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.
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The shRNA screen sequencing data have been deposited to the NCBI Sequence Archieve under the accession number GSE120813. Crystal structure data for HBO1–BPRF2 in complex with WM-3835 and acetyl-CoA have been submitted to the PDB under accession numbers 6MAJ (WM-3835) and 6MAK (acetyl-CoA). Source Data are provided for Figs. 1–4. Any other relevant data are available from the corresponding author upon reasonable request.
All code used in this study is publically available.
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We thank C. Lovitt, J. Wingerd, S. Jackson and E. Allan for their technical contributions to this project. The work in the Dawson, Blewitt and Burns laboratories was supported by the Cancer Council Victoria Venture Grant Scheme, and Dawson laboratory work was supported by project grant funding from the National Health and Medical Research Council of Australia (1085015). We thank the following funders for fellowship and grant support: Leukaemia Foundation Australia senior fellowship, Cancer Council Victoria Dunlop Fellopship and Howard Hughes Medical Institute international research scholarship (M.A.D.); Victoria Cancer Agency early-career (L.M.) and mid-career (E.Y.N.L.) fellowships; CSL Centenary fellowship (S.-J.D.), Snowdome Foundation (P.Y.), Maddie Riewoldt’s Vision Foundation (Y.-C.C.), Bellberry-Viertel Senior Medical Research Fellowship (M.E.B), Novo Nordisk Foundation Hallas Møller Fellowship NNF14OC0008541 (C.C.), National Health and Medical Research Council of Australia through project grants 1081421 (J.B.B. and T.T.), 575558, 1084248 (A.K.V. and T.T.), research fellowship 1081421 (A.K.V.) and postgraduate scholarship (K.-L.C.). Salary support for M.Z., M.d.S., H.F., C.C., P.S.K., P.A.S., I.P.S. and B.J.M. was provided by the Cancer Therapeutics CRC, funded through the Australian Government’s Cooperative Research Centre programme. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001). This work was made possible through the Victorian State Government Operation Infrastructure Support and Australian National Health and the Medical Research Council Research Institute Infrastructure Support Scheme. We thank the Australian Synchrotron and beamline scientists for help with data collection; this research was undertaken in part using the MX2 beamline at the Australian Synchrotron and made use of the ACRF detector.
M.A.D. has been a member of advisory boards for CTX CRC, Storm Therapeutics, Celgene and Cambridge Epigenetix. The Dawson, Thomas and Voss laboratories receive research funding from CTx CRC. The remaining authors declare no competing financial interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Stefan Knapp and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, RNA interference screen multidimensional scaling plot of shRNA sequences from bulk leukaemic blasts (bulk) and LSCs over 14 days of screening. Screens were performed in duplicate (R1 and R2). b, Expression of Ezh2, Lsd1 or Prmt5 mRNA, relative to non-targeting shRNA in LSCs that express shRNAs. n = 3.Mean + s.e.m. c, Negative-selection competition assays in bulk leukaemic blasts and LSCs that express shRNAs against Ezh2, Lsd1, Prmt5 or Dot1l. n = 3. Mean + s.e.m. Validation of the shRNAs against Dot1l have previously been published54. d. Expression of Hbo1 mRNA, relative to non-targeting shRNA, in LSCs that express shRNAs. n = 3. Mean + s.e.m.
a. Box plot of HBO1 expression levels in cancer types from The Cancer Genome Atlas (TCGA)55. The upper limit, centre and lower limit of each box denotes the upper quartile, median and lower quartile of the data, respectively. Cohort abbreviations refer to TCGA study abbreviations (https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). b, Dot plot of levels of HBO1 expression in normal haematopoietic and AML cells from BloodSpot56; horizontal line indicates mean expression.
