BET inhibitor resistance emerges from leukaemia stem cells

Journal name:
Nature
Volume:
525,
Pages:
538–542
Date published:
DOI:
doi:10.1038/nature14888
Received
Accepted
Published online

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks1, 2. Early clinical trials have shown promise, especially in acute myeloid leukaemia3, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL–AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

At a glance

Figures

  1. Establishment of a model of BET inhibitor resistance.
    Figure 1: Establishment of a model of BET inhibitor resistance.

    a, Strategy for the generation of resistant clones. HSPC, haematopoietic stem and progenitor cells. b, Resistance to I-BET demonstrated in cell proliferation assays performed in biological triplicate (mean ± s.d.). c, Resistance to JQ1 demonstrated in cell proliferation assays performed in biological triplicate (mean ± s.d.). d, Resistance to I-BET in clonogenic assays performed in biological duplicate (mean ± s.e.m.). e, Resistance to I-BET-mediated induction of apoptosis in biological triplicate experiments (mean ± s.e.m.). f, Resistant clones do not demonstrate cell cycle arrest in biological triplicate experiments (mean ± s.e.m.). g, Resistance to shRNA-mediated knockdown of Brd4 in biological duplicate experiments (mean ± s.e.m.). h, i, Kaplan–Meier curve of secondary syngeneic transplant of sensitive (h) and resistant (i) clones (n = 6 per group, statistical significance calculated using a log-rank test). Dotted line denotes treatment starting on day 9.

  2. Resistance to BET inhibitors arises from the LSC compartment.
    Figure 2: Resistance to BET inhibitors arises from the LSC compartment.

    a, Intracellular and extracellular concentrations of I-BET as assessed by quantitative mass spectrometry in biological duplicates (mean ± s.e.m., statistical significance calculated using a two-tailed Student’s t-test). NS, not significant. b, Resistant clones demonstrate an immature immunophenotype (Gr1/CD11b). c, Clonogenic capacity of the Gr1/CD11b and Gr1+/CD11b+ populations in resistant clones performed in biological duplicate (mean ± s.e.m.). d, Limiting dilution transplantation analyses Kaplan–Meier curves of C57BL/6 mice injected with indicated number of cells; detailed cohort and survival data can be found in Extended Data Fig. 3a. e, LSC frequency from limiting dilution transplantation analyses. Dotted lines indicate 95% confidence intervals (CI). f, L-GMP frequency in whole mouse bone marrow (mean ± s.e.m.) after serial transplantation of I-BET-exposed leukaemias from in vivo resistance model. Statistical significance of survival outcomes determined using log-rank test of Kaplan–Meier survival estimates. g, Proportion of human leukaemic CD34+ cells, GMPs and LMPPs in whole mouse bone marrow (mean ± s.e.m.) after I-BET exposure in an AML PDX model (n = 5). h, I-BET-naive L-GMPs do not demonstrate intrinsic resistance to I-BET in clonogenic assays in biological duplicate experiments (mean ± s.e.m.), see also Extended Data Fig. 5b. M1, mouse 1.

  3. Genetic, epigenetic and transcriptional characterization of BET-inhibitor-resistant cells.
    Figure 3: Genetic, epigenetic and transcriptional characterization of BET-inhibitor-resistant cells.

    a, Proliferation assays in sensitive, resistant cells maintained in I-BET and after 8 weeks drug withdrawal (mean ± s.d., n = 12 per group). b, Cell cycle profile in resistant clones after drug withdrawal performed in biological triplicate experiments (mean ± s.e.m.). c, Immunophenotype of sensitive, resistant and drug-withdrawal cells. d, Whole-exome capture sequencing data from vehicle-treated and resistant clones normalized to the parental cell line; red regions denote copy number loss and green regions denote copy number gain. e, Brd4 binding profiled across all annotated transcriptional start sites (TSSs). f, Brd4 binding and histone 3 Lys 27 acetylation (H3K27ac) at Myc enhancer elements. g, Principle component (PC) analysis of parental cells, vehicle-treated clones (n = 4), resistant clones (n = 9) and resistant clones after drug withdrawal (n = 2). Parentheses denote concentration of I-BET (nM) in which resistant clones have been stably maintained. h, GSEA identifies enrichment of a published LSC signature in resistant clones. Upregulated and downregulated genes in the published LSC signature are shown in red and blue, respectively, and correlate with upregulated and downregulated (false discovery rate (FDR) < 5.0 × 10−5) genes in the I-BET-resistant clones. i, Statistically significant upregulation (shaded red) of the WNT/β-catenin and TGF-β pathways and downregulation (shaded blue) of the NF-κB pathway is observed in all resistant clones (n = 11).

  4. WNT/[bgr]-catenin signalling regulates sensitivity to BET inhibition.
    Figure 4: WNT/β-catenin signalling regulates sensitivity to BET inhibition.

