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In a screen of 243,000 diverse small-molecule compounds12, we obtained the acylsulfonylhydrazide compound CTx-0124143, a competitive KAT6A inhibitor (half-maximal inhibitory concentration (IC50) 0.49 µM) in biochemical assays12. Medicinal chemistry optimization yielded the compound WM-8014 with an IC50 value of 8 nM (Fig. 1a, Supplementary Table 1), representing a 60-fold increase in inhibitory activity towards KAT6A. This was consistent with the binding affinity measured by surface plasmon resonance (SPR; equilibrium dissociation constant (KD) 5 nM; Fig. 1a, Extended Data Fig. 1). WM-8014 inhibits predominantly the closely related proteins KAT6A and KAT6B (IC50 8 nM and 28 nM, respectively), and is more than tenfold less active against KAT7 and KAT5 (IC50 342 nM and 224 nM, respectively; Fig. 1b, Supplementary Table 1). Kinetic binding curves obtained from SPR demonstrated that the interaction of this class of compounds with immobilized proteins was fully reversible and consistent with a single-site binding interaction. The interaction of WM-8014 with KAT6A and KAT7, although relatively strong, was in both cases driven by fast association kinetics (association rate constant (ka) >1 × 106 M−1 s−1), whereas the dissociation kinetics (dissociation rate constant (kd) ~ 4 × 10−2 for KAT6A and 17 × 10−2 s−1 for KAT7) were indicative of a relatively short lifespan of the binary complex (Extended Data Fig. 1). WM-8014 displayed an order of magnitude weaker binding to KAT7 than to KAT6A (KD 52 nM versus 5.1 nM, respectively) (Fig. 1b, Extended Data Fig. 1). We also generated an inactive analogue, WM-2474 (Fig. 1a, Supplementary Table 1). Notably, these compounds were almost inactive against KAT8, and no inhibition was observed for the more distantly related lysine acetyltransferases KAT2A, KAT2B, KAT3A and KAT3B (Fig. 1b, c, Supplementary Table 1).

Fig. 1: Development of an inhibitor of the MYST family of lysine acetyltransferases.
figure 1

a, Schematic summary of the medicinal chemistry optimization of high-throughput screening hit CTx-0124143, which resulted in WM-8014 and the inactive compound WM-2474. The IC50 values (determined by biochemical assays) and equilibrium dissociation constants (KD, determined by SPR) are shown for KAT6A. b, Histone acetyltransferase inhibition assay (competition of compound with acetyl-CoA) of CTx-0124143, WM-8014 and WM-2474. The areas of the circles reflect the IC50 values as indicated, assayed at the Michaelis constant (Km) of acetyl-CoA for each KAT tested. c, Dendrogram showing the relationship between major KAT families based on sequence differences in the acetyltransferase domain. d–g, Crystal structures of WM-8014 and acetyl-CoA bound to the MYST lysine acetyltransferase domain (MYSTCryst; see Extended Data Fig. 1). PDB codes: 6BA2 and 6BA4, respectively. d, Space-filling model showing WM-8014 in the acetyl-CoA-binding pocket of MYSTCryst. e, Ribbon diagram of MYSTCryst (blue) showing WM-8014 (yellow, with element colouring) bound to the acetyl-CoA-binding site. f, Ribbon diagram of MYSTCryst showing key amino acids interacting with WM-8014 (yellow, with element colouring). Hydrogen bonds are shown as dashed lines. g, Ribbon diagram showing acetyl-CoA (yellow, with element colouring) bound to MYSTCryst. Means of two experiments are shown for the IC50 values in a and b. SPR experiments in a were repeated four times.

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WM-8014 has desirable, drug-like physicochemical properties (Supplementary Table 2). It is completely stable in cell culture medium (10% fetal calf serum); however, relatively high protein binding (97.5%) in this medium reduces its free concentration. Although WM-8014 has relatively low solubility in water (8–16 μM), it could readily permeate Caco-2 cells (apparent permeability coefficient (Papp) 78 ± 13 × 10−6 cm s−1). Testing of WM-8014 at 1 µM and 10 µM revealed no notable affinity for a pharmacological panel of 158 diverse biological targets; only eight targets were affected by more than 50% (Supplementary Table 3).