Extended Data Fig. 3 HBO1 depletion increases apoptosis, cell-cycle arrest and myeloid differentiation in mouse LSCs.
a, Negative-selection competition assays in LSCs that express shRNAs against Moz, Qkf or Hbo1. n = 3. Mean + s.e.m. b, Negative-selection competition assays in LSCs that express Cas9, transduced with sgRNAs targeting Moz or Qkf. n = 3. Mean + s.e.m. c, Expression of members of the HBO1 complex, relative to non-targeting shRNA in LSCs that express shRNAs targeting individual members of the complex. n = 3. Mean + s.e.m. d, Apoptosis of LSCs that express shRNAs targeting Hbo1. n = 3. Mean + s.e.m. e, Cell-cycle profile of LSCs that express shRNAs targeting Hbo1. n = 3. Mean + s.e.m. f, Surface expression of GR1 and CD11b in LSCs that express shRNAs against Hbo1. n = 3. g. Apoptosis of LSCs that express Cas9 and sgRNAs targeting Hbo1. n = 3. Mean + s.e.m. h, Surface expression of KIT (also known as CD117) in LSCs that express sgRNAs targeting Hbo1. Representative of n = 2 biological replicates.
a, Kaplan–Meier curves of NSG mice transplanted with quinary MLL–AF9 leukaemic cells that express shRNAs targeting Hbo1 or Moz. n = 6 mice per group. b, Schematic of wild-type and Hbo1-mutant alleles with numbered black boxes representing exons. Genotyping primers are indicated (adapted from ref. 10). c, Kaplan–Meier curves of C57BL/6 mice injected with 106 Hbo1flox/+ Mx1-cre primary MLL–AF9 leukaemic cells. Mice were treated with saline or pIpC. n = 12 mice per group. Right, representative genotyping of recipient bone marrow at end point. d, Kaplan–Meier curves of C57BL/6 mice injected with Moz+/+, Moz+/− or Moz−/− MLL–AF9 leukaemic cells. n = 5 mice per group. e, Kaplan–Meier curves of BALB/c mice injected with Moz+/+ or Moz−/− HOXA9 and MEIS1 leukaemic cells. n = 5 mice per group.
a, Negative-selection competition assays in human AML cell lines that express Cas9 and sgRNAs targeting HBO1. n = 3. Mean + s.e.m. Driver mutations are in parentheses. b, Immunoblot of HBO1 and H3K14ac in OCI-AML3 cells that express Cas9 and sgRNAs targeting HBO1. n = 3. Mean + s.e.m. c, Cell-cycle profile of OCI-AML3 cells that express Cas9 and sgRNAs targeting HBO1. n = 3. Mean + s.e.m. d, Apoptosis of OCI-AML3 cells that express Cas9 and sgRNAs targeting HBO1. n = 3. Mean + s.e.m. e, Surface expression of CD11b in OCI-AML3 cells that express Cas9 and sgRNAs targeting HBO1. n = 3.
Negative-selection competition assays in human cancer cell lines that express Cas9 and sgRNAs targeting HBO1. n = 3. Mean + s.e.m. Cancer type is in parentheses.
a, Barcode plot evaluating changes in myeloid development signature after HBO1 depletion with Hbo1 e11.1 and Hbo1 e12.2 sgRNAs in LSCs. n = 3. b, Bar plot of changes in gene expression after Hbo1 deletion in LSCs, ranked by expression levels. Green bars show the top 25 most-downregulated genes after Hbo1 deletion. c, HOXA9 and HOXA10 mRNA expression in Molm13 and OCI-AML3 cells that express Cas9 and sgRNA targeting HBO1. n = 3. Mean ± s.e.m. d, Surface expression of CD11b in LSCs that overexpress Hoxa9 or Hoxa10, and sgRNAs targeting Hbo1. e, Immunoblot of overexpressed HOXA9 or HOXA10 in LSCs that express Cas9. Representative of n = 3 biological replicates. f, Hoxa9- and Hoxa10-rescue negative-selection competition assays. Representative of n = 3 biological replicates. g, ChIP–seq profiles of H3K14ac and RNA Pol-II at the Pbx3 locus in LSCs that express Cas9 and an sgRNA targeting Hbo1. Representative of n = 3 biological replicates. h, RNA Pol-II travelling ratio distribution for highly expressed genes, divided according to H3K14ac levels from ChIP–seq. i, Surface expression of GR1 in LSCs that overexpress sgRNAs targeting Smarca5. Representative of n = 3 biological replicates.
