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Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

[This article][1] ([1][2]) has been retracted at the request of the editors. Following an institutional review by Peter MacCallum Cancer Center (Victoria, Australia), the primary affiliation for several of the authors, it was not possible to confidently identify and assign the primary datasets to

In human papillomavirus (HPV)-infected cells, the p53 tumor suppressor is tightly regulated by the HPV-E6-E6AP complex, which promotes it for proteasomal degradation. We previously demonstrated that c-Abl tyrosine kinase protects p53 from HPV-E6-E6AP complex-mediated ubiquitination and degradation under stress conditions. However, the underlying mechanism was not defined. In this study, we explored the possibility that c-Abl targets E6AP and thereby protects p53. We demonstrated that c-Abl interacts with and phosphorylates E6AP. We determined that the E3 ligase activity of E6AP is impaired in response to phosphorylation by c-Abl. We mapped the phosphorylation site to tyrosine 636 within the HECT catalytic domain of E6AP, and using substitution mutants, we showed that this residue dictates the E3 ligase activity of E6AP, in a substrate-specific manner. On the basis of the crystal structure of the HECT domain of E6AP, we propose a model in which tyrosine 636 plays a regulatory role in the oligomerization of E6AP, which is a process implicated in its E3 ubiquitin ligase activity. Our results suggest that c-Abl protects p53 from HPV-E6-E6AP complex-mediated degradation by phosphorylating E6AP and impairing its E3 ligase activity, thus providing a molecular explanation for the stress-induced protection of p53 in HPV-infected cells.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

Recently, we showed that the ubiquitin ligase E6AP stabilizes β-catenin and activates its transcriptional activity. These activities were enhanced by the human papillomavirus (HPV) E6 protein. In the present study, we explored the function of E6AP, which increases β-catenin stabilization and transcriptional activation. Here, we report that E6AP interacts with β-catenin and mediates its nonproteolytic ubiquitylation, as evidenced in transiently transfected cell-based and in vitro reconstitution ubiquitylation assays. Overexpression of E6AP increased β-catenin polyubiquitylation and, consistent with that, knockdown or knock-out of E6AP expression reduced β-catenin polyubiquitylation. The ubiquitylation of β-catenin by E6AP was dependent on its E3 ubiquitin ligase activity, but it was proteasome-independent and did not require HPV-E6, phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK3β) or activity of the β-catenin ‘destruction complex’. We also show that transcriptional activation of β-catenin by E6AP is coupled with β-catenin protein stabilization, but not its ubiquitylation. In contrast to β-catenin ubiquitylation, β-catenin protein stability and its transcriptional activity were absolutely dependent on the activity of the destruction complex and phosphorylation by GSK3β. Collectively, our data uncover a dual role for E6AP in the regulation of β-catenin ubiquitylation, stability and transcriptional activity, with HPV-E6 enhancing only part of E6AP activities.

Hydroxylated fatty acids are valuable compounds in the chemical industry, being used in various products. ω-Hydroxylated fatty acids are versatile oleochemicals that can be used in polyester and polyamide production. Although a significant number of conventional synthetic chemistry production routes have been developed for ω-hydroxy acids, considerable challenge relates to their production in price-competitive, renewable, and sustainable production systems. The development and utilisation of biological systems for ω-hydroxy acid production may allow the transition of these compounds from specialty chemical to commodity material. The viability of biological routes to ω-hydroxylated fatty acids is dependent on the identification of enzymes and systems capable of such modifications. Cytochrome P450 monooxygenases are currently the only characterised enzymes known to ω-hydroxylate fatty acids and yeast provides valuable systems to perform the ω-hydroxylation biotransformations.

SWI/SNF chromatin-remodeling complexes have crucial roles in transcription and other chromatin-related processes. The analysis of the two members of this class in Saccharomyces cerevisiae, SWI/SNF and RSC, has heavily contributed to our understanding of these complexes. To understand the in vivo functions of SWI/SNF and RSC in an evolutionarily distant organism, we have characterized these complexes in Schizosaccharomyces pombe. Although core components are conserved between the two yeasts, the compositions of S. pombe SWI/SNF and RSC differ from their S. cerevisiae counterparts and in some ways are more similar to metazoan complexes. Furthermore, several of the conserved proteins, including actin-like proteins, are markedly different between the two yeasts with respect to their requirement for viability. Finally, phenotypic and microarray analyses identified widespread requirements for SWI/SNF and RSC on transcription including strong evidence that SWI/SNF directly represses iron-transport genes.

Metastatic disease is the major cause of breast cancer–related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganization to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonize distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonization, and metastasis in mice, and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for basal breast cancer. E6AP regulates actin cytoskeletal remodeling via regulation of Rho GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodeling through the control of ECT2 and Rho GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer. Cancer Res; 76(14); 4236–48. ©2016 AACR.

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery campaign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening campaign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.