We solved the crystal structures of a modified MYST histone acetyltransferase domain (MYSTCryst) in complex with WM-8014 (1.85 Å resolution, Fig. 1d–f, Extended Data Fig. 1, Supplementary Table 4) or acetyl coenzyme A (acetyl-CoA; 1.95 Å resolution, Fig. 1g). The WM-8014 molecule occupies the acetyl-CoA-binding site on MYSTCryst, being partially enclosed between the α-helix formed by residues D685 to R704 and the loop extending from Q654 to G657. The MYSTCryst–acetyl-CoA complex adopts a globular fold (Fig. 1g), as seen in previously reported structures13, with a root mean square deviation (r.m.s.d.) of 0.6 Å, and is nearly identical to the MYSTCryst–WM-8014 complex (r.m.s.d. of 0.3 Å for all aligned atoms). Accordingly, the core acylsulfonylhydrazide moiety of WM-8014 makes similar hydrogen bonds to MYSTCryst as does the diphosphate group of acetyl-CoA (Fig. 1f, g). This includes hydrogen bonds to the main-chain atoms of R655, G657 and R660—identical to acetyl-CoA—as well as additional hydrogen bonds to G659 and S690 (Extended Data Fig. 1). The biphenyl group of WM-8014 extends further into the acetyl-CoA-binding pocket, which enables van der Waals interactions with residues L601, I647, I649, S684 and L686 of MYSTCryst (Extended Data Fig. 1). WM-8014 therefore competes directly with acetyl-CoA in the substrate-binding domain.

Because KAT6A suppresses senescence9,10, we examined the ability of WM-8014 to induce cell cycle arrest in embryonic day (E)14.5 mouse embryonic fibroblasts (MEFs). Cells treated with WM-8014 failed to proliferate after 10 days of treatment (Fig. 2a; IC50 2.4 µM), with similar kinetics to Cre-recombinase Kat6a recombination (Fig. 2b). Higher doses of WM-8014 (up to 40 µM) did not accelerate growth arrest, which after 8 days of treatment was irreversible (Extended Data Fig. 2). The inactive compound WM-2474 did not affect cell proliferation. Cell cycle analysis showed an increase in the proportion of cells in G0/G1 after 6 days of treatment and a corresponding reduction in cells in G2/M and S phases, both in Fucci cells14 and in 5-bromo-2′-deoxyuridine (BrdU) incorporation assays (Fig. 2c, Extended Data Fig. 2).

Fig. 2: Treatment of MEFs with WM-8014 leads to cellular senescence.
figure 2

a, Left, effects of WM-8014 compared with the inactive compound WM-2474 or DMSO vehicle control on cell growth of MEFs grown in 3% O2. Right, effects of the dose of WM-8014 and the duration of treatment. b, Effects of acute genetic deletion of Kat6a on the growth of MEFs. Loss of KAT6A function was induced by nuclear translocation of Cre-recombinase using tamoxifen on MEFs isolated from Kat6alox/loxRosaCreERT2 and control RosaCreERT2 embryos. c, Left, epifluorescence phase-contrast images of Fucci MEFs after 6 days of treatment with 20 µM WM-2474 (top) and 20 µM WM-8014 (bottom). Right, the percentage of Fucci MEFs in each stage of the cell cycle after 6 days of treatment with 10 µM WM-8014, 10 µM WM-2474 or DMSO vehicle control, as quantified by flow-cytometry analysis. DN, double negative. d, mRNA levels of Cdkn2a (coding for cell cycle regulators p16INK4A and p19ARF) (left) and the KAT6A target gene Cdc6 (right) in MEFs treated for 4 days and 10 days with 10 µM WM-8014 or 10 µM WM-2474 control, assessed by RNA-seq. RPKM, reads per kilobase per million reads. e, Flow-cytometry assessment (mean ± s.e.m. of median fluorescence intensity (MFI)) of senescence-associated β-galactosidase activity in MEFs after 4 and 10 days of treatment with 10 µM WM-8014, 10 µM WM-2474 or DMSO vehicle control. f, Growth of MEFs lacking p16INK4A and p19ARF (left) and of MEFs lacking p53 (right) compared with wild type after treatment with WM-8014, DMSO vehicle control or WM-2474. n = 3 independent MEF isolates per treatment group and genotype. Data are mean ± s.e.m. Data were analysed by one-way ANOVA followed by Bonferroni post hoc test (a (left), b, c, e), non-linear regression curve fit (a, right) or two-way ANOVA (f) with treatment and with or without treatment duration as the independent factors. RNA-seq data (d) were analysed as described in the Supplementary Methods.

