Fig 2: AF10 expression maintains somatic cell identity. a Sample distance matrix of RNA-sequencing replicate samples. b Gene set enrichment analysis (GSEA) of transcriptome data of sgAF10 cells with respect to pluripotency-related and fibroblast-related gene sets. NES: normalized enrichment score, q: false discovery rate (FDR) q-value. c Gene set enrichment analysis (GSEA) of transcriptome data of sgAF10 cells with respect to iDOT1L_DOWN and iDOT1L_UP gene sets. NES: normalized enrichment score, q: false discovery rate (FDR) q-value. d mRNA levels for a set of DOT1L-regulated genes in AF10 knock-out fibroblasts as determined by qRT-PCR. ß-actin was used as an internal control. Gene expression levels were normalized to sgControl expressing fibroblasts for sgAF10 samples and DMSO treated cells for iDOT1L samples. Two biological replicates are indicated for sgAF10 samples and bar graph indicates the average of replicates. e Fold change in the number of Tra-1-60-positive colonies derived from AF10 sgRNA expressing cells in the presence of DMSO or a DOT1L inhibitor (iDOT1L; EPZ004777). P values were 0.001 for sgAF10-1 and 0.004 for sgAF10-2. n.s. not significant. f Fold change in the number of Tra-1-60-positive colonies derived from control or AF10 knock-down cells (shAF10) treated with either vehicle (DMSO) or iDOT1L (EPZ004777). P values were 0.034 for shAF10-1 and 0.007 for shAF10-2. n.s., not significant. g Immunoblot for H3K79me2 in double KO cells. Total H3 levels were used as loading control. Replicate of this experiment in Additional file 1: Figure S3c. Quantifications were normalized to gNT sample. h Fold change in the number of Tra-1-60-positive colonies derived from double knock-out cells expressing DOT1L and AF10 targeting sgRNAs. P values were 0.031 for sgAF10-1 and 0.014 for sgAF10-2. n.s. not significant

Fig 3: Association between BAZ1B, Dot1L and menin and analysis of BAZ1B expression in BC tumors. A Network reprentation interaction between Dot1L, menin, BAZ1B and the proteins of B-WICH (blue) and WINAC (green) complexes found among Dot1L and menin common interactors. B Western blot showing BAZ1B, Dot1L and menin co-immunoprecipitation MCF-7 nuclear extracts. BAZ1B mRNA C and protein D expression levels in luminal-like breast cancer samples from The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets analyzed with UALCAN. Kaplan–Meier curves, generated using the Kaplan–Meier Plotter, showing the probability of overall (E; 437 low and 283 high samples, respectively) and relapse-free (F: 457 low and 274 high samples, respectively) survival of ERa + BC patients according to BAZ1B mRNA expression levels

Fig 4: HMGA2 downregulation contributes to the anticancer effects of DOT1L inhibition.a Lentiviral HMGA2 knockdown in Y79 cells determined by qRT-PCR (N = 2). b, c Immunoblots for the indicated proteins in Y79 shHMGA2 cells treated with 10 µM etoposide for 24 h (b) and in Weri-Rb1 shHMGA2 cells treated with 0.5 µM etoposide for 20 h (c). d Dose-response study for the IC50 of CPX in Y79 cells treated for 72 h by MTT assays. The IC50 is shown as the mean ± SD (N = 6). e, f MTT assays for relative cell viability and proliferation. Y79 cells were subjected to single or combined treatment of 0.5 µM CPX and 0.2 µM etoposide for 72 h to determine the relative cell viability (e) or up to 4 days to monitor the cell proliferation (f). The results are shown as the mean ± SD (N = 6). ****P < 0.0001. g Immunoblots for indicated proteins in Weri-Rb1 cells treated with 2 µM CPX and 0.5 µM etoposide for 20 h. h Immunostaining of DOT1L and HMGA2 in human RB. Serial sections of the indicated tumor tissues were immunostained for DOT1L and HMGA2. The same region of the serial sections (marked by a red square) was magnified on the right (×40). Arrowheads in ZOC-168 mark DOT1L+ HMGA2- tumor foci. Scale bar: 50 µm.

Fig 5: HMGA2 expression is downregulated by DOT1L inhibition.a Volcano plot of DEGs in EPZ5676-treated Y79 cells in comparison to vehicle-treated cells. b KEGG pathway enrichment analyses for upregulated and downregulated DEGs. Cancer-related pathways are shown from the top 20 enriched pathways ranked by p-values before correction. The Rich Factor indicates the gene count ratio of DEG/annotated genes belonging to each pathway term listed. c Heat map of DEGs belonging to the KEGG pathway term of transcriptional misregulation in cancer. Each column represents an independent replicate. d qRT-PCR analysis for the relative expression of indicated DEGs in Y79 cells treated with 10 µM EPZ5676 for 6 days. The bar graph is shown as the mean ± SD of fold changes, relative to the vehicle-treated group (N = 3). e, f Expression of HMGA2 determined by qRT-PCR (e) and immunoblot (f) analyses for Y79 cells treated with 10 µM EPZ5676 for the indicated days. The bar graph represents the mean ± SD of fold changes (N = 4).