Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Illustration of the experimental strategy to isolate DOT1L-ko clones. B Western blot showing global H3K79me2 abundance in histone extracts (right) or DOT1L protein levels in nuclear extracts (left) from DOT1L-ko HEL clones with partial or total loss of DOT1L activity (sgDOT1L-clone 1; sgDOT1L-clone 2). C Bar graphs showing the results of a fluorochrome-based cell competition assay. Relative abundance of empty vector control or DOT1L-sgRNA expressing HEL cells as compared to non-transduced competitor cells in a chimeric mixture are shown. D Kaplan–Meyer curves showing survival of NXG mice engrafted with 50,000 HEL cells harboring a complete DOT1L-ko (clone 2) or empty vector control. Pie charts show the fraction of sgRNA vector expressing cells in human CD45+ cells in the bone marrow of mice that developed disease, indicating counterselection of the BFP+ cells in the animals of the DOT1L-ko cohort. Statistics: log-rank test, *** p < 0.0001. E Volcano-plot showing differentially expressed genes (DEGs) from RNAseq in HEL cells after knockout of DOT1L (clone 2). F Geneset-enrichment analysis plots from RNAseq in HEL cells after knockout of DOT1L. G Tornado-plots visualizing the global occupancy of DOT1L and deposition of H3K79me2 across all transcription start sites (TSS) in ChIPseq of HEL cells. H Heatmap showing the fractions of UP- and DOWN-regulated DEGs after DOT1L-ko among those genes associated with the top 3000 DOT1L-bound TSS. Selected genes from the downregulated cluster are annotated.

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Clone Assay, Western Blot, Activity Assay, Competitive Binding Assay, Plasmid Preparation, Control, Expressing, Knock-Out

Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Dose-response curve showing the sensitivity of HEL cells to Bomedemstat treatment at 6 days of incubation determined by MTS assay; n = 3 independent experiments, unpaired t-test; *<0.05, **<0.01, ***<0.001. B Cell growth of HEL cells (empty vector vs. DOT1L-ko, clone 2) in a growth-over-time assay under exposure to Bomedemstat (150 nM); unpaired t-test; comparison of DOT1L-ko+Bomedemstat with WT+Bomedemstat; *<0.05, **<0.01, ***<0.001. C Western blot showing global H3K79me2 abundance in histone extracts from SET2 cells after CRISPR-Cas9-mediated inactivation of DOT1L with two different sgRNAs. D Cell growth of SET2 cells (Luciferase (LUC) sgRNA negative control vs. DOT1L-sgRNA2) in a growth-over-time assay under exposure to Bomedemstat (100 nM); unpaired t-test; comparison of DOT1L-ko+Bomedemstat with WT+Bomedemstat; ***<0.001. E Bar graphs showing the total number of primary cells from two different MPN-BP patients or G-CSF mobilized normal cells after shRNA-mediated knockdown of DOT1L (shRNA5/6) or scrambled (scr) control and treatment with 100 nM Bomedemstat or DMSO for 7 days in methylcellulose; one-way ANOVA; **<0.01, ***<0.001. F Bar graphs showing the fraction of apoptotic cells (HEL empty vector vs. DOT1L-ko, clone 2) using an AnnexinV / Sytox blue assay under exposure to Bomedemstat (150 nM) or DMSO at 14 d; n = 3 independent experiments, One-way ANOVA; **<0.01, ***<0.001. G Representative cytospins (May-Grünwald/Giemsa staining) from HEL cells (empty vector or DOT1L-ko, clone 2) under exposure to Bomedemstat (150 nM) or DMSO at 14 d. Imaging of whole slides was performed on a Carl Zeiss AxioScan. n = 3 independent experiments. Scale bar indicates 50 µM. H Stacked bar graphs showing the fraction of cells in G0/G1, G2/M and S-phase in HEL cells (empty vector vs. DOT1L-ko, clone 2) determined by Click-it EdU cell cycle assay under exposure to Bomedemstat (150 nM) or DMSO at 14 d; n = 3 independent experiments, One-way ANOVA; **<0.01, ***<0.001.

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Incubation, MTS Assay, Plasmid Preparation, Comparison, Western Blot, CRISPR, Luciferase, Negative Control, shRNA, Knockdown, Control, Staining, Imaging, Cell Cycle Assay

Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Heatmap showing differentially expressed genes (adjusted p-value < 0.05, fold-change >2) in RNAseq from HEL cell lines (empty vector or DOT1L-ko, clone 2) under exposure to Bomedemstat (150 nM) or DMSO at 24 h or 5 d. Based on the expression pattern, genes were assigned to 8 unique clusters (Kmeans clustering). Selected genes from clusters 4, 5 and 8 are annotated. B Enrichment analysis of REACTOME among genes strongly DOWN-regulated by Bomedemstat in DOT1L-ko cells (cluster 5). The size of dots is proportional to the confidence in each term (log10FDR).

