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EZH2 noncanonically binds cMyc and p300 through a cryptic transactivation domain to mediate gene activation and promote oncogenesis

Nature Cell Biology volume 24, pages 384–399 (2022)Cite this article

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Abstract

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2–PRC2) and noncanonical (EZH2-TAD–cMyc–coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2–PRC2 and noncanonical EZH2–cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

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Fig. 1: EZH2 exhibits noncanonical PRC2-independent solo-binding in leukaemias.
Fig. 2: Cooperative EZH2 and cMyc recruitment to common targets leads to gene activation in leukaemia.
Fig. 3: EZH2 directly interacts with cMyc and coactivators via EZH2-TAD to promote malignant growth of leukaemia cells.
Fig. 4: Characterization of the EZH2-targeting PROTAC MS177.
Fig. 5: MS177 exhibits on-target inhibition of EZH2–PRC2.
Fig. 6: MS177 represses the cMyc-related oncogenic node.
Fig. 7: MS177 efficiently induces leukaemia cell growth inhibition, apoptosis and cell cycle progression arrest.
Fig. 8: MS177 represses AML growth in vivo.

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Data availability

The genomic dataset of this study, including ChIP-seq, CUT&RUN and RNA-seq, have been deposited in NCBI Gene Expression Omnibus (GEO) database under accession code GSE180448. Human AML datasets were derived from TCGA Research Network (http://cancergenome.nih.gov/). Publicly available datasets used in this work were from NCBI GEO accession numbers GSE113042 (ChIP-seq of H3K4me3, H3K27ac, SMARCA4 and SMARCC1 in EOL-1 cells), GSE82116 (ChIP-seq of H3K27ac, H3K9ac and POL2 in MV4;11 cells), GSE73528 (ChIP-seq of MLLn and MLLc in MV4;11 cells), GSE101821 (ChIP-seq of BRD4 in MV4;11 cells), GSE29611 (ChIP-seq of EZH2 and H3K27me3 in GM12878, HUVEC and K562 cells, as well as SUZ12 in K562 cells), GSE30226 (ChIP-seq of cMyc in HUVEC and K562 cells) and GSE33213 (ChIP-seq of cMyc in GM12878 cells). Other data supporting the findings of this study are available upon request. Source data are provided with this paper.

Code availability

We did not use custom code. All software and packages used in this study are listed in Nature Research Reporting Summary and are publicly available.

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Acknowledgements

We thank members of the Wang and Jin laboratories, H. Uryu and K. Suzuki for technical support and helpful discussions; X. Shi, H. Wen, E. Lichtman, P. Armistead, H. Huang, J. Bradner, W. Kaelin, E. Guccione, S. Frye, L. James, A. Chinnaiyan, H. Lin, W. Wei and Q. Zhang for providing reagents and cell lines; staff at the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) for generating the selectivity data of MS177 over GPCRs, ion channels and transporters; staff at the UNC’s core facilities, including the Imaging Core, the High-throughput Sequencing Facility (HTSF), the Bioinformatics Core, the Flow Cytometry Core, the Tissue Culture Facility, the Tissue Procurement Facility and the Animal Studies Core, for their professional assistance of this work. The cores affiliated to the UNC Cancer Center are supported in part by the UNC Lineberger Comprehensive Cancer Center Core Support Grant P30-CA016086. This work was supported in part by R01CA218600 (to J.J. and G.G.W.), R01CA268519 (to G.G.W. and J.J.), R01CA211336 (to G.G.W.), R01CA215284 (to G.G.W.), R01CA230854 (to J.J.) and R01GM122749 (to J.J.) grants from the US National Institutes of Health; a Kimmel Scholar Award (to G.G.W.); Gabrielle’s Angel Foundation for Cancer Research (to G.G.W.); When Everyone Survives (WES) Leukemia Research Foundation (to G.G.W.); and UNC Lineberger Cancer Center UCRF Stimulus Initiative grants (to G.G.W. and L.C.). G.G.W. is an American Cancer Society (ACS) Research Scholar, a Leukemia and Lymphoma Society (LLS) Scholar, and an American Society of Hematology (ASH) Scholar in Basic Science. This work utilized the NMR Spectrometer Systems at Mount Sinai acquired with funding from National Institutes of Health SIG grants 1S10OD025132 and 1S10OD028504.