    a, Dkk1 results in re-expression of differentiation markers (Gr1+/CD11b+). b, Kaplan–Meier curve of vehicle and I-BET-treated mice after syngeneic transplantation of resistant clone stably transduced with Dkk1 (n = 10 per group, statistical significance calculated using a log-rank test). Dotted line denotes treatment starting on day 16. c, Heat map representation of Bliss interaction index across five-point dose range of pyrvinium and I-BET performed in biological quadruplicate. d, Kaplan–Meier curve of I-BET with/without pyrvinium after syngeneic transplantation of resistant cells. Shaded area denotes active treatment between days 9 and 26 (vehicle n = 7, I-BET n = 5, pyrvinium n = 5, combination n = 5, statistical significance calculated using a log-rank test). e, Viable, shRNA-positive cells after treatment with either vehicle or I-BET normalized to day 0 performed in biological quadruplicate (mean ± s.d.). f, Binding of β-catenin at Myc TSS and enhancer elements in vehicle-treated cells, resistant cells and resistant cells with Dkk1. Mean enrichment relative to input (±s.e.m.) in chromatin immunoprecipitation (ChIP) analysis from biological triplicate experiments. g, Myc expression from biological triplicate experiments (mean ± s.d.). h, Heat map representation of Brd4 and β-catenin chromatin occupancy ranked according to amount of Brd4 binding in vehicle-treated clones. i, Correlation between aggregate relative expression of examined β-catenin pathway genes with responsiveness to I-BET therapy. Statistical significance determined using Pearson’s correlation. j, Schematic model proposing the mechanism of resistance to BET inhibitor therapy in AML.

  5. Establishment of a model of BET inhibitor resistance.
    Extended Data Fig. 1: Establishment of a model of BET inhibitor resistance.

    a, Resistance to I-BET demonstrated in cell proliferation assays. Representative dose–response curve of a vehicle treated clone and a resistant clone stably maintained in 1,000 nM I-BET after 72 h of growth (mean ± s.d., n = 4 per group). b, Cross-resistance to chemically distinct BET inhibitors demonstrated in cell proliferation assays. Representative dose–response curve of a vehicle-treated clone and a resistant clone after 72 h of growth (mean ± s.d., n = 4 per group). c, Abrogation of response to I-BET-mediated cell cycle arrest. This is more evident in resistant clones stably maintained in higher concentrations of I-BET. Data from biological duplicate experiments (mean ± s.e.m.). d, Representative flow plots of cell lines stably transduced with an inducible shRNA vector. Vector-positive cells constitutively express Venus. After the introduction of doxycycline, shRNA-expressing cells co-express Venus and dsRED. Selective disadvantage consequent to shRNA expression results in drop out of dsRED-positive cells from culture over time, which is assessed by flow cytometry. e, Independent inducible shRNAs specifically reduce the expression of Brd4, but not Brd2 or Brd3, after 48–72 h of doxycycline. Messenger RNA levels in shRNA-positive cells normalized to mRNA expression in shRNA-negative cells in biological duplicate experiments (mean ± s.e.m.). f, Brd4 protein levels are reduced in shRNA-positive cells. Uncropped blots are shown in Supplementary Fig. 1. g, In addition to resistance to selective knockdown of Brd4, BET-inhibitor-resistant cells are also refractory to RNAi-mediated dual knockdown of Brd3 and Brd4. shRNA-mediated knockdown of Brd2 has minimal effect on both vehicle-treated and resistant clones. dsRED-positive cells normalized to day 1 after doxycycline exposure in biological duplicate experiments (mean ± s.e.m.). h, Reduction of Brd3/4 mRNA expression and Brd2 mRNA expression with two independent shRNAs after 48–72 h of doxycycline. mRNA levels in shRNA-positive cells normalized to mRNA expression in shRNA-negative cells in biological duplicate experiments (mean ± s.e.m.). i, Examination of vehicle-treated and resistant clones demonstrates no major morphological differences. j, Resistant clones are smaller and demonstrate homogeneity in size and complexity (FSCmid/SSClow) by flow cytometry. k, Resistant clones are enriched for L-GMPs (Lin, Sca, cKit+, CD34+, FcγRII/RIII+). Representative FACS analysis of vehicle-treated and resistant clones, percentages represent proportion of parent gate.

  6. Resistance to BET inhibitors also arises from an immature cell compartment in MLL-ENL leukaemia.
    Extended Data Fig. 2: Resistance to BET inhibitors also arises from an immature cell compartment in MLL–ENL leukaemia.

    a, Strategy for the generation of resistant cell lines from primary HSPC after retroviral transduction with the oncogene MLL–ENL. The parental cell line was serially re-plated in cytokine supplemented semi-solid media containing either vehicle (0.1% DMSO) or 400 nM I-BET (~IC40 of parental cell line). Cells in each plate were then washed and transferred to liquid culture to generate cell lines. Resistant cell lines were subsequently exposed to increasing selection pressure in liquid culture. Vehicle-treated cell lines and the parental cell line were identically passaged. b, Resistant cell lines bearing MLL–ENL are smaller and demonstrate homogeneity in size and complexity (FSCmid/SSClow) in addition to exhibiting an immature immunophenotype (Gr1/CD11b). Representative FACS analysis of vehicle-treated and resistant cell lines. c, Resistant cell lines bearing MLL–ENL demonstrate increased expression of WNT/β-catenin pathway genes. qRT–PCR data performed in biological triplicate (mean ± s.d.).

  7. Resistance to BET inhibitors emerges from leukaemia stem cells.
    Extended Data Fig. 3: Resistance to BET inhibitors emerges from leukaemia stem cells.

    a, Transplant cohorts and survival of mice injected with vehicle-treated and resistant clones in limit dilution analyses of primary syngeneic transplants displayed in Fig. 2d, e. b, Experimental strategy for derivation of in vivo resistance to BET inhibitors in a MLL–AF9 leukaemia model. After primary transplant of a vehicle-treated clone, serial transplant of either I-BET-exposed or I-BET-naive leukaemias, derived from whole bone marrow of diseased mice, was undertaken until loss of I-BET-mediated survival advantage was observed. Treatment was started on days 11–13. c, Progressive loss of I-BET-mediated survival advantage observed in serial transplant generations. Kaplan–Meier curves of serial transplant generations. Second transplant: I-BET naive n = 6, I-BET exposed n = 6. Third transplant: I-BET naive n = 2, I-BET exposed n = 3. Fourth transplant: I-BET naive n = 2, I-BET exposed n = 3. Fifth transplant: I-BET naive n = 4, I-BET exposed n = 5. d, Limit dilution analyses of leukaemias derived from bone marrow of diseased mice chronically exposed to I-BET after the fourth transplant demonstrates that less than 10 cells are reliably able to transfer leukaemia. Kaplan–Meier curves of C57BL/6 mice injected with indicated number of cells. e, Chronic I-BET exposure significantly enriches for leukaemia stem cells in vivo. f, Transplant cohorts and survival of limit dilution analyses of data displayed in d and e. g, Gating strategy for identification of L-GMPs in whole mouse bone marrow.