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RNA sequencing (RNA-seq) of MEFs treated with WM-8014 revealed a signature of cellular senescence, including upregulated expression of Cdkn2a mRNA and decreased expression of Cdc6, which is a KAT6A target gene9 and a regulator of DNA replication15 (Fig. 2d; day 10: false discovery rate (FDR) < 10−6). A substantial increase in β-galactosidase activity—a marker of senescent cells—was also observed (Fig. 2e), accompanied by morphological changes typical of senescence (Extended Data Fig. 2). WM-8014 caused a concentration-dependent reduction in the level of E2f2 mRNA (adjusted (adj.) R2 = 0.73; P < 0.0005) and Cdc6 mRNA (adj. R2 = 0.5; P = 0.002), 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 Data Fig. 4), which suggests that cell cycle arrest was not a consequence of DNA damage. No increase in apoptosis or necrosis was seen (Extended Data Fig. 4). Treatment of either Trp53-null MEFs (Trp53−/−) or Cdkn2a-null (Ink4a−/−Arf−/−) MEFs with WM-8014 had a minor effect and no effect on cell proliferation, respectively (Fig. 2f, Extended Data Fig. 2). These results show that WM-8014 acts through the p16INK4A–p19ARF pathway, causing irreversible cell cycle exit leading to senescence, and does not have a general cytotoxic effect.

KAT7 is essential for global histone 3 lysine 14 (H3K14) acetylation16. By contrast, KAT6A regulates H3K9 acetylation only at target loci17,18. We determined the effects of WM-8014 on global levels of acetylation at H3K9 and H3K14 by western blot after 5 days of treatment. Treatment with 10 µM WM-8014 caused a 49% decrease in the global levels of H3K14ac but, as expected on the basis of the locus-specific roles of KAT6A17,18, did not significantly affect the global levels of H3K9ac (Fig. 3a, b; all gel source data in Supplementary Fig. 1). The effects of WM-8014 on global H3K14ac levels were concentration-dependent (Fig. 3b; H3K14ac/H4 ratio regressed on log concentration of WM-8014; adj. R2 = 0.76, P < 0.001; IC50 1.2 µM). RNA-seq showed a strong correlation between the changes in gene expression seen in Kat6a−/− MEFs compared with Kat6a+/+ MEFs and the genes differentially expressed after WM-8014 treatment (WM-8014 compared with inactive WM-2474), with a 2.6-fold enrichment in upregulated genes (FDR = 0.0001; Fig. 3c) and a 2.1-fold enrichment in downregulated genes (FDR = 0.0001; Fig. 3c), and gene expression signatures characteristic of cellular senescence (Extended Data Fig. 5). Loss of KAT6A results in the downregulation of E2f2, Ezh2 and Melk9. Similarly, treatment with WM-8014 caused significant downregulation of Ezh2, Melk and E2f2 mRNA levels compared with controls (Fig. 3d), as determined by RNA-seq (Extended Data Fig. 5) and confirmed by quantitative reverse-transcription PCR (RT–qPCR) (Extended Data Fig. 3). After treatment with WM-8014, there was a reduction of H3K9ac at the transcription start sites of these genes (Fig. 3e). Therefore, the treatment of cells with high concentrations of WM-8014 directly inhibits global H3K14 acetylation catalysed by KAT7, as well as KAT6A-specific H3K9 acetylation at transcription start sites.

