Abstract
Mutations of ASXL1, encoding a component of the BAP1 histone H2A deubiquitinase complex, occur in human myeloid neoplasms and are uniformly associated with poor prognosis. However, the precise molecular mechanisms through which ASXL1 mutations alter BAP1 activity and drive leukemogenesis remain unclear. Here we demonstrate that cancer-associated frameshift mutations in ASXL1, which were originally proposed to act as destabilizing loss-of-function mutations, in fact encode stable truncated gain-of-function proteins. Truncated ASXL1 increases BAP1 protein stability, enhances BAP1 recruitment to chromatin and promotes the expression of a pro-leukemic transcriptional signature. Through a biochemical screen, we identified BAP1 catalytic inhibitors that inhibit truncated-ASXL1-driven leukemic gene expression and impair tumor progression in vivo. This study represents a breakthrough in our understanding of the molecular mechanisms of ASXL1 mutations in leukemia pathogenesis and identifies small-molecular catalytic inhibitors of BAP1 as a potential targeted therapy for leukemia.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
ChIP-seq and RNA-seq data generated for this study are available at the Gene Expression Omnibus under accession number GSE166305. Source data for Figs. 1,4–6 and Extended Data Figs. 1,5 and 6 have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
Code availability
Custom scripts for analyzing NGS data will be made available upon request. The source code of Ceto pipeline used for analyzing the NGS data from this study is available at the GitHub site: https://github.com/ebartom/NGSbartom
References
Lawrence, M. S. et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499, 214–218 (2013).
Wang, L. et al. Resetting the epigenetic balance of Polycomb and COMPASS function at enhancers for cancer therapy. Nat. Med. 24, 758 (2018).
Wang, L. & Shilatifard, A. UTX mutations in human cancer. Cancer Cell 35, 168–176 (2019).
Morgan, M. A. & Shilatifard, A. Chromatin signatures of cancer. Genes Dev. 29, 238–249 (2015).
Rasmussen, K. D. & Helin, K. Role of TET enzymes in DNA methylation, development, and cancer. Gene Dev. 30, 733–750 (2016).
Zhang, J. Y. et al. Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis. Nat. Med. 21, 1190 (2015).
Ortega-Molina, A. et al. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development. Nat. Med. 21, 1199–1208 (2015).
Szczepanski, A. P. et al. ASXL3 bridges BRD4 to BAP1 complex and governs enhancer activity in small cell lung cancer. Genome Med. 12, 63 (2020).
Szczepanski, A. P. & Wang, L. Emerging multifaceted roles of BAP1 complexes in biological processes. Cell Death Discov. 7, 20 (2021).
Dey, A. et al. Loss of the tumor suppressor BAP1 causes myeloid transformation. Science 337, 1541–1546 (2012).
Misaghi, S. et al. Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol. Cell. Biol. 29, 2181–2192 (2009).
Gelsi-Boyer, V. et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Brit. J. Haematol. 145, 788–800 (2009).
Bejar, R. et al. Clinical effect of point mutations in myelodysplastic syndromes. N. Engl. J. Med. 364, 2496–2506 (2011).
Patel, J. P. et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N. Engl. J. Med. 366, 1079–1089 (2012).
Abdel-Wahab, O. et al. ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell 22, 180–193 (2012).
Balasubramani, A. et al. Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex. Nat. Commun. 6, 7307 (2015).
Yang, H. et al. Gain of function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies. Blood 131, 328–341 (2018).
Campagne, A. et al. BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation. Nat. Commun. 10, 348 (2019).
Guo, Y. et al. Reduced BAP1 activity prevents ASXL1 truncation-driven myeloid malignancy in vivo. Leukemia 32, 1834–1837 (2018).
Yu, H. et al. The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression. Mol. Cell. Biol. 30, 5071–5085 (2010).
Asada, S. et al. Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis. Nat. Commun. 9, 2733 (2018).
Inoue, D. et al. Myelodysplastic syndromes are induced by histone methylation-altering ASXL1 mutations. J. Clin. Invest. 123, 4627–4640 (2013).
Carbone, M. et al. Tumour predisposition and cancer syndromes as models to study gene-environment interactions. Nat. Rev. Cancer 20, 533–549 (2020).
Carbone, M. et al. Biological mechanisms and clinical significance of BAP1 mutations in human cancer. Cancer Discov. 10, 1103–1120 (2020).