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Author notes

  1. These authors contributed equally: Jun Wang, Xufen Yu.

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Jun Wang, Weida Gong, Xijuan Liu, Yi-Hsuan Tsai, David F. Allison, Ling Cai & Gang Greg Wang

  2. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Jun Wang, David F. Allison & Gang Greg Wang

  3. Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Xufen Yu, Kwang-Su Park, Anqi Ma, Yudao Shen, Jing Liu & Jian Jin

  4. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, USA

    Takashi Onikubo & Robert G. Roeder

  5. Institute of Biochemistry and Molecular Biology, National Yang Ming Chiao Tung University, Taipei, Taiwan

    Wen-Chieh Pi & Wei-Yi Chen

  6. Department of Genetics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Ling Cai

  7. Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA

    Gang Greg Wang

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  1. Jun Wang
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Contributions

J.W., X.Y., X.L., K.-S.P., A.M., Y.S., W.-C.P., D.F.A., T.O., W.-Y.C., J.L. and L.C. performed experiments. J.W. performed functional studies (including ChIP-seq, RNA-seq and CUT&RUN, as well as cancer biology works) and data analyses (under the direction of G.G.W.). X.Y. performed chemical biology studies (under the direction of J.J.). W.G. and Y-H.T. performed RNA-seq data analysis (under the direction of G.G.W. and L.C.). X.Y., A.M., J.L. and J.J. analysed the structure–activity relationship results. K.-S.P., T.O., W.-C.P. and J.W. conducted EZH2-TAD and protein complex characterizations (under the direction of J.J., W.-Y.C., R.G.R. and G.G.W.). J.J. and G.G.W. conceived the project, organized and led the study. J.W., X.Y., J.J. and G.G.W. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Jian Jin or Gang Greg Wang.

Ethics declarations

Competing interests

Icahn School of Medicine at Mount Sinai filed a patent application (WO 2018/081530 A1) covering EZH2 degraders that lists J.J. and A.M. as inventors. The Jin Laboratory received research funds from Celgene Corporation, Levo Therapeutics, Cullgen, Inc. and Cullinan Oncology. J.J. is a co-founder, scientific advisory board member and equity shareholder in Cullgen Inc., and is a consultant for Cullgen Inc., EpiCypher Inc. and Accent Therapeutics Inc. The remaining authors declare no competing interests.

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Nature Cell Biology thanks Martin Eilers, Thomas Milne and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In MLL-rearranged (MLL-r) acute leukaemia, EZH2 exhibits the noncanonical ‘solo’-binding pattern at sites enriched for the gene-activation-related histone marks and co-binding of RNA polymerase II (Pol II), (co)activators and cMyc, in addition to its canonical EZH2:PRC2 sites showing H3K27me3 co-binding.

(a-b) Heatmaps showing the K-means clustered EZH2 and H3K27me3 ChIP-seq signal intensities ± 3 kb around peak centers in MV4;11 (a) and EOL-1 (b) cells. EZH2-‘solo’ and EZH2-‘ensemble’ refer to noncanonical EZH2 + /H3K27me3- peaks (cluster 9 in MV4;11 and cluster 8 in EOL-1 cells) and canonical EZH2 + /H3K27me3+ ones (clusters 1-8 in MV4;11 and clusters 1-7 in EOL-1 cells), respectively. (c) Averaged EZH2 and H3K27me3 ChIP-seq signals around ± 3 kb from the centers of the EZH2-‘solo’-binding peaks in MV4;11 (top) and EOL-1 (bottom) cells. (d) Venn diagram showing the overlap between the called EZH2 and H3K27me3 peaks in MV4;11 (top) and EOL-1 (bottom) cells. (e) Motif search analysis of the EZH2-‘solo’-binding peaks in MV4;11 cells by using the SeqPos tool in Cistrome. (f) Co-immunoprecipitation (co-IP) using anti-HA antibodies for assaying interaction between Flag-EZH2 and HA-p300 in 293 T cells. (g) Co-IP for endogenous EZH2 and MAX using anti-cMyc antibody in EOL-1 or MOLM-13 cells after the treatment of benzonase and ethidium bromide. (h) Co-IP for interaction between endogenous cMyc and EZH2 in 293 T cells by using either anti-cMyc (upper) or anti-EZH2 (bottom) antibodies. (i) Co-IP using anti-HA antibodies for interaction between endogenous EZH2 and the transiently expressed HA-cMyc in 293 T cells. (j) Co-IP using anti-cMyc antibodies for interaction between the transiently expressed Flag-EZH2 and endogenous cMyc in 293 T cells. (k) Pearson correlation analysis of cMyc ChIP-seq profiles generated by using two independent anti-cMyc antibodies in MV4;11 and EOL-1 cells. (l) Pie-chart plot showing the genomic distribution of peaks with both EZH2-‘solo’-binding and cMyc-binding in MV4;11 (top) or EOL-1 (bottom) cells. (m) Heatmaps of EZH2, SUZ12, H3K27me3 and cMyc ChIP-seq signal intensities ± 5 kb from the centers of the called EZH2 peaks in the GM12878 lymphoblast cells (left), human umbilical vein endothelial cells (HUVEC; middle) and K562 chronic myeloid leukaemia (CML) cells (right).

