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Inhibitors of Histone Acetyltransferases KAT6A/B Induce Senescence and Arrest Tumour Growth Jonathan B

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Inhibitors of Histone Acetyltransferases KAT6A/B Induce Senescence and Arrest Tumour Growth Jonathan B LETTER https://doi.org/10.1038/s41586-018-0387-5 Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth Jonathan B. Baell1,2*, David J. Leaver1, Stefan J. Hermans3, Gemma L. Kelly4,5, Margs S. Brennan4,5, Natalie L. Downer4, Nghi Nguyen1, Johannes Wichmann4,5, Helen M. McRae4,5, Yuqing Yang4,5, Ben Cleary1, H. Rachel Lagiakos1,6, Stephen Mieruszynski4,5, Guido Pacini4, Hannah K. Vanyai4,5, Maria I. Bergamasco4,5, Rose E. May4, Bethany K. Davey4,6, Kimberly J. Morgan4,5, Andrew J. Sealey4,5, Beinan Wang4,5,7, Natasha Zamudio4,5, Stephen Wilcox4,5, Alexandra L. Garnham4,5, Bilal N. Sheikh4,5, Brandon J. Aubrey4,5, Karen Doggett4,5, Matthew C. Chung3, Melanie de Silva4,6, John Bentley8, Pat Pilling8, Meghan Hattarki8, Olan Dolezal8, Matthew L. Dennis8, Hendrik Falk4,5,6, Bin Ren8, Susan A. Charman9, Karen L. White9, Jai Rautela4,5, Andrea Newbold10, Edwin D. Hawkins4,5, Ricky W. Johnstone10, Nicholas D. Huntington4,5, Thomas S. Peat8, Joan K. Heath4,5, Andreas Strasser4,5, Michael W. Parker3,11, Gordon K. Smyth4,12, Ian P. Street4,5,6, Brendon J. Monahan4,5,6, Anne K. Voss4,5,13* & Tim Thomas4,5,13* Acetylation of histones by lysine acetyltransferases (KATs) is and KAT6B (IC50 8 nM and 28 nM, respectively), and is more than 1 essential for chromatin organization and function . Among tenfold less active against KAT7 and KAT5 (IC50 342 nM and 224 nM, the genes coding for the MYST family of KATs (KAT5–KAT8) respectively; Fig. 1b, Supplementary Table 1). Kinetic binding curves are the oncogenes KAT6A (also known as MOZ) and KAT6B obtained from SPR demonstrated that the interaction of this class (also known as MORF and QKF)2,3. KAT6A has essential roles in of compounds with immobilized proteins was fully reversible and normal haematopoietic stem cells4–6 and is the target of recurrent consistent with a single-site binding interaction. The interaction of chromosomal translocations, causing acute myeloid leukaemia7,8. WM-8014 with KAT6A and KAT7, although relatively strong, was Similarly, chromosomal translocations in KAT6B have been in both cases driven by fast association kinetics (association rate 8 6 −1 −1 identified in diverse cancers . KAT6A suppresses cellular senescence constant (ka) >1 × 10 M s ), whereas the dissociation kinetics 9,10 −2 −2 −1 through the regulation of suppressors of the CDKN2A locus , a (dissociation rate constant (kd) ~ 4 × 10 for KAT6A and 17 × 10 s function that requires its KAT activity10. Loss of one allele of KAT6A for KAT7) were indicative of a relatively short lifespan of the binary extends the median survival of mice with MYC-induced lymphoma complex (Extended Data Fig. 1). WM-8014 displayed an order of mag- 11 from 105 to 413 days . These findings suggest that inhibition of nitude weaker binding to KAT7 than to KAT6A (KD 52 nM versus KAT6A and KAT6B may provide a therapeutic benefit in cancer. 5.1 nM, respectively) (Fig. 1b, Extended Data Fig. 1). We also gener- Here we present highly potent, selective inhibitors of KAT6A ated an inactive analogue, WM-2474 (Fig. 1a, Supplementary Table 1). and KAT6B, denoted WM-8014 and WM-1119. Biochemical and Notably, these compounds were almost inactive against KAT8, and structural studies demonstrate that these compounds are reversible no inhibition was observed for the more distantly related lysine competitors of acetyl coenzyme A and inhibit MYST-catalysed acetyltransferases KAT2A, KAT2B, KAT3A and KAT3B (Fig. 1b, c, histone acetylation. WM-8014 and WM-1119 induce cell cycle exit Supplementary Table 1). and cellular senescence without causing DNA damage. Senescence WM-8014 has desirable, drug-like physicochemical properties is INK4A/ARF-dependent and is accompanied by changes in gene (Supplementary Table 2). It is completely stable in cell culture medium expression that are typical of loss of KAT6A function. WM-8014 (10% fetal calf serum); however, relatively high protein binding potentiates oncogene-induced senescence in vitro and in a zebrafish (97.5%) in this medium reduces its free concentration. Although model of hepatocellular carcinoma. WM-1119, which has increased WM-8014 has relatively low solubility in water (8–16 µM), it could bioavailability, arrests the progression of lymphoma in mice. We readily permeate Caco-2 cells (apparent permeability coefficient (Papp) anticipate that this class of inhibitors will help to accelerate the 78 ± 13 × 10−6 cm s−1). Testing of WM-8014 at 1 µM and 10 µM development of therapeutics that target gene transcription regulated revealed no notable affinity for a pharmacological panel of 158 diverse by histone acetylation. biological targets; only eight targets were affected by more than 50% In a screen of 243,000 diverse small-molecule compounds12, we (Supplementary Table 3). obtained the acylsulfonylhydrazide compound CTx-0124143, a We solved the crystal structures of a modified MYST histone competitive KAT6A inhibitor (half-maximal inhibitory concentra- acetyltransferase domain (MYSTCryst) in complex with WM-8014 12 tion (IC50) 0.49 µM) in biochemical assays . Medicinal chemistry (1.85 Å resolution, Fig. 1d–f, Extended Data Fig. 1, Supplementary optimization yielded the compound WM-8014 with an IC50 value of Table 4) or acetyl coenzyme A (acetyl-CoA; 1.95 Å resolution, Fig. 1g). 