Bononi, A. et al. BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation. Nature 546, 549 (2017).
Morris, M. R. & Latif, F. The epigenetic landscape of renal cancer. Nat. Rev. Nephrol. 13, 47–60 (2017).
Verdijk, R. et al. Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Virchows Arch. 465, S44–S44 (2014).
Murali, R., Wiesner, T. & Scolyer, R. A. Tumours associated with BAP1 mutations. Pathology 45, 116–126 (2013).
Qin, J. Y. et al. BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. Nat. Commun. 6, 8471 (2015).
He, M. et al. Intrinsic apoptosis shapes the tumor spectrum linked to inactivation of the deubiquitinase BAP1. Science 364, 283–285 (2019).
Sahtoe, D. D., van Dijk, W. J., Ekkebus, R., Ovaa, H. & Sixma, T. K. BAP1/ASXL1 recruitment and activation for H2A deubiquitination. Nat. Commun. 7, 10292 (2016).
Daou, S. et al. The BAP1/ASXL2 histone H2A deubiquitinase complex regulates cell proliferation and is disrupted in cancer. J. Biol. Chem. 290, 28643–28663 (2015).
Mashtalir, N. et al. Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol. Cell 54, 392–406 (2014).
Fang, Y., Fu, D. & Shen, X. Z. The potential role of ubiquitin C-terminal hydrolases in oncogenesis. Bba-Rev. Cancer 1806, 1–6 (2010).
D’Arcy, P. et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat. Med. 17, 1636–U1150 (2011).
Anders, S., Pyl, P. T. & Huber, W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Saldanha, A. J. Java Treeview-extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
Tripathi, S. et al. Meta- and Orthogonal integration of influenza ‘OMICs’ data defines a role for UBR4 in virus budding. Cell Host Microbe 18, 723–735 (2015).
Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008).
Shen, L., Shao, N. Y., Liu, X. C. & Nestler, E. ngs.plot: quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genomics 15, 284 (2014).
Acknowledgements
We thank M. Mrksich for providing the compound library for our screening. We thank A. P. Szczepanski for editorial assistance. We thank F. Zhang for the kind gifts of the lentiCRISPR v2 vectors. Z.Z. is supported in part by National Institutes of Health/National Cancer Institute training grant T32 CA070085 and Alex’s Lemonade Stand Foundation. Studies in the Shilatifard laboratory related to this project are supported by National Cancer Institute’s Outstanding Investigator Award R35CA197569.
Author information
Authors and Affiliations
Contributions
L.W. and A. Shilatifard designed the study; L.W. performed the majority of the biochemistry and in vitro experiments; N.W.B., C.M.N., A.K., A. Shilati and A.B. performed all of the animal experiments; E.J.R., D.Z. and C.A.R. generated and sequenced the NGS libraries; Z.Z. and P.A.O. performed bioinformatics analysis; L.W., Z.Z., M.A.J.M. and A. Shilatifard revised the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Cancer thanks David Vetrie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Generation of anti-ASXL1-NTD antibody.
a, Mutation status of K562 cell line. b, Schematic of human ASXL1 protein and the antigen peptide for generation of the polyclonal antibody. c, Mutations status of ASXL1 gene in K562, 293T-ASXL1-WT, 293T-ASXL1-Y591* mutant cells. d, The HEK293T-ASXL1-WT and –Y591* cells were treated with 50 μg/ml CHX for different time. The BAP1 protein was determined by western blot, n=2 independent experiments (d) and further quantified by imageJ e. f, An N-terminal 591 amino acid portion of human ASXL1 gene was expressed as a GFP-tagged fusion protein in HEK293T cells and subjected to GFP-purification from nuclear extracts and used for mass spectrometry analysis. Peptide numbers of each subunit of BAP1 complex purified by ASXL1-NTD were shown. g, Heat map showing the common downstream genes in ASXL1-Y591* 293T cells. h, The venn diagram shows the up-regulated and downregulated genes in HEK293T-ASXL1-WT/Y591* and HEK293T-ASXL1-Y591*/Y591* comparing with HEK293T-ASXL1-WT cells, n=2 independent experiments. i, Representative tracks showing the expression level of BAP1 in 293T-ASXL1-WT and mutant cells. j, GSEA analysis shows the most enriched gene expression signature in ASXL1-Y591* 293T cells.