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Extended Data Fig. 2 Cooperative recruitment of EZH2 and cMyc to common targets leads to gene activation in leukaemia.

(a-d) Immunoblotting for EZH2 (a) or cMyc (c) and growth of MV4;11 cells following the doxycycline (Dox)-induced EZH2 knockdown (KD; shEZH2, b) or cMyc knockout (KO; by either sgcMyc-1 or sgcMyc-5, d), relative to respective empty vector (EV) controls. Y-axis shows growth after normalization to controls (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). iCas9, Dox-inducible Cas9. (e) Venn diagram using downregulated DEGs, identified by RNA-seq with (blue) or without (red) the spike-in control normalization, in MV4;11 cells following EZH2 KD or cMyc KO. (f) Venn diagram using downregulated DEGs, identified by RNA-seq with spike-in control normalization, in MV4;11 cells post-depletion of EZH2 or cMyc. (g) Venn diagram using the EZH2/cMyc co-upregulated genes, identified by RNA-seq with (blue) or without (red) spike-in control normalization, in MV4;11 cells. (h) Averaged signals of the indicated protein bound at the EZH2/cMyc co-upregulated genes (n = 129; shown in f). TSS, transcriptional start site; TES, transcriptional end site. (i) Gene Ontology (GO) analysis (top) and enrichment of the DisGeNet category (bottom) using the EZH2/cMyc co-upregulated genes in f. (j-k) Box plot showing overall expression of all genes (left) and those associated with EZH2-‘solo’ (middle) or -‘ensemble’ (right) peaks in EOL-1 (j) or MV4;11 (k) cells. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers (dashed) indicate 1.5× the interquartile range. Paired two-sided t-test. (l) Box plots showing log2-converted ratios for the indicated sample comparisons by using the 204 genes identified in main Fig. 2a. Box plot was defined the same as above. (m) IGV views of the indicated factor at GADD45B, CD55 and ADAM9 in MV4;11 cells. (n-p) Immunoblotting of the indicated protein in MV4;11 cells post-treatment with C24 (n), UNC6852 (o) or A-485 (p) for 24 hours. (q) EZH2 immunoblot following EZH2 KO. *, **, and *** denote P < 0.05, 0.01 and 0.005, respectively. NS denotes not significant. Numerical source data, statistics, exact P values and unprocessed blots are available as source data.

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Extended Data Fig. 3 A cryptic transactivation domain (TAD) of EZH2 (EZH2-TAD) directly associates with cMyc and coactivator (p300), promoting malignant growth of leukaemia cells.

(a) Analysis with a prediction software, 9aaTAD, showing two putative TAD sequences (TAD1 and TAD2) within EZH2. Algorithm for the 9aaTAD amino acid pattern was applied in the search, and region clustering conformity was assessed by percentage. (b) Hydrophobicity profile of EZH2-TAD. (c) Luciferase reporter assay using the Gal4 DNA-binding domain (DBD) fusion of EZH2-TAD or VP16-TAD (a potent TAD as a positive control), compared to EV. Y-axis shows relative reporter activation after normalization of signals from an internal control (Renilla luciferase) and then to those of EV-transduced mock (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). *, **, and *** denote the P value of < 0.05, 0.01 and 0.005, respectively. NS denotes not significant. Numerical source data, statistics and exact P values are available as source data.

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Extended Data Fig. 4 Compared to C24 or MS177N1, MS177 is much more potent in inhibiting tumor cell growth.

(a-c) Plots showing the growth inhibitory effect of various used concentrations (x-axis; in the log10 converted values) of either C24 (a), MS177 (b) or MS177N1 (c) using a panel of six MLL-r acute leukaemia cell lines (that is, MV4;11, RS4;11, MOLM-13, KOPN-8, THP-1 and EOL-1 cells), treated for 2, 4 or 6 days. Y-axis shows relative cell growth after normalization to DMSO-treated controls (n = 3; mean ± SD).

