Fig 2: 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 3: AutoCUT&Tag profiling reveals therapeutic sensitivity of KMT2Ar leukemia samples to DOT1L inhibition.a, Heatmaps comparing the co-occupancy of the transcriptional cofactors DOT1L and ENL over 10-kb windows centered on fusion oncoprotein sites (top) and sample-matched wild-type sites (bottom) in the 1° MPAL-1 sample. b, DOT1L is significantly enriched at fusion oncoprotein-binding sites in all KMT2Ar leukemia samples. The KMT2A–AF9-bearing 1° AML-3 and 1° MPAL-1 samples as well as the KMT2A–ENL-bearing cell line KOPN-8 show the strongest enrichment of DOT1L at fusion oncoprotein sites. For all box plots, the center line indicates the median, box limits represent the first and third quartiles, and whiskers show all data within 1.5 times the IQR of the lower and upper quartiles; outliers are not shown. P values were computed using a two-sample t test (two sided). n values are listed as narrow sites and wide sites for control samples and as wild-type sites and oncoprotein sites for KMT2Ar samples: CD34+: n = 6,204, 131; SEM: n = 8,168, 91; RS4;11: n = 10,287, 92; KOPN-8: n = 9,723, 192; 1° ALL-1: n = 3,747, 156; 1° AML-1: n = 15,915, 349; 1° AML-2: n = 26,148, 423; 1° AML-3: n = 3,943, 103; 1° AML-4: n = 7,218, 270: 1° AML-5: n = 11,256, 186; 1° MPAL-1: n = 8,641, 248; 1° MPAL-2: n = 15,179, 189. c, ENL is significantly enriched at fusion oncoprotein-binding sites in all KMT2Ar leukemia samples. n values are the same as in b. d, Cell survival curves of the SEM, RS4;11 and KOPN-8 cell lines in response to increasing concentrations of the DOT1L inhibitor EPZ-5676 (DOT1Li). Error bars indicate the s.d. of three replicates. e, In response to treatment with 30 µM of the DOT1L inhibitor for 3 d, H3K27ac is depleted at fusion oncoprotein-binding sites in KOPN-8 cells but not SEM or RS4;11 cells. n values are the same as in b.

Fig 4: DOT1L interacts with monomeric NPM1 on chromatin. A DOT1L (DOT1L-FLAG-HA) was overexpressed in N2a cells and immunoprecipitated using HA antibody (left panel) or NPM1 antibody (right panel). IgG antibody was used as control. For DOT1L immune-detection FLAG antibody was used. B Proximity ligation assay (in situ PLA) to visualize in vivo NPM1/DOT1L interaction (red signal) when both proteins are overexpressed in N2a cells. LMNB1 (LAMINB1) and FBL (FIBRILLARIN) were used as controls to mark the nuclear membrane and the nucleoli respectively. DOT1L/NPM1 interaction occurred inside the nucleus (LMNB1) and outside the nucleolus (FBL). (Scale bar 10 µm). C Immunoblot analysis of DOT1L/NPM1 co-IP as in A) from nucleoplasm or chromatin of N2a cell fractions. Total histone 3 (H3) was used to control the purity of the cell fractionation. For DOT1L immune-detection FLAG antibody was used. D Immunoblot analysis of DOT1L/NPM1 interaction in N2a cell extract as in A) in the presence of DNase or RNase, respectively

Fig 5: MLL oncoprotein levels scale with target gene expression.a Genome Browser tracks show the difference in MLL N-terminal (red) and C-terminal (blue) signals in three MLL::AF4 bearing leukemias, over the most common oncoprotein-target locus: MBNL1 (bottom black bar = oncoprotein-target peak). The USP5 promoter is an example of a wild-type MLL target site (bottom black bar = wild-type MLL peak). AF4 (brown) DOT1L (cyan) ENL (magenta) and H3K4me3 (green) signal is also shown for the p454 sample. b Oncoprotein scores over MBNL1 (quantified over the bottom-black bar in (a)) are representative of the average oncoprotein scores in each UMAP cluster; p values were computed using a two-tailed independent samples t-test; Cluster 1 n = 13 samples, Cluster 2 n = 6 samples, Cluster 3 n = 15 samples, Cluster 4 n = 5 samples; boxplot center lines = median, box limits = first and third quartiles, whiskers = 1.5 times the interquartile range (IQR). c The MBNL1 oncoprotein scores are correlated with the relative expression of MBNL1 as measured by qPCR; dotted lines indicate the regression and R2 measures the fit to the data, error bars indicate the standard deviation of the mean of three qPCR biological replicates. d ENL is significantly enriched over the oncoprotein-target sites. p values were computed using a two-tailed independent samples t-test; n values are listed as wild-type site #, oncoprotein site #: SEM = 15611, 491; p318 = 22014, 246; p279 = 25672, 272; p454 = 18509, 438; boxplot center lines = median, box limits = first and third quartiles, whiskers = 1.5 times the interquartile range (IQR). Source data are provided as a Source Data file.