Fig. 3: Treatment of cells with WM-8014 leads to a reduction in acetylation of specific histone lysine residues and changes in gene expression that resemble the genetic loss of KAT6A.
figure 3

a, Western blot detection of H3K14ac or H3K9ac in MEFs treated with 10 µM WM-8014, 10 µm WM-2474 or DMSO for 5 days. The densitometric analysis is presented on the right. n = 6 (H3K14ac) and n = 9 (H3K9ac) independent cultures per treatment group. b, Western blot of MEFs treated with increasing doses of WM-8014 and controls as indicated. Densitometric analysis is presented on the right. n = 3 independent experiments. Histone acetylation levels were regressed on the log10 of the WM-8014 concentration. H3K14ac and H3K9ac levels were normalized to pan-H4 levels and DMSO treatment. c, Barcode plot in which genes that are differentially up- or downregulated in Kat6a−/− versus Kat6a+/+ MEFs (that is, after genetic deletion of KAT6A) are compared with genes differentially expressed in MEFs treated with WM-8014 versus WM-2474. Combined results of day 4 and day 10 treatment, ROAST P = 0.0001; MEF isolates from individual E12.5 embryos, namely from n = 3 Kat6a−/− and 2 Kat6a+/+ embryos, as well as 3 MEF isolates from 3 wild-type embryos treated with either WM-8014 or WM-2474. d, Ezh2, Melk and E2f2 mRNA levels measured by RNA-seq in MEFs treated for 4 days and 10 days with 10 µM WM-8014 or 10 µM control WM-2474 (n = 3 MEF isolates from 3 wild-type embryos treated with either WM-8014 or WM-2474). e, Anti-H3K9ac chromatin immunoprecipitation followed by qPCR detection of transcription start sites of genes after treatment with DMSO 10 µM WM-8014 or WM-2474 for 3 days. The results of one of four experiments are shown; total n = 16 cultures per treatment group in 4 experiments. Data are mean ± s.e.m. (with the exception of e, mean ± s.d.) and were analysed by one-way ANOVA followed by Bonferroni post hoc test (a), by regression analysis (b) or by t-test comparing WM-8014 to WM-02474 (e). The RNA-seq analysis (c, d) is described in the Supplementary Methods.

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Because WM-8014 induced cellular senescence, we reasoned that it might exacerbate oncogenic RAS-induced senescence. Accordingly, MEFs that express HRASG12V, a constitutively active form of RAS, were more sensitive to the induction of cell cycle arrest by WM-8014 (Extended Data Fig. 6). We then examined the effects of WM-8014 in a zebrafish model19 of KRASG12V-driven hepatocellular overproliferation. We observed a significant, concentration-dependent reduction in liver volume in response to treatment with WM-8014, and a substantial reduction in hepatocytes in S phase (Extended Data Fig. 6). Notably, WM-8014 did not impair the growth of the normal liver, demonstrating that the inhibitory effects of WM-8014 were specific to hepatocytes that express oncogenic RAS. Treatment with WM-8014 was found to robustly upregulate the cell cycle regulators Cdkn2a and Cdkn1a in hepatocytes that express oncogenic KRASG12V, but not control hepatocytes. Therefore, WM-8014 potentiates oncogene-induced senescence, but it does not affect normal hepatocyte growth.