  8. Enrichment of a LMPP population in AML PDX.
    Extended Data Fig. 4: Enrichment of a LMPP population in AML PDX.

    a, Experimental treatment strategy for treatment of NOD/SCID/Il2rg−/− (NSG) mice bearing AML PDXs. Treated mice (with either vehicle or I-BET) belonged to identical transplant generations. b, Cytogenetic and genetic information of PDX models used. c, Gating strategy for identification of LMPP-like LSCs and GMP-like LSCs from mouse bone marrow. mTer119/mCD45 denotes mouse Ter119/CD45; hCD45/hCD3/hCD19/hCD33/hCD34/hCD38/hCD123/hCD45RA/hCD90 denotes human CD45/CD3/CD19/CD33/CD34/CD38/CD123/CD45RA/CD90. d, Representative FACS analysis of bone marrow obtained from vehicle- and I-BET-treated mice demonstrating enrichment of LMPP-like LSCs in I-BET-treated mice. Events displayed are gated on mTer119/hCD45+/hCD33+ cells and are expressed as a percentage of total hCD45+ cells.

  9. Intrinsic resistance to BET inhibition is not a feature of L-GMPs.
    Extended Data Fig. 5: Intrinsic resistance to BET inhibition is not a feature of L-GMPs.

    a, Experimental strategy for testing intrinsic resistance of L-GMPs to BET inhibition. After syngeneic transplant of a vehicle-treated clone, L-GMPs were FACS-isolated from whole mouse bone marrow of diseased mice and cultured in cytokine supplemented semi-solid media containing either vehicle (0.1% DMSO) or 1 μM I-BET. b, L-GMPs do not demonstrate intrinsic resistance to I-BET. Colony counts after 7 days of growth in biological triplicate (see also Fig. 2h) experiments (mean ± s.e.m.) of FACS-isolated L-GMPs after primary transplant of vehicle-treated clones in two additional independent mice. M2, mouse 2; M3, mouse 3.

  10. Further genetic characterization of BET-inhibitor-resistant cells.
    Extended Data Fig. 6: Further genetic characterization of BET-inhibitor-resistant cells.

    a, Comparison of whole-exome sequencing (WES) data from early and late time points identifies non-advantageous passenger mutations. Data from WES of samples obtained at an earlier time point to that presented in Fig. 3d is shown. Call out box identifies genes within a small region on chromosome 13 in one resistant clone which demonstrate copy number gain and are associated with increased mRNA expression relative to non-resistant cells. b, Venn diagram demonstrating gene mutations shared between vehicle-treated and resistant clones. Highlighted in the call out box are 24 gene mutations shared between resistant clones but not found in vehicle-treated clones. c, Resistant clones do not exhibit marked genetic instability with low mutation frequency observed. d, No specific mutation signature is identified in resistant clones. e, Correlation of genes identified in copy number gain region on chromosome 13 with gene expression data from the two resistant clones examined by WES. Fold change in gene expression compared to vehicle-treated clones obtained from microarray analysis is shown. f, Mutations detected by WES can be validated with data obtained from RNA sequencing (RNA-seq) of the same clones. Selected examples of mutations unique to resistant clones and shared between vehicle-treated and resistant clones is shown in integrative genomics viewer (IGV) tracks.

  11. Further epigenetic and gene expression characterization of BET-inhibitor-resistant cells.
    Extended Data Fig. 7: Further epigenetic and gene expression characterization of BET-inhibitor-resistant cells.