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Extended Data Fig. 5 Biochemical characterization of the EZH2-targeting PROTAC degrader, MS177.

(a) Scheme showing the expected effect by MS177, MS177N1 (which contains a moiety that does not bind CRBN; indicated by a cross-mark) and MS177N2 (which contains a moiety that does not bind EZH2). (b) A radioactive methyltransferase assay (3H-labeled S-Adenosyl methionine [SAM] as methyl donor) showing that MS177 exhibits a high inhibition potency for EZH2 and a high selectivity for EZH2 over EZH1. X-axis and y-axis show the used concentration of MS177 (in Log scale) and the rate of inhibition (treatment versus mock), respectively (n = 3; mean ± SD). IC50, half maximal inhibitory concentration. (c) Selectivity of MS177 (10 μM, relative to mock) against a panel of 23 different lysine, arginine or DNA methyltransferases in radioactive methyltransferase assays (n = 3; mean ± SD). (d) Immunoblotting of the indicated histone modification in Hela cells after a 24-hour treatment with different concentrations of C24, MS177, MS177N1 or MS177N2, in comparison to mock (DMSO). (e) Immunoblotting of PRC2 subunits (GAPDH as a loading control) and global H3K27 methylation levels (H3 as a loading control) in EOL-1 cells post-treatment with DMSO, the indicated concentrations of MS177, or 2.5 μM of C24, MS177N1 or MS177N2 for 16 hours. (f) Immunoblotting of PRC2 subunits (GAPDH as a loading control) in MV4;11 cells after a 24-hour treatment with the increasing concentration of MS177, relative to mock (DMSO). (g) Measurement of half-maximal degradation concentration (DC50) value of MS177 in MV4;11 cells, based on EZH2 immunoblotting signals in the MS177-treated and mock-treated cells (n = 2 independent experiments; mean ± SD; quantified with ImageJ).

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Extended Data Fig. 6 Integrated ChIP-seq and RNA-seq analysis showing the EZH2:PRC2 on-target effect of MS177.

(a) Immunoblotting of EZH2, either nucleoplasmic (left) or chromatin-bound (right), in EOL-1 cells after a 16-hour treatment with DMSO or 2.5 µM of C24, MS177N1, MS177N2 or MS177. GAPDH and histone H3 serve as the cell fractionation controls. (b) IGV views of EZH2 and H3K27me3 ChIP-seq peaks at the indicated EZH2:PRC2 target gene post-treatment of EOL-1 (upper; for 16 hours) and MV4;11 cells (bottom; for 24 hours) with either DMSO or 0.5 μM of C24 or MS177. (c) Unsupervised clustering analysis using the RNA-seq-based transcriptome profiles of the three independent MLL-r acute leukaemia cell lines after the treatment with DMSO or 0.5 μM of MS177, C24 or MS177N1. MV4;11 and MOLM13 cells were treated for 24 hours, and EOL-1 cells for 16 hours (n = 2 replicated samples). (d) Bar plot showing the number of DEGs associated with the direct EZH2 binding in MV4;11 (left) or EOL-1 (right) cells. Up and down refer to those up- and down-regulated DEGs, respectively, based on RNA-seq analysis. (e) Volcano plot showing differential expression analysis of genes based on RNA-seq profiles of EOL-1 cells with EZH2 KO versus mock control (n = 2 replicated samples). The x-axis shows the log2 value of fold-change in gene expression (in KO versus vector-treated cells) and the y-axis shows the -log10 value of adjusted P (q) value, with the dashed lines indicating the cut-off of significance. (f) GSEA revealing that, relative to DMSO, MS177 treatment is positively correlated with upregulation of the indicated genes repressed by PRC2:EED (left) or bound by H3K27me3 (right) in MOLM-13 (top) or MV4;11 (bottom) cells. (g) DAVID functional annotation reveals that the DEGs up-regulated after EZH2 KO (sgEZH2_up) or MS177 treatment (MS177_up) in EOL-1 cells have similar enrichment for the immunity-related genes. (h) GSEA shows that, relative to their respective controls, both MS177 treatment (top) and EZH2 KO (bottom) in EOL-1 cells are positively correlated with upregulation of the indicated immunity-related genesets.

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Extended Data Fig. 7 MS177 represses Myc-related oncogenic nodes by inducing Myc protein ubiquitination and degradation.