8 nM (Fig. 1a, Supplementary Table 1), representing a 60-fold increase The WM-8014 molecule occupies the acetyl-CoA-binding site on in inhibitory activity towards KAT6A. This was consistent with the MYSTCryst, being partially enclosed between the α-helix formed by binding affinity measured by surface plasmon resonance (SPR; equilib- residues D685 to R704 and the loop extending from Q654 to G657. The Cryst rium dissociation constant (KD) 5 nM; Fig. 1a, Extended Data Fig. 1). MYST –acetyl-CoA complex adopts a globular fold (Fig. 1g), as seen WM-8014 inhibits predominantly the closely related proteins KAT6A in previously reported structures13, with a root mean square deviation 1Medicinal Chemistry Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. 2School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China. 3ACRF Rational Drug Discovery Centre, St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia. 4The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria, Australia. 5Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia. 6Cancer Therapeutics CRC, Parkville, Victoria, Australia. 7School of Pharmaceutical Sciences, Tsinghua University, Beijing, China. 8Commonwealth Scientific and Industrial Research Organisation (CSIRO), Biomedical Program, Parkville, Victoria, Australia. 9Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. 10The Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 11Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia. 12Department of Mathematics 13 and Statistics, University of Melbourne, Parkville, Victoria, Australia. These authors jointly supervised this work: Anne K. Voss, Tim Thomas. *e-mail: [email protected]; [email protected]; [email protected] 9 AUGUST 2018 | VOL 560 | NATURE | 253 © 2018 Springer Nature Limited. All rights reserved. RESEARCH LETTER IC = 0.49 M 9,10 a 50 μ Because KAT6A suppresses senescence , we examined the ability of K = 0.38 M D μ WM-8014 to induce cell cycle arrest in embryonic day (E)14.5 mouse O H embryonic fibroblasts (MEFs). Cells treated with WM-8014 failed to S N N proliferate after 10 days of treatment (Fig. 2a; IC50 2.4 µM), with similar O H O F kinetics to Cre-recombinase Kat6a recombination (Fig. 2b). Higher CTx-0124143 N N doses of WM-8014 (up to 40 µM) did not accelerate growth arrest, IC = 0.008 M IC > 125 M 50 μ 50 μ which after 8 days of treatment was irreversible (Extended Data Fig. 2). K = 0.005 μM K SPR: no binding D D The inactive compound WM-2474 did not affect cell proliferation. Cell O O cycle analysis showed an increase in the proportion of cells in G0/G1 H H S N S N after 6 days of treatment and a corresponding reduction in cells in G2/M N N 14 O H O H and S phases, both in Fucci cells and in 5-bromo-2′-deoxyuridine O F O F (BrdU) incorporation assays (Fig. 2c, Extended Data Fig. 2). WM-8014 WM-2474 (inactive control) RNA sequencing (RNA-seq) of MEFs treated with WM-8014 b IC50 (μM) c KAT6A revealed a signature of cellular senescence, including upregulated KAT6A 100 KAT6B expression of Cdkn2a mRNA and decreased expression of Cdc6, which KAT6B 10 KAT7 9 15 KAT7 1 is a KAT6A target gene and a regulator of DNA replication (Fig. 2d; KAT8 0.1 KAT8 day 10: false discovery rate (FDR) < 10−6). A substantial increase KAT5 0.01 KAT5 KAT2A KAT2A in β-galactosidase activity—a marker of senescent cells—was also KAT2B KAT2B KAT3A observed (Fig. 2e), accompanied by morphological changes typical of KAT3B KAT3A senescence (Extended Data Fig. 2). WM-8014 caused a concentration- KAT3B CTx-143 WM-2474 dependent reduction in the level of E2f2 mRNA (adjusted (adj.) WM-8014 R2 = 0.73; P < 0.0005) and Cdc6 mRNA (adj. R2 = 0.5; P = 0.002), d e accompanied by upregulation of both splice products of the Cdkn2a locus, Ink4a and Arf (day 10: P < 0.0005 and P = 0.005, respectively; Extended Data Fig. 3). Notably, MEFs treated for 4 days or 10 days with 10 µM WM-8014, the control compound WM-2474 or DMSO vehicle control showed no change in the levels of γH2A.X (Extended f WM-8014 g Acetyl-CoA Ser690 Data Fig. 4), which suggests that cell cycle arrest was not a conse- quence of DNA damage. No increase in apoptosis or necrosis was Gly659 seen (Extended Data Fig.
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