    a, BRD4 binding profile at Polr2a enhancer elements demonstrates no significant loss of BRD4 binding or H3K27ac levels in resistant clones. b, Genome wide profiling of BRD2 and BRD3 binding at TSSs comparing vehicle-treated and resistant clones is demonstrated in heat maps centred on the TSS of annotated genes with 5 kb flanking sequence either side. Red indicates higher density of reads in ChIP-seq data. c, Heat map of differential mRNA expression data from a vehicle-treated and resistant clone performed by RNA-seq in biological triplicate experiments. d, RNA-seq and microarray data are highly correlated. Correlation of log2 fold change (logFC) between RNA-seq and microarray data across all genes. No genes show opposing expression changes. Dotted line indicates y = x, blue dots represent genes that are significantly differentially expressed (gene expression log(FC) at least ±1.0, FDR corrected P < 0.05). eg, GSEA shows enrichment of LSC signature in I-BET-resistant cells, with resistant clones stably maintained in progressively higher concentrations of I-BET demonstrating increased enrichment of differentially expressed genes associated with a L-GMP self-renewal program. Barcode plot compares differential expression of genes in vehicle-treated and resistant clones to published microarray data comparing L-GMPs and MLL–AF9 cells propagated in liquid culture. Shaded area in the centre of plot shows genes ranked by fold change in expression in resistant relative to vehicle clones. Pink and blue shading represent significantly up- and downregulated genes, respectively. Upregulated and downregulated genes in the previously published LSC gene expression signature are shown in red and blue, respectively. Resistant (400) upregulated FDR = 1.2 × 10−1, downregulated FDR = 9.3 × 10−3. Resistant (600) upregulated FDR <1.0 × 10−4, downregulated FDR <2.5 × 10−4. Resistant (800) upregulated FDR <5.0 × 10−5, downregulated FDR <5.0 × 10−5. h, GSEA demonstrates that resistant clones show significant enrichment for genes associated with a self-renewal program identified from L-GMPs arising from haematopoietic stem cells (L-GMP HSCs). Upregulated FDR = 4.69 × 10−2, downregulated FDR = 1.3 × 10−3. i, RNA-seq identifies enrichment of LSC gene expression signature following chronic in vivo BET inhibitor exposure. Heat map of differential mRNA expression data from RNA-seq of leukaemias from the bone marrow of I-BET-exposed (n = 2) and I-BET-naive (n = 2) mice after the fourth transplant. j, GSEA of RNA-seq data identifies enrichment of a previously published LSC gene expression signature in leukaemias chronically exposed to I-BET in vivo. Barcode plot compares differential expression of genes in I-BET-exposed and I-BET-naive leukaemias to published data comparing L-GMPs and MLL–AF9 cells propagated in liquid culture. Shaded area in the centre of plot shows genes ranked by fold change in expression in I-BET-exposed relative to I-BET-naive leukaemias. Pink and blue shading represent significantly up- and downregulated genes, respectively. Upregulated and downregulated genes in the previously published LSC gene expression signature are shown in red and blue, respectively, and correlate with expression of genes in the I-BET-exposed leukaemias (FDR = 0.05).

  12. Negative regulation of Wnt/[bgr]-catenin signalling in resistant clones re-establishes sensitivity to BET inhibition.
    Extended Data Fig. 8: Negative regulation of Wnt/β-catenin signalling in resistant clones re-establishes sensitivity to BET inhibition.

    a, Schematic representation of the Wnt/β-catenin pathway. Highlighted by green stars are components of the pathway identified from transcriptome data which are significantly upregulated (>1.5-fold change, FDR <0.05) in resistant clones relative to vehicle-treated clones. b, GSEA of previously published human LSC gene expression data demonstrates enrichment of the WNT/β-catenin pathway. c, Dkk1 expression in the resistant cells before and after retroviral transduction of mouse Dkk1. qRT–PCR data from biological triplicate experiments (mean ± s.d.). d, Partial restoration of sensitivity to BET inhibition is observed in resistant clones after transduction with Dkk1. Dose–response curve of a vehicle-treated clone and resistant clone with and without expression of Dkk1 after 72 h of growth (mean ± s.e.m., n = 16 per group). e, Restoration of BET inhibitor induced cell-cycle arrest in resistant clones stably transduced with Dkk1. Flow cytometric analysis after 48 h exposure to either vehicle or 1,000 nM I-BET in biological triplicate experiments (mean ± s.e.m.). f, Resistant clones stably expressing Dkk1 do not show immunophenotypic enrichment for L-GMPs (see Extended Data Fig. 1k for comparison). Representative FACS analysis of resistant clone expressing Dkk1, percentages represent proportion of parent gate. g, Abrogation of Myc mRNA and protein expression in vehicle-treated clones after treatment with I-BET. qRT–PCR data of Myc expression in a vehicle-treated clone after 6 h of treatment with 1 μM I-BET151 in biological triplicate experiments (mean ± s.d.). Uncropped blots are found in Supplementary Fig. 1. h, Negative regulation of Wnt/β-catenin signalling by Dkk1 in resistant clones results in decreased expression of Myc. qRT–PCR data from biological triplicate experiments (mean ± s.d.). i, Small molecule inhibition of Wnt/β-catenin pathway expression re-establishes sensitivity to BET inhibition. Exposure of resistant clones to the Wnt/β-catenin pathway inhibitor pyrvinium also results in re-expression of Gr1+ and CD11b+. Representative FACS analysis of resistant clone in the presence or absence of pyrvinium. j, Pyrvinium synergises with I-BET to induce a modest cell cycle arrest and an induction of cell death (sub-G0 cell fraction). Data from biological triplicate experiments (mean ± s.e.m.). k, l, Pyrvinium reduces the expression of Wnt/β-catenin target genes such as Myc and Ccnd2 in vehicle-treated and resistant cells. qRT–PCR data from biological duplicate experiments (mean ± s.e.m.). m, These findings are similar to those seen for resistant cells stably expressing Dkk1.

  13. shRNA-mediated knockdown of Apc confers resistance to sensitive clones
    Extended Data Fig. 9: shRNA-mediated knockdown of Apc confers resistance to sensitive clones

    a, shRNA-mediated knockdown of Apc, a negative regulator of Wnt/β-catenin signalling, confers resistance to vehicle-treated clones. BET inhibitor treatment enriches for shRNA-containing (mCherry-positive) cells. Representative FACS plots after 7 days of cumulative drug exposure to either vehicle (0.1% DMSO) or 1 μM I-BET in a vehicle clone transduced with an Apc shRNA. b, c, Independent shRNAs directed against Apc confer resistance to vehicle-treated clones. Viable, shRNA-positive cells after treatment with either vehicle or I-BET normalized to day 0 performed in biological triplicate (mean ± s.d.). qRT–PCR data from FACS-isolated shRNA-containing cells performed in biological duplicate (mean ± s.e.m.). d, I-BET treatment of vehicle-treated clones transduced with a non-targeting shRNA does not enrich for shRNA-containing cells. Viable, shRNA-positive cells after treatment with either vehicle or I-BET normalized to day 0 performed in biological triplicate (mean ± s.e.m.). e, shRNA-mediated knockdown of Apc results in increased expression of Wnt/β-catenin target gene Myc. qRT–PCR data from FACS isolated shRNA containing cells performed in biological duplicate (mean ± s.e.m.).