The progression of lymphoma is highly dependent on KAT6A, as Kat6a heterozygous mice are protected from early-onset MYC-driven lymphoma11. However, the high levels of plasma-protein binding exhibited by WM-8014 (Supplementary Table 2) precluded in vivo studies in mice. Development of derivatives of WM-8014 resulted in WM-1119, which has reduced plasma-protein binding (Fig. 4a; Supplementary Table 2). The interaction of WM-1119 with KAT6A is similar to that of WM-8014: it is characterized by strong reversible binding (KD 2 nM, compared with 5 nM for WM-8014; Extended Data Fig. 7) that is competitive with acetyl-CoA, and driven by fast association kinetics (ka > 1 × 106 M−1 s−1; Extended Data Fig. 7). The structure of MYSTCryst in complex with WM-1119 was solved (Extended Data Fig. 7, Supplementary Table 5) and was found to be almost identical to that of MYSTCryst–WM-8014, with an r.m.s.d. for aligned main-chain atoms of 0.2 Å. There are two key differences between the complexes: an additional hydrogen bond is formed between the WM-1119 pyridine nitrogen and the main chain at I649 that is not present in the complex with WM-8014 (Extended Data Fig. 7), and the hydrophobic interaction that exists between the meta-methyl of the biphenyl group of WM-8014 and I663 is not present in the complex with WM-1119. WM-1119 is 1,100-fold and 250-fold more active against KAT6A than against KAT5 or KAT7, respectively (Fig. 4a, Extended Data Fig. 7), and so shows greater specificity for KAT6A than does WM-8014. The testing of WM-1119 at 1 µM and 10 µM against a pharmacological panel of 159 diverse biological targets revealed no affinity (Supplementary Table 6). Treatment of MEFs with WM-1119 resulted in cell cycle arrest in G1 and a senescence phenotype similar to that seen upon treatment with WM-8014 (Extended Data Fig. 8). Notably, the activity of WM-1119 in this cell-based assay is an order of magnitude greater than WM-8014 and WM-1119 is able to induce cell cycle arrest at 1 µM.

Fig. 4: Treatment with WM-1119 arrests lymphoma growth.
figure 4

a, Medicinal chemistry optimization of WM-8014 resulted in compound WM-1119. The binding data (obtained by SPR) for the interaction of WM-1119 with immobilized KAT6A, KAT7 and KAT5 are compared with the interaction data for WM-8014. b, Growth inhibition assays of Eµ-Myc lymphoma cell line EMRK1184 treated with WM-1119 and WM-8014 at the doses indicated. c, Bioluminescence images of EMRK1184 lymphoma cells expressing luciferase before (day 3) and after (day 14) 11 days of treatment with WM-1119 (50 mg kg−1 four times per day) or PEG400 vehicle control. The red boxes show the regions used for quantification (imaging at days 7, 10 and 12 in Extended Data Fig. 10). d, Quantification of the signals measured in all experiments: two cohorts of mice treated with WM-1119 three times per day, combined n = 6; two cohorts of mice treated with WM-1119 four times per day, combined n = 9; vehicle controls, n = 15. One mouse did not respond to WM-1119 treatment, shown in grey. e, Dissected spleens obtained after imaging on day 14, taken from the mice shown in c. f, Spleen weights of mice treated with WM-1119 or vehicle. n values as stated in d. i.p., intraperitoneal. g, Flow-cytometry analysis of spleen cells from vehicle-treated mice and mice treated with WM-1119 (four times per day). The tumour cells were CD19+IgM−, and normal splenic B cells were CD19+IgM+. Quantification of flow-cytometry analysis in bone marrow (BM), spleen and peripheral white blood cells (PWBC). n = 4 independent experiments for WM-1119 and 2 for WM-8014 in b, and number of mice as indicated in d, f, g in three independent experiments. Data are mean ± s.e.m. and were analysed by nonlinear regression dose–response curve fit, least squares fit, inhibitor versus response, variable slope (b); one-way ANOVA followed by Bonferroni post hoc test with treatment as the independent factor (d, g), or two-tailed t-tests (f).

