BET inhibitor resistance emerges from leukaemia stem cells

Abstract

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.

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Figure 1: Establishment of a model of BET inhibitor resistance.
Figure 2: Resistance to BET inhibitors arises from the LSC compartment.
Figure 3: Genetic, epigenetic and transcriptional characterization of BET-inhibitor-resistant cells.
Figure 4: WNT/β-catenin signalling regulates sensitivity to BET inhibition.

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Primary accessions

Gene Expression Omnibus

Data deposits

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

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Acknowledgements

We thank A. Bannister for critical reading of the manuscript. The Leukaemia Foundation Australia, Haematology Society of Australia and New Zealand, Royal Australasian College of Physicians and the Victorian Comprehensive Cancer Centre have supported CYF with PhD scholarships. M.A.D. is a Senior Leukaemia Foundation Australia Fellow, VESKI Innovation Fellow and Herman Clinical Fellow. The National Health and Medical Research Council of Australia (1085015; 1066545) and Leukaemia Foundation Australia fund the Dawson laboratory.

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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.

Corresponding author

Correspondence to Mark A. Dawson.

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Competing interests

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

Extended data figures and tables

Extended Data Figure 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.

Extended Data Figure 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.).

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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).

Extended Data Figure 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.

Extended Data Figure 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.).

Extended Data Figure 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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Fong, C., Gilan, O., Lam, E. et al. BET inhibitor resistance emerges from leukaemia stem cells. Nature 525, 538–542 (2015). https://doi.org/10.1038/nature14888

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