  14. WNT/[bgr]-catenin pathway expression correlates with responsiveness to I-BET in primary human AML samples.
    Extended Data Fig. 10: WNT/β-catenin pathway expression correlates with responsiveness to I-BET in primary human AML samples.

    a, Assessment of β-catenin pathway gene expression in eight primary human AML samples with associated response to I-BET exposure. Each panel represents an individual primary human AML sample, with genetic abnormality denoted. Waterfall plot of relative qRT–PCR expression data of key β-catenin pathway genes (AXIN2, CCND1, CTNNB1, FZD5, MYC, TCF4 (also known as E2-2)) is displayed. Each bar is labelled 1–6 according to gene represented. Relative apoptosis observed after 48 h exposure to 500 nM I-BET versus vehicle (0.1% DMSO) is denoted in square parenthesis and is also represented as a heat map background shading in each panel. b, log2-transformed expression levels of selected genes in the WNT/β-catenin pathway were measured using qRT–PCR. A corrgram shows the genes are highly correlated with each other. The colour and thinness of the ellipse indicate the strength of correlation (a line is perfect correlation; a circle is uncorrelated). The ellipse direction indicates the sign of the correlation (correlated: right/blue, inversely correlated: left/red). c, Expression of selected genes is correlated with apoptosis. Scatterplots show apoptosis versus the log2 expression level of each gene. Expression of five genes (CCND1, CTNNB1, FZD5, MYC and TCF4) predicts apoptosis. The relationship is highlighted in a plot of apoptosis predicted using a multiple linear regression model with the five genes versus the actual data. d, Apoptosis observed after 48 h exposure to either vehicle (0.1% DMSO) or 500 nM I-BET across eight primary human AML samples. e, Relative viability of primary human AML samples after treatment with I-BET.

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Affiliations

  1. Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia

    • Chun Yew Fong,
    • Omer Gilan,
    • Enid Y. N. Lam,
    • Sarah Ftouni,
    • Dean Tyler,
    • Kym Stanley,
    • Devbarna Sinha,
    • Paul Yeh,
    • Robert G. Ramsay,
    • Ricky W. Johnstone,
    • Sarah-Jane Dawson,
    • Anthony T. Papenfuss &
    • Mark A. Dawson
  2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, East Melbourne, Victoria 3002, Australia

    • Chun Yew Fong,
    • Omer Gilan,
    • Dean Tyler,
    • Paul Yeh,
    • Robert G. Ramsay,
    • Ricky W. Johnstone,
    • Sarah-Jane Dawson,
    • Anthony T. Papenfuss &
    • Mark A. Dawson
  3. Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia

    • Chun Yew Fong,
    • Paul Yeh &
    • Mark A. Dawson
  4. Bioinformatics Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia

    • Alan F. Rubin &
    • Anthony T. Papenfuss
  5. Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia

    • Alan F. Rubin &
    • Anthony T. Papenfuss
  6. Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust-MRC Stem Cell Institute, Cambridge CB2 0XY, UK

    • Jessica Morison,
    • George Giotopoulos &
    • Brian J. P. Huntly
  7. Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK

    • Dave Lugo,
    • Philip Jeffrey,
    • Richard Gregory &
    • Rab K. Prinjha
  8. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Stanley Chun-Wei Lee &
    • Omar Abdel-Wahab
  9. Cancer Epigenetics DPU, Oncology R&D, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA

    • Christopher Carpenter
  10. QIMR Berghofer Medical Research Institute, University of Queensland, Brisbane, Queensland 4029, Australia

    • Steven W. Lane
  11. Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK

    • Tony Kouzarides

Contributions

C.Y.F. and M.A.D. designed the research, interpreted data and wrote the manuscript. C.Y.F., O.G., E.Y.N.L., A.F.R., S.F., D.T., K.S., D.S., P.Y., J.M., G.G., D.L., R.G., A.T.P. and M.A.D. performed experiments and/or analysed data. E.L., A.F.R., P.J., R.G.R., S.C.-W.L., C.C., S.W.L., O.A.-W., T.K., R.W.J., S.-J.D., B.J.P.H., R.K.P. and A.T.P. provided critical reagents, interpreted data and aided in manuscript preparation.

Competing financial interests

D.L., P.J., C.C., R.G. and R.K.P. are employees of GlaxoSmithKline.

Corresponding author

Correspondence to:

The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE63683.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Establishment of a model of BET inhibitor resistance. (502 KB)

    a, Resistance to I-BET demonstrated in cell proliferation assays. Representative dose–response curve of a vehicle treated clone and a resistant clone stably maintained in 1,000 nM I-BET after 72 h of growth (mean ± s.d., n = 4 per group). b, Cross-resistance to chemically distinct BET inhibitors demonstrated in cell proliferation assays. Representative dose–response curve of a vehicle-treated clone and a resistant clone after 72 h of growth (mean ± s.d., n = 4 per group). c, Abrogation of response to I-BET-mediated cell cycle arrest. This is more evident in resistant clones stably maintained in higher concentrations of I-BET. Data from biological duplicate experiments (mean ± s.e.m.). d, Representative flow plots of cell lines stably transduced with an inducible shRNA vector. Vector-positive cells constitutively express Venus. After the introduction of doxycycline, shRNA-expressing cells co-express Venus and dsRED. Selective disadvantage consequent to shRNA expression results in drop out of dsRED-positive cells from culture over time, which is assessed by flow cytometry. e, Independent inducible shRNAs specifically reduce the expression of Brd4, but not Brd2 or Brd3, after 48–72 h of doxycycline. Messenger RNA levels in shRNA-positive cells normalized to mRNA expression in shRNA-negative cells in biological duplicate experiments (mean ± s.e.m.). f, Brd4 protein levels are reduced in shRNA-positive cells. Uncropped blots are shown in Supplementary Fig. 1. g, In addition to resistance to selective knockdown of Brd4, BET-inhibitor-resistant cells are also refractory to RNAi-mediated dual knockdown of Brd3 and Brd4. shRNA-mediated knockdown of Brd2 has minimal effect on both vehicle-treated and resistant clones. dsRED-positive cells normalized to day 1 after doxycycline exposure in biological duplicate experiments (mean ± s.e.m.). h, Reduction of Brd3/4 mRNA expression and Brd2 mRNA expression with two independent shRNAs after 48–72 h of doxycycline. mRNA levels in shRNA-positive cells normalized to mRNA expression in shRNA-negative cells in biological duplicate experiments (mean ± s.e.m.). i, Examination of vehicle-treated and resistant clones demonstrates no major morphological differences. j, Resistant clones are smaller and demonstrate homogeneity in size and complexity (FSCmid/SSClow) by flow cytometry. k, Resistant clones are enriched for L-GMPs (Lin, Sca, cKit+, CD34+, FcγRII/RIII+). Representative FACS analysis of vehicle-treated and resistant clones, percentages represent proportion of parent gate.

  2. Extended Data Figure 2: Resistance to BET inhibitors also arises from an immature cell compartment in MLL–ENL leukaemia. (356 KB)

    a, Strategy for the generation of resistant cell lines from primary HSPC after retroviral transduction with the oncogene MLL–ENL. The parental cell line was serially re-plated in cytokine supplemented semi-solid media containing either vehicle (0.1% DMSO) or 400 nM I-BET (~IC40 of parental cell line). Cells in each plate were then washed and transferred to liquid culture to generate cell lines. Resistant cell lines were subsequently exposed to increasing selection pressure in liquid culture. Vehicle-treated cell lines and the parental cell line were identically passaged. b, Resistant cell lines bearing MLL–ENL are smaller and demonstrate homogeneity in size and complexity (FSCmid/SSClow) in addition to exhibiting an immature immunophenotype (Gr1/CD11b). Representative FACS analysis of vehicle-treated and resistant cell lines. c, Resistant cell lines bearing MLL–ENL demonstrate increased expression of WNT/β-catenin pathway genes. qRT–PCR data performed in biological triplicate (mean ± s.d.).

  3. Extended Data Figure 3: Resistance to BET inhibitors emerges from leukaemia stem cells. (435 KB)

    a, Transplant cohorts and survival of mice injected with vehicle-treated and resistant clones in limit dilution analyses of primary syngeneic transplants displayed in Fig. 2d, e. b, Experimental strategy for derivation of in vivo resistance to BET inhibitors in a MLL–AF9 leukaemia model. After primary transplant of a vehicle-treated clone, serial transplant of either I-BET-exposed or I-BET-naive leukaemias, derived from whole bone marrow of diseased mice, was undertaken until loss of I-BET-mediated survival advantage was observed. Treatment was started on days 11–13. c, Progressive loss of I-BET-mediated survival advantage observed in serial transplant generations. Kaplan–Meier curves of serial transplant generations. Second transplant: I-BET naive n = 6, I-BET exposed n = 6. Third transplant: I-BET naive n = 2, I-BET exposed n = 3. Fourth transplant: I-BET naive n = 2, I-BET exposed n = 3. Fifth transplant: I-BET naive n = 4, I-BET exposed n = 5. d, Limit dilution analyses of leukaemias derived from bone marrow of diseased mice chronically exposed to I-BET after the fourth transplant demonstrates that less than 10 cells are reliably able to transfer leukaemia. Kaplan–Meier curves of C57BL/6 mice injected with indicated number of cells. e, Chronic I-BET exposure significantly enriches for leukaemia stem cells in vivo. f, Transplant cohorts and survival of limit dilution analyses of data displayed in d and e. g, Gating strategy for identification of L-GMPs in whole mouse bone marrow.

  4. Extended Data Figure 4: Enrichment of a LMPP population in AML PDX. (243 KB)

    a, Experimental treatment strategy for treatment of NOD/SCID/Il2rg−/− (NSG) mice bearing AML PDXs. Treated mice (with either vehicle or I-BET) belonged to identical transplant generations. b, Cytogenetic and genetic information of PDX models used. c, Gating strategy for identification of LMPP-like LSCs and GMP-like LSCs from mouse bone marrow. mTer119/mCD45 denotes mouse Ter119/CD45; hCD45/hCD3/hCD19/hCD33/hCD34/hCD38/hCD123/hCD45RA/hCD90 denotes human CD45/CD3/CD19/CD33/CD34/CD38/CD123/CD45RA/CD90. d, Representative FACS analysis of bone marrow obtained from vehicle- and I-BET-treated mice demonstrating enrichment of LMPP-like LSCs in I-BET-treated mice. Events displayed are gated on mTer119/hCD45+/hCD33+ cells and are expressed as a percentage of total hCD45+ cells.