(a) Heatmaps showing the EZH2 ChIP-seq signal intensities (normalized against spike-in control and sequencing depth) ± 5 kb around the centers of EZH2/H3K27me3-cobound ‘ensemble’ peaks in EOL-1 (left) and MV4;11 cells (right), treated for 16 and 24 hours respectively with DMSO (left), C24 (middle) or MS177 (right). (b) Bar plot showing the number of DEGs, down-regulated in EOL-1 (left) or MV4;11 (right) cells following the MS177 versus DMSO treatment, that displayed either the EZH2-‘solo’ or EZH2:PRC2 (‘ensemble’) binding. (c) Immunoblotting for cMyc in MLL-r leukemia cells treated with the indicated compound. (d) cMyc immunoblotting using the nucleoplasmic (left) and chromatin-bound (right) fractions of EOL-1 cells, treated with DMSO or 2.5 µM of C24, MS177N1, MS177N2 or MS177 for 16 hours. (e) RT-PCR for the indicated E3 ligase in EOL-1 and MV4;11 cells post-treatment with DMSO or 5 µM of MS177 for 4 hours (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). (f) RT-PCR for the indicated E3 ligase in MOLM-13 cells, stably expressed with an E3 ligase-targeting shRNAs or EV (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). (g) Immunoblotting for cMyc in MOLM-13 cells, stably expressed with an E3 ligase-targeting shRNA or EV, after the treatment with DMSO or 0.5 µM of MS177 for 24 hours. (h-i) Immunoblotting of EZH2, EED and N-Myc using lysate of Kelly cells after a 24-hour treatment with the increasing concentration of MS177 (h), in comparison to C24 and MS177’s non-PROTAC analogs (i). (j) N-Myc ubiquitination immunoblotting in Kelly cells, treated for 48 hours with 2.5 µM of DMSO, C24 or MS177. (k) Colony formation of Kelly cells treated with the indicated compound. (l) GSEA revealing significant enrichments of the indicated cMyc-upregulated (up) or cMyc-repressed (down) genesets in the MS177-treated (left/middle) or cMyc-depleted (sgcMyc; right) cells. (m) GSEA revealing a lack of significant correlation between cMyc-regulated genes and C24 treatment in EOL-1 cells. *, **, and *** denote P < 0.05, 0.01 and 0.005, respectively. NS denotes not significant. Numerical source data, statistics, exact P values and unprocessed blots are available as source data.

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Extended Data Fig. 8 MS177 exhibits potent effect on inducing leukaemia cell growth inhibition, apoptosis, and cell cycle progression arrest.

(a) Immunoblotting for EZH2 and GAPDH in four MLL-r leukaemia cell lines, treated with the indicated compound (0.5 µM) for 24 hours. (b) Effect of a 24-hour treatment with different concentrations of MS177 on proliferation of the two indicated primary samples from de-identified AML patients. Y-axis shows mean ± SD after normalization to DMSO-treated (n = 3). (c) Proliferation of primary AML cells treated with 1 µM of C24 or MS177, relative to DMSO, for 48 hours (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). (d-e) Immunoblotting for EZH2, PRC2 subunits and cMyc (d) and RT-qPCR for EZH2:H3K27me3-cobound genes (e) in primary AML cells, treated with the indicated compound (0.5 µM) for 24 hours. For e, y-axis shows RT-qPCR signals after normalization to those of GAPDH and to DMSO-treated cells (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). (f) Growth of K562 cells treated with MS177, relative to DMSO, for the indicated time. Y-axis shows mean ± SD after normalization to DMSO-treated (n = 3). (g) Representative view of colonies formed by murine HSPCs in the presence of DMSO or MS177. (h-i) Immunoblotting for EZH2 and IKZF1/3 (h) and growth (i) of EOL-1 cells after the indicated treatment of compound (0.5 µM for all). Pom, pomalidomide. For i, y-axis shows relative growth after normalization to DMSO-treated (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). (j) Representative flow cytometry-based histograms showing the DNA content in MOLM-13 cells, treated with indicated compound for 24 hours. (k-l) Immunoblotting for apoptotic markers in EOL-1 (k) or MV4;11 (l) cells after the indicated compound treatment. (m) Proliferation of the EZH2-depleted (sgEZH2) or control (sgEV) MOLM-13 cells, treated with DMSO or MS177 (0.1, 0.5 or 1 µM) for 48 hours (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). *, **, and *** denote the P value of < 0.05, 0.01 and 0.005, respectively. NS denotes not significant. Numerical source data, statistics, exact P values and unprocessed blots are available as source data.