Extended Data Fig. 2 Genome-wide binding of ASXL1 mutants.
a, Schematic of the human ASXL1 gene locus and the CRISPR gRNA designed to target promoter and Exon 1 of the ASXL1 gene. b, RNA-seq was performed in ASXL1-WT and ASXL1-KO cells, and the representative tracks show the depletion of ASXL1 gene in CAL51 cells, n=2 independent experiments. c, ChIP-seq track example shows the specificity of ASXL11 antibodies in ASXL1- WT and ASXL1-KO cells. d, ChIP-seq track example shows the occupancy of H3K27me3 level in ASXL1-WT, ASXL1-WT-GSK126-treated and ASXL1-KO cells. e, The average plot (upper panel) and the heat map (lower panel) show the H3K27me3 levels in ASXL1-WT, ASXL1-WT- GSK126-treated and ASXL1-KO cells. f, The metaplot shows ASXL1 and H2K119Ub peaks from ASXL1-WT and ASXL1-Y591* cells are centered on BAP1 peaks at Cluster 1-2 (left) and Cluster 3-5 (right) loci. g, ChIP-seq analysis of H2AUb level in ASXL1-WT and ASXL1- Y591* cells at BAP1 binding regions.
Extended Data Fig. 3 GOF ASXL1 mutants stabilizes BAP1 and increases BAP1 recruitment.
a, Exon 12 of the ASXL1 gene was chosen for targeting with CRISPR-CAS9. The sequence of mutated allele is shown containing frame-shift mutations. b, Whole-cell lysates were used for western blot with ASXL1-NTD antibody in THP1-ASXL1-WT and THP1-ASXL1-Y591fs cells, n=3 independent experiments. c, Whole-cell lysates were used for immunoprecipitation with ASXL1-NTD antibodies followed by immunoblotting for BAP1 in cells expressing wild-type ASXL1 or frame shift ASXL1 truncations, n=3 independent experiments. d, Pathway analysis of the significantly up-regulated genes in ASXL1-Y591fs cells was performed with Metascape.
Extended Data Fig. 4 Small-molecule screening for BAP1 inhibitor.
a, Purification of recombinant BAP1 from bacteria. b, Optimization of Ub-AMC assay with recombinant BAP1, n=3 independent experiments. c, CAL51 cells were treated with 10 μM iBAP for 24 hours. The protein level of total histone H2A and H2AK119Ub level was determined by western blot, n=3 independent experiments. d, 293T-BAP1-WT and –KO cells were treated with 0.1, 1 and 10 μM of iBAP for 24 hours. The gene expression in each group was determined by RNA-seq, n=2 independent experiments. The venn-diagram shows the number of genes that are significantly up-regulated or down-regulated by iBAP. e, BAP1 peaks in CAL51 was divided into five clusters by K-means clustering. The fold-change heat map shows H2AK119Ub level change at BAP1 peaks (Left). The right panel shows the log2 (fold change) of nearby gene expression in BAP1-KO cells vs BAP1-WT cells, or iBAP treated vs DMSO. f, CAL51-BAP1-WT/KO cells were treated with iBAP for four days. The time-dependent cell viability was determined by cell counting assay (n=2 independent experiments). g, Pathway analysis by Metascope shows the co-regulated pathways in BAP1-KO cells or iBAP treated cells. h, The mRNA level of genes involved in MYC and p53 pathways were validated by real-time PCR (n = 3 technical replicates).
Extended Data Fig. 5 iBAP selectively inhibits cells with ASXL1 GOF mutations.
a, Whole-cell lysates were used for western blot with BAP1 and ASXL1 NTD antibody for BAP1 and ASXL1 in THP1-ASXL1-WT (infected with non-targeting gRNA) and THP1- ASXL1-G710fs cells. HSP90 was used as loading control, n=3. b, THP1-ASXL1-WT, THP1- ASXL1-G710fs cells were treated with different concentrations of iBAP for 72 hours, the cell viability was determined by cell counting assay (n = 3 independent experiments). c, The cell proliferation ability of THP1-WT, THP1-ASXL1-Y591fs, and THP1-ASXL1-G710fs cells were determined by cell counting assay (n = 3 independent experiments) (left panel). The cell proliferation ability of K562 cells transduced with either non-targeting shRNA or two distinct ASXL1 shRNA were determined by cell counting assay (n = 3 independent experiments). Data are presented as mean ± s.d. *P < 0.05; **P < 0.01 by two-tailed unpaired Student’s t-test. d, MCF7 and CAL51 cells were treated with different concentrations of iBAP for three days. The cell viability was determined by cell counting assay. DMSO was used as negative control (n = 3 independent experiments). e, Representative tracks showing the expression level of CDC42 and MFF genes in THP1-ASXL1-WT and THP1-ASXL1-Y591fs treated with either DMSO or iBAP.