a, KAT biochemical and SPR values for WM-3835 compared to WM-1119. Biochemical assay was done at 1 μM acetyl-CoA, the Km of HBO1. b, Proliferation assays of human AML cells treated with 1 μM WM-2474. n = 3. Mean + s.e.m. c, Cellular H3K14ac biomarker assay dose–response curves for WM-3835 (blue) and WM-1119 (red). n = 6. Mean ± s.e.m. d, Growth inhibition assays of the MLL–AF9 AML cell line Molm13 treated with WM-3835 at the doses indicated. Boxes represent minimum and maximum values. n = 11. Mean ± s.e.m. e, Cell-cycle profile of OCI-AML3 cells treated with WM-3835 or vehicle. n = 3. Mean + s.e.m. f, Apoptosis of OCI-AML3 cells treated with WM-3835 or vehicle. n = 3. Mean + s.e.m. g, Surface expression of CD11b in OCI-AML3 cells treated with WM-3835 or vehicle. n = 3. h, HOXA10 mRNA expression in LSCs and Molm13 cells treated with WM-3835 or vehicle. n = 3. Mean + s.e.m. i, HOXA9 and HOXA10 mRNA expression in OCI-AML3 cells treated with WM-3835. n = 3. Mean + s.e.m. j, Hoxa9 and Hoxa10 rescue proliferation assays with 1 μM WM-3835 in LSCs. n = 3. Mean ± s.e.m.
a, Data collection and refinement statistics of WM-3835 HBO1–BRPF2 co-crystal structure. b, WM-3835 binding site in HBO1–BRPF2. WM-3835 is shown in silver with element colouring and the omit electron density map, contoured to 3σ, is shown in green. c, Overlay of WM-3835 and acetyl-CoA (purple with element colouring), showing that WM-3835 binds in the acetyl-CoA binding site of HBO1. d, Ribbon diagram of HBO1–BRPF2 showing WM-3835 bound to the acetyl-CoA binding site. e, Space-filling model showing WM-3835 (yellow with element colouring) in the acetyl-CoA binding pocket of HBO1–BRPF2.
a, Proliferation assays of human AML cell lines treated with 1 μM WM-3835. b, WM-3835 demonstrates high clearance in both human and mouse liver microsome assays. The use of dual cofactors (UDPGA and NADPH) results in an increased rate of clearance in human liver microsomes, which is consistent with glucuronidation having a role in the clearance of this compound. c, BALB/c female mice were dosed with WM-3835 at 100 mg kg−1 taken by mouth twice a day, formulated in 20% PEG400/10% solutol or vehicle. Four hours after the third dose, blood samples were collected. An average total drug concentration of 1,860 nM was observed. The free drug level was determined to be 2.6 nM after accounting for mouse plasma protein binding (fraction unbound 0.0014). This free drug level was considered too low to affect the acetylation of H3 at K14, on the basis of the in vitro H3K14ac cellular biomarker data (Fig. 4). An additional chromatographic peak eluting earlier than WM-3835 was detected in the plasma samples from the treatment group. Subsequent analyses using predicted multiple-reaction monitoring and accurate mass measurement indicated that this peak is likely to be a glucuronide conjugate of WM-3835, consistent with the in vitro metabolism data.
This file contains supplementary methods detailing the chemistry for WM-3835.
Contains scans of uncropped images of western blot gels.
Number of shRNAs depleted by more than 5-fold in leukaemic stem cell and blast cell populations from screen.
A list of acetylation sites quantified in core histones. Site-specific acetylation of core histones was quantified in HBO1 knockout and control cells using SILAC-based mass spectrometry. The data shows median SILAC ratio (HBO1 knockout/Control) from three independent biological replicates.
shRNA, sgRNA and mRNA primer sequences.
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MacPherson, L., Anokye, J., Yeung, M.M. et al. HBO1 is required for the maintenance of leukaemia stem cells. Nature 577, 266–270 (2020). https://doi.org/10.1038/s41586-019-1835-6
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