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Extended Data Fig. 9 MS177 represses MLL-r leukaemia growth in multiple animal models established by human cell line xenograft or PDX.

(a) Violin plots showing complete blood counting (CBC) of white blood cells (WBC), red blood cells (RBC), neutrophils and lymphocytes, as well as hematocrit or hemoglobin, in C57BL/6 mice treated with either vehicle (n = 3) or MS177 (with a dose of 100 mg/kg, BID, 6 days/week, i.p. [n = 3]; or 200 mg/kg, BID, 3 days/week, i.p. [n = 2]) on day 5, 10, 15 and 21. The boundaries of the violin plots indicate the 25th and 75th percentiles. (b) The body weight change of C57BL/6 mice treated with either vehicle (n = 3; mean ± SD) or MS177 (100 mg/kg, BID, 6 days/week, i.p. [n = 3; mean ± SD]; or 200 mg/kg, BID, 3 days/week, i.p. [n = 2; mean ± SD]) over a course of 21 days. (c-d) Flow cytometry-based analysis for the expression of human-specific cell surface antigens (~93% as hCD45 + /hCD33 + , c) and GFP (from a cell-labeling construct, d) among the splenic cells harvested from leukemic mice, established by intravenous (i.v.) injection of an MLL-r AML PDX line carrying the stably expressed luciferase/GFP reporter (PDX # 68555-Luc). (e-f) Body weight change of NSG-SGM3 mice bearing the MLL-r AML PDX tumors, xenografted either intravenously (e) or subcutaneously (s.c.; f), as measured from the starting time point of treatment with the indicated dose of vehicle or MS177 over a course of 21 days. Mean ± SD. (g) Body weight change of NSG mice bearing the subcutaneous RS4;11 cell xenografts, as measured from the starting point of treatment with the indicated dose of vehicle or MS177 over a course of 21 days. Mean ± SD. (h) Immunoblotting for the indicated proteins using a collection of the s.c. xenografted EOL-1 tumors, which were freshly isolated from NSG mice treated with vehicle or 200 mg/kg of MS177 (BID per day) for a total of 5 days. Numerical source data and unprocessed blots are available as source data.

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Supplementary information

Supplementary Note 1

This file contains part of the methods and the general scheme of the synthetic route for the compounds used in this study.

Reporting Summary

Supplementary Tables

Supplementary Table 1: RNA-seq identifies DEGs significantly downregulated in MV4;11 cells after EZH2 KD or cMyc KO, relative to the respective mock treatment. Supplementary Table 2: Selectivity of MS177 against 45 kinases. Supplementary Table 3: Selectivity of MS177 against 33 GPCRs, ion channels and transporters. Supplementary Table 4: Summary of total read tag counts from the spike-in controlled ChIP-seq experiments after mapping to the hg19 or dm3 genome. Supplementary Table 5: RNA-seq identifies DEGs showing significant upregulation in the three independent MLL-r AML cell lines (EOL-1, MV4;11 or MOLM-13 cells) after treatment with MS177, C24 or MS177N1, relative to DMSO mock. Supplementary Table 6: Summary of the total number of downregulated or upregulated DEGs identified by RNA-seq in MV4;11, MOLM-13 or EOL-1 cells after treatment with MS177, C24 or MS177N1, in comparison to DMSO. Supplementary Table 7: RNA-seq identifies DEGs showing significant upregulation in EOL-1 cells with EZH2 KO relative to mock treatment. Supplementary Table 8: Summary of a panel of primary samples from the de-identified patients with AML, newly diagnosed, in remission (primarily healthy cells based on real-time PCR-based analysis of gene mutations) or relapsed. Supplementary Table 9: EC50 values of MS177, MS177N1 and C24 in the indicated AML cell lines and primary AML patient samples after 4 days of treatment. Supplementary Table 10: Pharmacokinetics parameters of MS177 in plasma. Supplementary Table 11: Sequence information for primers, shRNA and sgRNA used in this study. Supplementary Table 12: Antibodies used in this study.

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Wang, J., Yu, X., Gong, W. et al. EZH2 noncanonically binds cMyc and p300 through a cryptic transactivation domain to mediate gene activation and promote oncogenesis. Nat Cell Biol 24, 384–399 (2022). https://doi.org/10.1038/s41556-022-00850-x

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