  5. Extended Data Figure 5: Intrinsic resistance to BET inhibition is not a feature of L-GMPs. (62 KB)

    a, Experimental strategy for testing intrinsic resistance of L-GMPs to BET inhibition. After syngeneic transplant of a vehicle-treated clone, L-GMPs were FACS-isolated from whole mouse bone marrow of diseased mice and cultured in cytokine supplemented semi-solid media containing either vehicle (0.1% DMSO) or 1 μM I-BET. b, L-GMPs do not demonstrate intrinsic resistance to I-BET. Colony counts after 7 days of growth in biological triplicate (see also Fig. 2h) experiments (mean ± s.e.m.) of FACS-isolated L-GMPs after primary transplant of vehicle-treated clones in two additional independent mice. M2, mouse 2; M3, mouse 3.

  6. Extended Data Figure 6: Further genetic characterization of BET-inhibitor-resistant cells. (552 KB)

    a, Comparison of whole-exome sequencing (WES) data from early and late time points identifies non-advantageous passenger mutations. Data from WES of samples obtained at an earlier time point to that presented in Fig. 3d is shown. Call out box identifies genes within a small region on chromosome 13 in one resistant clone which demonstrate copy number gain and are associated with increased mRNA expression relative to non-resistant cells. b, Venn diagram demonstrating gene mutations shared between vehicle-treated and resistant clones. Highlighted in the call out box are 24 gene mutations shared between resistant clones but not found in vehicle-treated clones. c, Resistant clones do not exhibit marked genetic instability with low mutation frequency observed. d, No specific mutation signature is identified in resistant clones. e, Correlation of genes identified in copy number gain region on chromosome 13 with gene expression data from the two resistant clones examined by WES. Fold change in gene expression compared to vehicle-treated clones obtained from microarray analysis is shown. f, Mutations detected by WES can be validated with data obtained from RNA sequencing (RNA-seq) of the same clones. Selected examples of mutations unique to resistant clones and shared between vehicle-treated and resistant clones is shown in integrative genomics viewer (IGV) tracks.

  7. Extended Data Figure 7: Further epigenetic and gene expression characterization of BET-inhibitor-resistant cells. (474 KB)

    a, BRD4 binding profile at Polr2a enhancer elements demonstrates no significant loss of BRD4 binding or H3K27ac levels in resistant clones. b, Genome wide profiling of BRD2 and BRD3 binding at TSSs comparing vehicle-treated and resistant clones is demonstrated in heat maps centred on the TSS of annotated genes with 5 kb flanking sequence either side. Red indicates higher density of reads in ChIP-seq data. c, Heat map of differential mRNA expression data from a vehicle-treated and resistant clone performed by RNA-seq in biological triplicate experiments. d, RNA-seq and microarray data are highly correlated. Correlation of log2 fold change (logFC) between RNA-seq and microarray data across all genes. No genes show opposing expression changes. Dotted line indicates y = x, blue dots represent genes that are significantly differentially expressed (gene expression log(FC) at least ±1.0, FDR corrected P < 0.05). eg, GSEA shows enrichment of LSC signature in I-BET-resistant cells, with resistant clones stably maintained in progressively higher concentrations of I-BET demonstrating increased enrichment of differentially expressed genes associated with a L-GMP self-renewal program. Barcode plot compares differential expression of genes in vehicle-treated and resistant clones to published microarray data comparing L-GMPs and MLL–AF9 cells propagated in liquid culture. Shaded area in the centre of plot shows genes ranked by fold change in expression in resistant relative to vehicle clones. Pink and blue shading represent significantly up- and downregulated genes, respectively. Upregulated and downregulated genes in the previously published LSC gene expression signature are shown in red and blue, respectively. Resistant (400) upregulated FDR = 1.2 × 10−1, downregulated FDR = 9.3 × 10−3. Resistant (600) upregulated FDR <1.0 × 10−4, downregulated FDR <2.5 × 10−4. Resistant (800) upregulated FDR <5.0 × 10−5, downregulated FDR <5.0 × 10−5. h, GSEA demonstrates that resistant clones show significant enrichment for genes associated with a self-renewal program identified from L-GMPs arising from haematopoietic stem cells (L-GMP HSCs). Upregulated FDR = 4.69 × 10−2, downregulated FDR = 1.3 × 10−3. i, RNA-seq identifies enrichment of LSC gene expression signature following chronic in vivo BET inhibitor exposure. Heat map of differential mRNA expression data from RNA-seq of leukaemias from the bone marrow of I-BET-exposed (n = 2) and I-BET-naive (n = 2) mice after the fourth transplant. j, GSEA of RNA-seq data identifies enrichment of a previously published LSC gene expression signature in leukaemias chronically exposed to I-BET in vivo. Barcode plot compares differential expression of genes in I-BET-exposed and I-BET-naive leukaemias to published data comparing L-GMPs and MLL–AF9 cells propagated in liquid culture. Shaded area in the centre of plot shows genes ranked by fold change in expression in I-BET-exposed relative to I-BET-naive leukaemias. Pink and blue shading represent significantly up- and downregulated genes, respectively. Upregulated and downregulated genes in the previously published LSC gene expression signature are shown in red and blue, respectively, and correlate with expression of genes in the I-BET-exposed leukaemias (FDR = 0.05).

