MORF and DOT1L cooperatively induces differentiation of CALM-AF10-leukemia cells. These results reveal roles for the MOZ/ENL complex as an essential recruiting factor of the AF10 fusion/DOT1L complex, providing a rationale for using MOZ/MORF KAT inhibitors in AF10 translocation-induced leukemia.

Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.

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.

Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity.

Proteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 toglutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs atdifferent lysine residues, regulates stability of Fe65. Thisis the first report describing a role for acetylation intheregulation of Fe65 transcriptional activity, with Tip60being the only KAT tested that supports AFT spot formation.

Role of histone acetyltransferase inhibitors in cancer therapy.

The development of cancer is a complex phenomenon driven by various extrinsic as well as intrinsic risk factors including epigenetic modifications. These post-translational modifications are encountered in diverse cancer cells and appear for a relatively short span of time. These changes can significantly affect various oncogenic genes and proteins involved in cancer initiation and progression. Histone lysine acetylation and deacetylation processes are controlled by two opposing classes of enzymes that modulate gene regulation either by adding an acetyl moiety on a histone lysine residue by histone lysine acetyltransferases (KATs) or via removing it by histone deacetylases (KDACs). Deregulated KAT activity has been implicated in the development of several diseases including cancer and can be targeted for the development of anti-neoplastic drugs. Here, we describe the predominant epigenetic changes that can affect key KAT superfamily members during carcinogenesis and briefly highlight the pharmacological potential of employing lysine acetyltransferase inhibitors (KATi) for cancer therapy.

Bioorthogonal Chemical Probes of Lysine Acetyltransferases

Acetylation of lysine residues is one of the most important posttranslational modifications that diversify protein functions. Thousands of acetylated lysine residues have been identified, suggesting that the effects of lysine acetyltransferases (KATs) go far beyond the chromatin kingdom. However, there is a missing link connecting the compositions of the cellular acetylome networks to the enzymatic activities of different KAT members. The outstanding challenge is how to dissect the subacetylomes of individual KATs and address their functions on the proteomic scale. We have explored a bioorthogonal profiling of protein acetylation strategy to label and interrogate substrates of KAT enzymes. In this strategy, a suite of Ac‐CoA analogs containing either alkynyl or azido functional group (e.g. 3AZ‐CoA, 4AZ‐CoA, 4PY‐CoA, 5HY‐CoA, 6HY‐CoA) were synthesized as potential cofactor surrogate for selective labeling of KAT substrates. Meanwhile, the active site of the KATs was engineered in order to expand the cofactor binding capability of the enzymes to accommodate the bulkier synthetic cofactors. The acylated substrates can be selectively linked through the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction with fluorescent reporter or biotin affinity tag for optical imaging or protein enrichment on streptavidin‐coated resin. We have successfully used this bioorthogonal technology to profile substrates of p300 and GCN5 in the context of complex cellular proteomes. Together with LC‐MS/MS technology, more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. Overall, we have invented a bioorthogonal, chemoproteomic technology to investigate KAT biology. This chemical biology strategy provides a powerful enabling technology for activity‐based lysine acylation profiling on proteomic scale. Indeed, our KAT activity profiling demonstrates extensive engagement of KATs in cellular pathways and provides new molecular insights into understanding their functions in epigenetics and other biological processes.bioorthogonal profilingFigure 1

The flip side of sirtuins: the emerging roles of protein acetyltransferases in aging.

Protein N-ε-lysine acetylation is is an important post-translational modification that plays critical roles in the regulation of many cellular processes. A role for this modification in the process of aging goes back two decades to the discovery that the yeast NAD+-dependent histone deacetylase Sir2 regulates lifespan in yeast. While the Sirtuin family of protein deacetylases has been intensively studied in many model systems and is definitively linked to aging, the enzymes responsible for protein acetylation, protein acetyltransferases (KATs), have not received a similar level of attention. However, a series of recent studies have directly explored the role of specific KATs in aging. These studies have shown that modulation of KAT activity can influence cellular pathways important for aging and directly effect organismal lifespan.

