a, Pooled negative-selection screening in MLL-AF9/Nras^G12D leukaemia, depicting changes in representation of 1,072 informative shRNAs during 14 days of culture. shRNA abundance ratios were calculated as the number of reads after 14 days of culture on doxycycline (T14) divided by the number of reads before doxycycline treatment (T0), and plotted as the mean of two replicates in ascending order. Completely depleted shRNAs (0 reads at T14, n = 71) were plotted as a ratio of 10⁻⁵; highlighted shRNAs in this group are shown with even spacing in alphabetical order. Positive controls included shRNAs targeting Rpa1, Rpa3, Pcna and Polr2b. Negative-control shRNAs targeted renilla luciferase or Braf. b, Percentage of GFP-positive MLL-AF9/Nras^G12D leukaemia cells 2 days and 12 days after transduction with LMN constructs expressing the indicated shRNAs (top panel). Western blotting of whole-cell lysates prepared from MEF cultures transduced with the indicated TtTMPV-shRNAs and induced with doxycycline for 5 days (bottom panel). shREN, renilla luciferase shRNA. Representative experiments are shown. c, d, Proliferation rates of JQ1-treated cells, calculated by measuring the increase in viable cell number after 72 h in culture and fitting data to an exponential growth curve. Results are normalized to the proliferation rate of vehicle/DMSO-treated cells, set to 1 (n = 3). CML-BC, chronic myeloid leukaemia blast crisis; T-ALL, T-cell acute lymphoblastic leukaemia. e, f, Percentage of cells in S-phase (bromodeoxyuridine (BrdU)⁺) after JQ1 treatment for 48 h at the indicated concentrations (n = 3). BrdU was pulsed for 30 min in all experiments shown. All error bars represent s.e.m.

a, Bioluminescent imaging of mice transplanted with MLL-AF9/Nras^G12D leukaemia cells harbouring the indicated TRMPV-shRNAs. Doxycycline was administered upon disease onset, 6 days after transplant. Day 0 indicates the time of doxycycline treatment. b, Quantification of bioluminescent imaging responses after doxycycline treatment. Mean values of four replicate mice are shown. c, Kaplan-Meier survival curves of recipient mice transplanted with the indicated TRMPV-shRNA leukaemia lines. The interval of doxycycline treatment is indicated by the arrow. Each shRNA group contained 6–8 mice. Statistical significance compared to shRen was calculated using a log-rank test; *, P = 0.0001; **, P < 0.0001. d, Representative flow cytometry plots of donor-derived (Cd45.2⁺) bone marrow cells in terminally diseased doxycycline-treated mice. The gate shown includes dsRed⁺/shRNA⁺ cells. e, Percentage of dsRed⁺/shRNA⁺ cells in the Cd45.2⁺ terminal leukaemia burden. Mean values of four replicate mice are shown. f, Bioluminescent imaging of MLL-AF9/Nras^G12D leukaemia recipient mice at the indicated day after initiation of treatment with JQ1 (50 mg⁻¹ kg⁻¹ d⁻¹) or DMSO carrier. g, Quantification of bioluminescent imaging responses to JQ1 treatment. Mean values of six DMSO- and seven JQ1-treated mice are shown. P values were calculated using a two-tailed Student’s t-test. h, Kaplan–Meier survival curves of control and JQ1-treated mice. Statistical significance was calculated using a log-rank test. In f, g and h, JQ1 treatment was initiated on day 1 after transplant of 50,000 leukaemia cells. i, Quantification of bioluminescent imaging responses to JQ1 treatment in established disease. Treatment of leukaemic mice was initiated 6 days after transplant, when disease first became detectable by imaging. Mean values of six DMSO- and seven JQ1-treated mice are shown. P values were calculated using a two-tailed Student’s t-test. All error bars represent s.e.m.

a, b, Light microscopy of May–Grünwald/Giemsa-stained MLL-AF9/Nras^G12D leukaemia cells after 2 days of doxycycline-induced shRNA expression or 2 days of JQ1 treatment (100 nM). Expression of shRNA was induced in TRMPV-transduced leukaemia cells. Imaging was performed with a ×40 objective. Representative images of three biological replicates are shown. c, d, FACS analysis of Mac-1 and Kit surface expression after 4 days of shRNA expression or 2 days of JQ1 treatment (100 nM). A representative experiment of three biological replicates is shown. e–h, GSEA plots evaluating changes in macrophage and LSC gene signatures upon Brd4 inhibition. In e and g, RNA for expression arrays was obtained from sorted dsRed⁺/shRNA⁺ cells (shRen versus three different Brd4 shRNAs) after 2 days of doxycycline induction. In f and h, microarray data were obtained from leukaemia cells treated for 2 days with DMSO or 100 nM JQ1. NES, normalized enrichment score; FDR q-val, false discovery rate q-value (the probability that a gene set with a given NES represents a false-positive finding).

a, Quantitative reverse transcription PCR (qRT–PCR) of relative Myc mRNA levels in the indicated mouse or human cells after a 48-h treatment with JQ1. Results were normalized to Gapdh, with the relative mRNA level in untreated cells set to 1 (n = 3). b, Western blotting of whole-cell lysates prepared from MLL-AF9/Nras^G12D leukaemia cells treated for 48 h with DMSO or 250 nM JQ1. A representative experiment of three biological replicates is shown. c, qRT–PCRtime course at indicated time points after treatment of MLL-AF9/Nras^G12D leukaemia cells with 250 nM JQ1. Results were normalized toGapdh, with the relative mRNA level in untreated cells set to 1 (n = 3). d, ChIP–qPCR performed in MLL-AF9/Nras^G12D leukaemia cells with the indicated antibodies and PCR primer locations (n = 6 for DMSO; n = 4 for JQ1-treated). TSS, transcription start site. e, Western blotting of whole-cell lysates prepared from MLL-AF9/Nras^G12D leukaemia cells transduced with empty vector or a Myc-cDNA-containing MSCV retrovirus. Cells were treated for 48 h with DMSO or 250 nM JQ1. A representative experiment of three biological replicates is shown. f, Light microscopy of May–Grünwald/Giemsa-stained MLL-AF9/Nras^G12D leukaemia cells transduced with an empty vector or with the Myc cDNA. Cells were treated for 5 days with 50 nM JQ1 and imaged using a×40 objective. A representative image of three biological replicates is shown. g, Quantification of BrdU incorporation after a 30-min pulse in MLL-AF9/Nras^G12D leukaemia cells transduced with empty control vector or the Myc cDNA. Cells were treated with JQ1 for 5 days at the indicated concentrations (n = 3). All error bars shown represent s.e.m.