Fig 2: Concurrent CDK9 inhibition disrupts IR-driven chromatin reorganization and abrogates transcriptional induction.a Immunoblot of p-Pol II (Ser2) and total Pol II at indicated doses of AZD4573. Quantification normalized to total Pol II shown below. Data represent two independent experiments. b Click-IT fluorescent assay of relative nascent RNA abundance at indicated doses of AZD4573 (bar = 100 µm), n = 4 biologically independent replicates. Box plots display interquartile range, median, and whisker (minimum to maximum). c. ATAC-seq heatmap of untreated SU-DIPG4 controls, 6 Gy IR exposed, or IR exposed with concurrent AZD4573 (40 nM) (n = 2). Genome is clustered by a change in ATAC-seq peaks following IR exposure. d H3K27ac, BRD, and ENL heatmap of untreated SU-DIPG4 controls, 6 Gy IR exposed, or IR exposed with concurrent AZD4573 (40 nM) (n = 2). Genome is clustered by a change in H3K27ac ChIP-seq peaks at enhancers or promoters following IR exposure. e. H3K27ac metagene profile at transcripts differentially upregulated following IR in the presence or absence of AZD4573. f p-Pol II heatmap (left) and metagene profile (right) of untreated controls, IR exposed, or IR exposed with concurrent AZD4573 (40 nM) (n = 3). g Histogram of IR-induced transcripts (ANOVA p < 0.05) LFC value in the presence or absence of AZD4573 (SU-DIPG4). h IR-induced gene (ANOVA p < 0.05) LFC value in the presence or absence of AZD4573 in SF8628 and HSJD-DIPG007 cells. Source data are provided as a Source Data file.

Fig 3: Amido-imidazopyridine scaffold with activity against ENL/AF9 YEATS domains. (a) Summary of screening campaign to identify and validate ligands for the ENL YEATS domain. (b) Structure of screening hit, 1, and compounds used for preliminary SAR-finding studies. (c) Activity against the ENL YEATS domain measured by HTRF. Mean percent inhibition ± s.e.m., n = 4. (d) Engagement of ENL(YEATS)-HiBiT measured by ligand-induced luminescence. Signal normalized to DMSO ± s.e.m., n = 4. (e) Chemical structure of SR-1114 (4). (f) Immunoblot confirmation of CRBN-dependent ENL degradation by SR-1114. (g, h) Volcano plots depicting DMSO-normalized changes in protein abundance in MV4;11 cells after 4 h (g) and 16 h (h) treatments of SR-1114 (10 μM). Depicted are 6972 proteins with at least 2 unique spectral counts detected. ENL target genes are shown in green. P values derived from two-tailed Student’s t-test, n = 3.

Fig 4: ENL remains bound to MLL oncoprotein target genes during treatment with the menin inhibitors.a Genome Browser tracks showing MLL, menin, DOT1L and ENL bound to the MBNL1 locus in the B-ALL sample (top). In the patient-matched AML sample treated with the menin inhibitor only ENL remains bound (bottom). b Oncogene expression is significantly reduced in the AML after lineage switching. Bar height is the average of three qPCR biological replicates; Error bars = standard deviation, and the p value was computed using a two-tailed independent samples t-test; n = 3 qPCR biological replicates. c Reverse transcriptase PCR with two primer sets confirms the MLL::ENL oncogene is expressed in the AML treated with the menin inhibitor (expected size for 1 = 159 bp, and 2 = 150 bp). d Scatterplot comparing the normalized menin levels in the B-ALL and the AML sample treated with the menin inhibitor over an internal control group of oncoprotein-target sites (gray) the B-ALL-specific program (gold) and the GMP-like program (pink). e Same as (d) but showing the ENL signal persists over many of the oncoprotein-target genes in the AML sample. f Boxplot comparing ENL signal over an internal control group of oncoprotein-target genes (gray, n = 1595) the B-ALL-specific program (gold, n = 52) and the GMP-like program (pink, n = 45). p values were computed using a two-sided Mann-Whitney U Test with a Bonferroni correction for multiple hypothesis testing; boxplot center lines = median, box limits = first and third quartiles, whiskers = 1.5 times the interquartile range (IQR). g The menin inhibitor disrupts MLL oncoproteins, but ENL accumulation persists on oncoprotein targets in the GMP-like program. Source data are provided as a Source Data file.

Fig 5: Ser5-P, Ser2-P, CDK9, negative elongation factor (NELF), and super-elongation complex (SEC) subunits on the Ad5 genome.(a, top) Global Run-On sequencing in cells expressing wt E1A; (bottom) ChIP-seq of C-terminal domain (CTD) Ser5-P, CTD Ser2-P, CDK9, and NELF. (b) CDK9, AF9, ENL, and BRD4 ChIP-seq in cells expressing wt or DM E1A. AF9 and ENL ChIP-seq data are plotted with two different y-axes. (c) Average plots of AF9 and ENL binding near transcription start sites on the human genome.