Kynurenine Aminotransferases I, II and III Are Present in Saliva.

Fluids of the human body such as serum, cerebrospinal fluid and saliva contain a wide variety of proteins. Because kynurenic acid (KYNA) has been detected in human saliva, we wondered if KYNA could be produced in saliva by KYNA-synthesising enzymes, namely the kynurenine aminotransferases KAT I, KAT II and KAT III. Thirty samples of human saliva from control volunteers were investigated. KAT activity was measured in the presence of 1 mM pyruvate and 2 µM or 100 µM L-kynurenine and KYNA production was assessed by high-performance liquid chromatography. Saliva dose- and time-dependently produced KYNA. KAT activity ranged between 900 and 1050 pmol/mg protein/h: 900 for KAT I, 950 for KAT III and 1050 for KAT II. KYNA was synthesised in saliva at a physiological concentration of 2 µM L-kynurenine and at a higher concentration of 100 µM. Investigation of the distributions of the enzymes in saliva revealed that KAT I, KAT II and KAT III activity in a centrifuge-obtained pellet ranged from ~100% to 120%; in the supernatant, the percentage was between 0% and 20%. We observed a nonsignificant tendency for lower KAT activity in women's saliva than in men's. KATs present in saliva were sensitive to the GABA-transaminase inhibitor γ-acetylenic GABA, with a concentration of 100 µM γ-acetylenic GABA significantly blocking the formation of KYNA (50% of control, p < 0.05). Furthermore, KATs in saliva were sensitive to anti-dementia drugs, such as D-cycloserine and cerebrolysin, in an in vitro study. Our data revealed for the first time the presence of KAT I, KAT II and KAT III proteins in human saliva. KAT activity was found mostly in pelleted cells, suggesting their presence in salivary gland cells. KAT proteins in saliva are sensitive to drugs blocking KYNA formation. Our data indicate the presence of cells in saliva involved in the biochemical machinery of the kynurenine pathway. Their role in the digestive process remains to be clarified. We speculate that modulation of KYNA formation in the mouth by food and/or drugs might affect glutamate neurotransmission and cholinergic activity in the CNS and/or periphery and play a role under physiological as well as pathological conditions.

Characterization of Lysine Acetyltransferase Activity of Recombinant Human ARD1/NAA10.

Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its’ role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.

Regulation of epigenetic state by non-histone chromatin proteins and transcription factors: Implications in disease

Besides the fundamental components of the chromatin, DNA and octameric histone, the non-histone chromatin proteins and non-coding RNA play a critical role in the organization of functional chromatin domains. The non-histone chromatin proteins therefore regulate the transcriptional outcome in both physiological and pathophysiological state as well. They also help to maintain the epigenetic state of the genome indirectly. Several transcription factors and histone interacting factors also contribute in the maintenance of the epigenetic states, especially acetylation by the induction of autoacetylation ability of p300/CBP. Alterations of KAT activity have been found to be causally related to disease manifestation, and thus could be potential therapeutic target.

EP300 Suppresses Leukemia Development in Myelodysplastic Syndrome through Myb Repression