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To test inhibitors of KAT6A in a cancer model, we investigated the effect of WM-1119 and WM-8014 on the proliferation of lymphoma cells. We selected the B cell lymphoma cell line EMRK1184, which was isolated from mice with a tumour resulting from the expression of Myc under the control of the IgH enhancer20, because it expressed the Cdnk2a-locus-encoded ARF and wild-type p53 (Extended Data Fig. 9). Treatment with WM-8014 or WM-1119 inhibited the proliferation of the EMRK1184 lymphoma cells in vitro (Fig. 4b); RNA-seq and western blot analysis showed that treatment with WM-1119 resulted in increased levels of Cdkn2a and Cdkn2b mRNA and p16INK4a and p19ARF proteins, as well as a delayed increase in Cdkn1a mRNA (Extended Data Fig. 9). WM-1119 (IC50 0.25 µM) was ninefold more active than WM-8014 (IC50 2.3 µM; Fig. 4b), as expected on the basis of reduced protein binding (Supplementary Table 2).

We tested the effectiveness of KAT6 inhibitors in the treatment of lymphoma in mice. Male C57BL/6-albino (B6(Cg)-Tyrc-2J/J) mice were injected intravenously with 100,000 EMRK1184 cells transfected with a luciferase-expression construct. Lymphoma growth was monitored using the IVIS imaging system. Three days after the lymphoma-cell transplant, all mice showed luciferase activity (Fig. 4c), which indicated the expansion of lymphoma cells. Mice were then divided randomly into WM-1119-treatment and vehicle-control groups. Because WM-1119 is rapidly cleared after intraperitoneal injection, with the plasma concentration decreasing to below 1 µM after 4–6 h (Extended Data Fig. 9), cohorts of mice were injected every 8 h (three times per day, two cohorts of three mice per treatment group) or every 6 h (four times per day, two cohorts of three and six mice per treatment group; Fig. 4d). Mice were imaged five times over the course of these experiments to monitor the growth of lymphoma. No significant difference between the treatment and control groups was seen before day 10 (Fig. 4d, Extended Data Fig. 10), which was expected as the inhibition of cell proliferation in vitro took approximately seven days. However, by day 14, the cohorts that were treated four times per day with WM-1119 had arrested tumour growth (Fig. 4c, Extended Data Fig. 10), with the exception of one mouse that did not respond (Fig. 4d). Spleen weights in the WM-1119-treatment group (treated four times per day) were substantially lower than spleen weights in the vehicle-treated group, and not significantly different from those of tumour-free eight-week-old mice (P < 0.0005 and P = 0.2, respectively; Fig. 4e, f). Treatment with WM-1119 three times per day led to a significant reduction in tumour burden and spleen weight, but was not as effective as treatment four times per day (Fig. 4d, f). WM-1119 was well-tolerated; mice showed no generalized ill effects and weight loss was not observed (Extended Data Fig. 10). WM-1119 treatment had no effect on haematocrit, erythrocytes or platelet numbers, but there was overall leukopenia (Extended Data Fig. 10). The proportion and overall number of tumour cells was substantially reduced by WM-1119 treatment (four times per day; Fig. 4g). Analysis by intracellular flow cytometry demonstrated a reduction in H3K9ac in tumour cells (P = 0.03; Extended Data Fig. 10). These results demonstrate that WM-1119 is effective in treating lymphoma in vivo.

In summary, using high-throughput screening followed by medicinal chemistry optimization, in-cell assays, biochemical assessment of target engagement and tumour models in mice and fish, we have developed a novel class of inhibitors for a hitherto unexplored category of epigenetic regulators. These inhibitors engage the MYST family of lysine acetyltransferases in primary cells, specifically induce cell cycle exit and senescence, and are effective in preventing the progression of lymphoma in mice.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

The RNA-seq data of MEFs treated with WM-8014, WM-2474 and DMSO, of MEFs from Kat6a−/− and wild-type embryos and of lymphoma cell line EMRK1184 treated with vehicle and WM-1119 have been submitted to the Gene Expression Omnibus (GEO) database under accession number GSE108244. The crystal structure data for the MYST domain in complex with WM-8014, acetyl-CoA and WM-1119 have been submitted to the Protein Data Bank (PDB) under accession numbers 6BA2, 6BA4 and 6CT2, respectively. Source Data for all graphs are provided.