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Epigenetic targeted therapy of stabilized BAP1 in ASXL1 gain-of-function mutated leukemia

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.

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Fig. 1: Endogenous truncated mutant ASXL1 is stable.
Fig. 2: Genome-wide binding of ASXL1 mutants.
Fig. 3: GOF ASXL1 mutants stabilize BAP1 and increase BAP1 recruitment in leukemia.
Fig. 4: Identification of BAP1 inhibitor by small-molecule screening.
Fig. 5: iBAP selectively inhibits cells with ASXL1 GOF mutations.
Fig. 6: iBAP delays the progression of ASXL1-mutant leukemia and improves survival.

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. 1,46 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

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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

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Authors

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

Correspondence to Ali Shilatifard.

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The authors declare no competing interests.

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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.

Source data

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.

Source data

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).

Source data

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.

Source data

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).

Source data

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

Reporting Summary

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

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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

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