Background Epigenetic dysregulation is a hallmark feature of myelodysplastic syndrome (MDS) as epigenetic regulator genes, such as ASXL1, TET2, and SRSF2, are highly mutated in MDS patients. Tet2 (ten-eleven translocation-2, a methylcytosine deoxygenase) mutations are found in ~20% of MDS patients; they are also seen in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Loss of Tet2 in hematopoietic cells leads hematopoietic defects including enhanced hematopoietic stem cell (HSC) self-renewal, myeloid expansion and an increased propensity to develop MDS or AML. The lysine acetyltransferase (KAT) p300 (encoded by the EP300 gene) plays pivotal roles in essential cellular functions including proliferation, differentiation and signal transduction. However, the function of p300 in hematopoietic malignancies is clearly cell-context dependent, as we have shown that p300 functions as an oncogene in AML1-ETO-driven AML, and a tumor suppressor gene in NUP98-HOXD13-driven MDS. The EP300 and the related CREBBP genes are infrequently mutated in MDS patients. Methods To investigate the role of p300 in MDS driven by lack of Tet2, we generated a mouse model carrying a hematopoietic-specific EP300 conditional knockout on a Tet2-null background and characterized the effect of p300 loss on HSC function and the pathogenesis of MDS and AML. We also explored the therapeutic potential of manipulating p300 KAT activity to affect the course of MDS driven by Tet2 loss. Results Compared to the Tet2-/- mice, the EP300Δ/ΔTet2-/- (DKO) mice had shortened survival and an accelerated onset of AML, with increased blasts in the bone marrow and peripheral blood, anemia, splenomegaly, and universal presence of granulocytic sarcomas, demonstrating that depletion of p300 enhances the leukemogenicity of Tet2-/- HSC. The DKO mice showed enhanced hematopoietic stem/progenitor cell (HSPC, defined as Lin-Sca1+Kit+ cell) proliferation and an increased HSPC pool only 2 weeks after p300 deletion, far in advance of the development of any disease manifestations. In contrast, deletion of p300 in wild type (wt) mice affected neither HSPC proliferation, nor pool size. To further define the mechanisms by which these two genetic events cooperate to drive the development of MDS/AML, we assessed the DNA methylation status and gene expression signatures of HSPCs isolated from wt, Tet2-/- and DKO mice. As expected, loss of Tet2 leads to a genome-wide decrease in 5-hmC, whereas loss of p300 does not change 5-hmC distribution across the genome. Interestingly, DKO HSPCs showed local gains of 5-hmC at a variety of genes (compared to Tet2-null HSPCs), including at the c-Myb gene, which is a known oncogenic driver of AML. Gene Set Enrichment Analysis (GSEA) revealed that p300 loss in Tet2-/- HSPCs interferes with leukocyte differentiation, enhances proliferation and inhibits apoptosis. ChIP-X Enrichment Analysis (ChEA) further also indicated that among the differentially expressed genes, between DKO HSPCs and Tet2-null HSPCs, are potential Myb target genes. Given this data and the RNA-Seq and Q-PCR results that showed elevated levels of Myb mRNA in the DKO HSPCs compared to the Tet2-/- HSPCs, we examined the biological effects of knocking down (KD) Myb. Myb KD reduced the colony-forming capacity of the DKO HSPCs, but not the Tet2 -/- HSPCs, identifying its potential role in mediating the effect of p300 on MDS prone HSPCs. These results indicate that loss of p300 in Tet2-null HSPCs enhances their leukemogenicity through elevated Myb expression and activity. To determine whether increasing p300 KAT activity could have an opposite effect on MDS cells, we utilized I-CBP112, which binds p300 and increases its KAT activity and ability to stimulate H3K18 acetylation. Exposure to I-CBP112 did in fact eliminate the indefinite self-renewal capacity of Tet2-/- HSPCs, as measured by serial replating assays, further highlighting the importance of p300 KAT activity in regulating the behavior of Tet2-/- HSPCs. Additional in vivo studies of the effects of I-CBP112 on the course of MDS/AML driven by the absence of Tet2 are ongoing. Conclusions This work clearly indicates that the EP300 gene can play a tumor suppressor role in MDS/AML driven by loss of Tet2- by repressing Myb expression and activity. It also suggests that the enhancement of p300's KAT activity by use of small molecules, could be an effective therapeutic strategy for MDS/AML. Disclosures Cole: Abbvie: Consultancy; Acylin Therapeutics: Equity Ownership.

Bioorthogonal Reporters for Detecting and Profiling Protein Acetylation and Acylation.

Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl-coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.