Increased expression or dysregulation of protein arginine methyltransferase 5 (PRMT5) activity is associated with poor prognosis in many cancers. Through increased methylation of epigenetic and non-epigenetic targets, the aberrant activity of PRMT5 has been associated with many pro-tumourigenic cellular changes such as, increased levels of protein synthesis, dysregulation of cell cycle, cellular adaptation to hypoxic conditions, and suppression of normal cell death pathways. Genetic studies suggest that suppression of PRMT5 activity can reverse many of these pro-tumourigenic effects making PRMT5 an attractive drug discovery target. We screened a library of 350,000 lead-like compounds with a biochemical assay measuring the methylation of a histone H4 peptide by the recombinant human PRMT5/MEP50 complex. Biochemical and biophysical profiling of the inhibitory compounds indicated that several distinct binding modes were exhibited by the different chemical scaffolds. Inhibitors displayed competitive, noncompetitive or uncompetitive interactions with respect to S-adenosyl methionine and the peptide substrate. Medicinal chemistry developed several classes of potent, highly selective inhibitors of PRMT5 methyltransferase activity from the hit set. The optimised tool compound, CTx-034, is a potent inhibitor of PRMT5 methyl transferase activity (KD = 2 nM), which is highly selective (>100-fold) versus a panel of 18 methyltransferases (including 6 PRMT family members), 11 lysine demethylases, and 15 safety related targets (GPCRs, ion channels, enzymes). Treatment of cancer cell lines with CTx-034 reduces cellular levels of symmetrically dimethylated H4 Arginine 3 (H4R3me2s), in a dose dependent manner (IC50 = 4 nM) to levels undetectable by Western blot. Furthermore, within this chemical series the ability of compounds to reduce cellular levels of H4R3me2s closely correlates with PRMT5 inhibitory activity supporting PRMT5 as the cellular target of these compounds, and suggesting that PRMT5 is the major writer of this histone mark in many cancer cell lines. CTx-034 also inhibits the symmetric dimethylation of arginine on other histone and non-histone cellular substrates of PRMT5, including H3R2me2s and SmD1. Conversely, CTx-034 treatment does not reduce levels of H4R3 asymmetric dimethylation, a histone mark catalysed by PRMT1. Finally, CTx-034 has good oral bioavailability and pharmacokinetic properties in rodents and twice-daily dosing (10 - 100 mg/kg) over 10-14 days produces a dose dependent reduction of the H4R3me2s mark in bone marrow cells and peripheral white blood cells. This treatment is well tolerated by the mice, with no significant reduction in body weight or changes in haematological parameters observed. CTx-034 provides an excellent tool compound for cellular and in vivo proof of concept studies. Citation Format: Hendrik Falk, Richard C. Foitzik, Elizabeth Allan, Melanie deSilva, Hong Yang, Ylva E. Bozikis, Marica Nikac, Scott R. Walker, Michelle A. Camerino, Ben J. Morrow, Alexandra E. Stupple, Rachel Lagiakos, Jo-Anne Pinson, Romina Lessene, Wilhelmus JA Kersten, Danny G. Ganame, Ian P. Holmes, Gill E. Lunniss, Matthew Chung, Stefan J. Hermans, Michael W. Parker, Alison Thistlethwaite, Karen White, Susan A. Charman, Brendon J. Monahan, Patricia Pilling, Julian Grusovin, Thomas S. Peat, Stefan Sonderegger, Emma Toulmin, Stephen M. Jane, David J. Curtis, Paul A. Stupple, Ian P. Street. PRMT5 inhibitors as novel treatment for cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2015-5371

Ammonium and the analogue methylammonium are taken into the cell by active transport systems which constitute a family of transmembrane proteins that have been identified in fungi, bacteria, plants, and animals. Two genes from Aspergillus nidulans , mepA and meaA , which encode ammonium transporters with different affinities have been characterized. The MepA transporter exhibits the highest affinity for methylammonium ( K m 0 , 44.3 μM); in comparison, the K m for MeaA is 3.04 mM. By use of targeted gene replacement strategies, meaA and mepA deletion mutants were created. Deletion of both meaA and mepA resulted in the inability of the strain to grow on ammonium concentrations of less than 10 mM. The single meaA deletion mutant exhibited reduced growth at the same concentrations, whereas the mepA deletion mutant displayed wild-type growth. Interestingly, multiple copies of mepA were found to complement the methylammonium resistance phenotype conferred by the deletion of meaA . The expression profiles for mepA and meaA differed; the mepA transcript was detected only in nitrogen-starved cultures, whereas meaA was expressed under both ammonium-sufficient and nitrogen starvation conditions. Together, these results indicate that MeaA constitutes the major ammonium transport activity and is required for the optimal growth of A . nidulans on ammonium as the sole nitrogen source and that MepA probably functions in scavenging low concentrations of ammonium under nitrogen starvation conditions.

Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.