  8. Extended Data Figure 8: Negative regulation of Wnt/β-catenin signalling in resistant clones re-establishes sensitivity to BET inhibition. (346 KB)

    a, Schematic representation of the Wnt/β-catenin pathway. Highlighted by green stars are components of the pathway identified from transcriptome data which are significantly upregulated (>1.5-fold change, FDR <0.05) in resistant clones relative to vehicle-treated clones. b, GSEA of previously published human LSC gene expression data demonstrates enrichment of the WNT/β-catenin pathway. c, Dkk1 expression in the resistant cells before and after retroviral transduction of mouse Dkk1. qRT–PCR data from biological triplicate experiments (mean ± s.d.). d, Partial restoration of sensitivity to BET inhibition is observed in resistant clones after transduction with Dkk1. Dose–response curve of a vehicle-treated clone and resistant clone with and without expression of Dkk1 after 72 h of growth (mean ± s.e.m., n = 16 per group). e, Restoration of BET inhibitor induced cell-cycle arrest in resistant clones stably transduced with Dkk1. Flow cytometric analysis after 48 h exposure to either vehicle or 1,000 nM I-BET in biological triplicate experiments (mean ± s.e.m.). f, Resistant clones stably expressing Dkk1 do not show immunophenotypic enrichment for L-GMPs (see Extended Data Fig. 1k for comparison). Representative FACS analysis of resistant clone expressing Dkk1, percentages represent proportion of parent gate. g, Abrogation of Myc mRNA and protein expression in vehicle-treated clones after treatment with I-BET. qRT–PCR data of Myc expression in a vehicle-treated clone after 6 h of treatment with 1 μM I-BET151 in biological triplicate experiments (mean ± s.d.). Uncropped blots are found in Supplementary Fig. 1. h, Negative regulation of Wnt/β-catenin signalling by Dkk1 in resistant clones results in decreased expression of Myc. qRT–PCR data from biological triplicate experiments (mean ± s.d.). i, Small molecule inhibition of Wnt/β-catenin pathway expression re-establishes sensitivity to BET inhibition. Exposure of resistant clones to the Wnt/β-catenin pathway inhibitor pyrvinium also results in re-expression of Gr1+ and CD11b+. Representative FACS analysis of resistant clone in the presence or absence of pyrvinium. j, Pyrvinium synergises with I-BET to induce a modest cell cycle arrest and an induction of cell death (sub-G0 cell fraction). Data from biological triplicate experiments (mean ± s.e.m.). k, l, Pyrvinium reduces the expression of Wnt/β-catenin target genes such as Myc and Ccnd2 in vehicle-treated and resistant cells. qRT–PCR data from biological duplicate experiments (mean ± s.e.m.). m, These findings are similar to those seen for resistant cells stably expressing Dkk1.

  9. Extended Data Figure 9: shRNA-mediated knockdown of Apc confers resistance to sensitive clones (234 KB)

    a, shRNA-mediated knockdown of Apc, a negative regulator of Wnt/β-catenin signalling, confers resistance to vehicle-treated clones. BET inhibitor treatment enriches for shRNA-containing (mCherry-positive) cells. Representative FACS plots after 7 days of cumulative drug exposure to either vehicle (0.1% DMSO) or 1 μM I-BET in a vehicle clone transduced with an Apc shRNA. b, c, Independent shRNAs directed against Apc confer resistance to vehicle-treated clones. Viable, shRNA-positive cells after treatment with either vehicle or I-BET normalized to day 0 performed in biological triplicate (mean ± s.d.). qRT–PCR data from FACS-isolated shRNA-containing cells performed in biological duplicate (mean ± s.e.m.). d, I-BET treatment of vehicle-treated clones transduced with a non-targeting shRNA does not enrich for shRNA-containing cells. Viable, shRNA-positive cells after treatment with either vehicle or I-BET normalized to day 0 performed in biological triplicate (mean ± s.e.m.). e, shRNA-mediated knockdown of Apc results in increased expression of Wnt/β-catenin target gene Myc. qRT–PCR data from FACS isolated shRNA containing cells performed in biological duplicate (mean ± s.e.m.).

  10. Extended Data Figure 10: WNT/β-catenin pathway expression correlates with responsiveness to I-BET in primary human AML samples. (300 KB)

    a, Assessment of β-catenin pathway gene expression in eight primary human AML samples with associated response to I-BET exposure. Each panel represents an individual primary human AML sample, with genetic abnormality denoted. Waterfall plot of relative qRT–PCR expression data of key β-catenin pathway genes (AXIN2, CCND1, CTNNB1, FZD5, MYC, TCF4 (also known as E2-2)) is displayed. Each bar is labelled 1–6 according to gene represented. Relative apoptosis observed after 48 h exposure to 500 nM I-BET versus vehicle (0.1% DMSO) is denoted in square parenthesis and is also represented as a heat map background shading in each panel. b, log2-transformed expression levels of selected genes in the WNT/β-catenin pathway were measured using qRT–PCR. A corrgram shows the genes are highly correlated with each other. The colour and thinness of the ellipse indicate the strength of correlation (a line is perfect correlation; a circle is uncorrelated). The ellipse direction indicates the sign of the correlation (correlated: right/blue, inversely correlated: left/red). c, Expression of selected genes is correlated with apoptosis. Scatterplots show apoptosis versus the log2 expression level of each gene. Expression of five genes (CCND1, CTNNB1, FZD5, MYC and TCF4) predicts apoptosis. The relationship is highlighted in a plot of apoptosis predicted using a multiple linear regression model with the five genes versus the actual data. d, Apoptosis observed after 48 h exposure to either vehicle (0.1% DMSO) or 500 nM I-BET across eight primary human AML samples. e, Relative viability of primary human AML samples after treatment with I-BET.

Supplementary information

PDF files

  1. Supplementary Information (262 KB)

    The file contains Supplementary Figure 1, uncropped scans with size marker indications for western blots.

Additional data