Extended Data Fig. 6 Toxicity test for iBAP in vivo.
a, Mice were treated with different dosage (10, 25, 50 and 100 mg/kg) of iBAP once per day for five days. The body weight of mice from each group was measured. b, The IC50 from different iBAP analogs was determined by cell counting assay and normalized with iBAP IC50. c, Comparison of iBAP analog ability to inhibit BAP1 catalytic activity in vitro and compound solubility in vehicle to guide further compound development. d, Mice were treated with different dosage iBAP analogs once per day for five days. The body weight of each mouse was measured as a change in percentage from baseline weight, n=3. e, Relative luminescence intensity is shown for representative mice treated with either vehicle control or 50mg/kg of iBAP or analog following fourteen daily treatments. f, Relative change in whole-body luminescence intensity of xenotransplanted mice during fourteen days of treatment with iBAP or analogs (n=10 for iBAP, n=8 for vehicle, and n=4 for all other treatment groups; p<0.05 by by two-tailed unpaired Student’s t-test for Veh vs. *iBAP and analogs †#2, ‡#4, ʇ#10, §#16, or ¶#17).
Extended Data Fig. 7 Structure of iBAP analogs.
The structure of analogs of iBAP molecule. Structures of all of these analogs were obtained as iBAP analogs directly from ChemBridge Corp (https://www.chembridge.com/). All of the compounds were purchased from Chembridge.
Supplementary information
Supplementary Tables 1 and 2
Supplementary Table 1. Small molecule screening data. The table summarizes the small-molecule compound screening platform, details for the screening assay and compound library information, as well as the candidate prioritization criteria. Supplementary Table 2. Structure–activity relationship (SAR) table. The core structure of iBAP and the side chain (R1 and R2) of each analog. The IC50 for BAP1 enzyme inhibition, IC50 for K562 cell viability was also provided for each of the analogs
Source data
Source Data Fig. 1
Statistical Source Data
Source Data Fig. 1
Unprocessed western blots
Source Data Fig. 3
Unprocessed western blots
Source Data Fig. 4
Statistical Source Data
Source Data Fig. 4
Unprocessed western blots
Source Data Fig. 5
Statistical Source Data
Source Data Fig. 6
Statistical Source Data
Source Data Fig. 6
Unprocessed western blots
Source Data Extended Data Fig. 1
Unprocessed western blots
Source Data Extended Data Fig. 3
Unprocessed western blots
Source Data Extended Data Fig. 4
Statistical Source Data
Source Data Extended Data Fig. 4
Unprocessed western blots
Source Data Extended Data Fig. 5
Statistical Source Data
Source Data Extended Data Fig. 5
Unprocessed western blots
Source Data Extended Data Fig. 6
Statistical Source Data
Rights and permissions
About this article
Cite this article
Wang, L., Birch, N.W., Zhao, Z. et al. Epigenetic targeted therapy of stabilized BAP1 in ASXL1 gain-of-function mutated leukemia. Nat Cancer 2, 515–526 (2021). https://doi.org/10.1038/s43018-021-00199-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s43018-021-00199-4
This article is cited by
-
A reappraisal of ASXL1 mutation sites and the cohesin-binding motif in myeloid disease
Blood Cancer Journal (2023)
-
Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase
Nature (2023)
-
Proposals for Clinical Trials in Chronic Myelomonocytic Leukemia
Current Treatment Options in Oncology (2023)
-
H2A monoubiquitination: insights from human genetics and animal models
Human Genetics (2023)
-
BAP1-related signature predicts benefits from immunotherapy over VEGFR/mTOR inhibitors in ccRCC: a retrospective analysis of JAVELIN Renal 101 and checkmate-009/010/025 trials
Cancer Immunology, Immunotherapy (2023)