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Plasmid Preparation, Expressing

Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Tornado-plots visualizing the occupancy of DOT1L at LSD1 binding sites in HEL cells. The map was created based on regions with LSD1-ChIPseq peaks (MACS2) and sorted by LSD1 signal intensity. DOT1L-ChIPseq signals were mapped to these regions showing a large degree of co-occupancy. B Pie-chart showing the assignment of LSD1 and DOT1L co-occupied sites to different regions in the genome (ChIPseeker output). C Tornado-plots visualizing the deposition of H3K79me2 and H3K4me1 at LSD1 and DOT1L co-occupied regions. Map was sorted based on H3K79me2 signal intensities. D Tornado-plots visualizing the occupancy of DOT1L at LSD1 binding sites in primary MPN-BP patient samples. The map was created based on regions with LSD1-ChIPseq peaks (MACS2) and sorted by LSD1 signal intensity. Regions with signal enrichment in the corresponding input samples were deemed artefacts and removed. DOT1L-ChIPseq signals were mapped to these regions showing a large degree of co-occupancy. E Pie-chart showing the assignment of LSD1 and DOT1L co-occupied sites in MPN-BP samples to different regions in the genome (ChIPseeker output). F Genome-browser (IGV / Integrative Genome Viewer) tracks showing DOT1L, LSD1 and H3K4me1 co-occupancy at selected regulatory regions in HEL cells. G Tornado-plots visualizing LSD1-signal intensity at LSD1 + DOT1L co-occupied genomic sites in DOT1L-WT and DOT1L-ko HEL cells.

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Binding Assay

Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Top-10 transcription factor motifs enriched at LSD1 and DOT1L co-occupied regions in HEL cells. Output of the “Homer” motif enrichment analysis software. B Left: Heatmap showing DEGs (adjusted p-value < 0.05, fold-change >2) in RNAseq from HEL cell lines (empty vector or DOT1L-ko, clone 2) under exposure to Bomedemstat (150 nM) or DMSO at 5 d. DEGs were filtered for genes associated with LSD1 and DOT1L co-occupied regions (ChIPseeker). Genes were assigned to 4 clusters (Kmeans clustering). Selected genes from clusters 1 and 2 are annotated. Right: Enrichment analysis results of REACTOME terms in cluster 1 (top) and cluster 2 (bottom) are shown as bar graphs.

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Software, Plasmid Preparation

Journal: Leukemia

Article Title: The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN

doi: 10.1038/s41375-025-02719-y

Figure Lengend Snippet: A Representative Western blot showing global H3K79me2 abundance in histone extracts from HEL and SET2 cells treated with different concentration of the DOT1L inhibitor Pinometostat or DMSO as control. B Curves showing the cell growth of HEL or SET2 cells in a growth-over-time assay under exposure to single- or combination treatments with Bomedemstat (100 nM) and Pinometostat (10 µM). C Simplified model of DOT1Ls known canonical function at promoters of transcriptionally active genes. D Proposed working model of a novel non-canonical function of DOT1L at LSD1-bound enhancer regions.

Article Snippet: For IPs we used antibodies against DOT1L (Cell Signaling Technologies, clone: D1W4Z), LSD1 (Abcam, clone: EPR6825), H3K4me1 (Abcam, clone: EPR16597 ) and H3K79me2 (Cell Signaling Technologies, clone: D15E8).

Techniques: Western Blot, Concentration Assay, Control

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of the screening strategy. epi, epigenetic; FACS, fluorescence-activated cell sorting; NGS, Next-generation sequencing. ( B and C ) Scatterplot of the screen results. Control sgRNAs (dark blue) are scattered randomly across the diagonal. EZH2 (light blue) and KDM1A (green) represent positive control sgRNAs. NT, non-targeting. ( D ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells transduced with the indicated sgRNAs. ( E ) CD47 or PD-L1 mRNA levels in HuH-7 cells upon DOT1L depletion with or without IFNγ treatment for 48 h. Results are shown as mean ± SD (n = 3). ( F ) CD47 or PD-L1 flow cytometry analyses of WT and DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Fluorescence, FACS, Next-Generation Sequencing, Control, Positive Control, Western Blot, Transduction, Flow Cytometry

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A and B ) Volcano plot of RNA-seq analysis in WT and DOT1L-depleted HuH-7 cells. NT, non-targeting. ( C ) Gene ontology (GO) analysis of RNA-seq data showing top 10 pathways that are up-regulated upon DOT1L depletion in HuH-7 cells. ( D ) Gene Set Enrichment Analysis (GSEA) of top 5 hallmark gene sets enriched in DOT1L-depleted HuH-7 cells. ( E ) GSEA of RNA-seq data showing IFNα (top) and IFN γ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( F and G ) Heatmaps of selected immune-related genes whose expression is higher in DOT1L-depleted HuH-7 cells with or without IFNγ treatment for 48 h. ( H ) Flow cytometry histograms of MHC I in sgDOT1L HuH-7 cells with or without IFNγ treatment for 48 h. ( I and J ) Binding density heatmap showing H3K4me3 distribution at the promoters of all genes (I) or the up-regulated genes (J) caused by DOT1L KO in HuH-7 cells. ( K and L ) Binding density heatmap showing H3K27ac distribution at the promoters of all genes (K) or the up-regulated genes (L) caused by DOT1L KO in HuH-7 cells.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from HuH-7 cells with EPZ-5676 treatment for 7 days. ( B ) RT-qPCR analysis of CD47 and PD-L1 mRNA levels of HuH-7 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( C ) Flow cytometry histograms of CD47 and PD-L1 in HuH-7 cells with EPZ-5676 treatment for 7 days. ( D ) Volcano plot of RNA-seq analysis in DMSO and EPZ-5676 treatment HuH-7 cells. ( E ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in HuH-7 cells with EPZ-5676 treatment. ( F ) Heatmaps of selected immune-related genes whose expression is higher in EPZ-5676 treatment for 7 days. ( G ) Expression levels of immune-related genes by RT-qPCR analysis. Results are shown as mean ± SD (n=3). ( H ) Flow cytometry histograms of MHC I in HuH-7 cells with EPZ-5676 treatment for 7 days. ( I ) GSEA of RNA-seq data showing IFNα (top) and IFNγ (bottom) gene sets that are up-regulated upon DOT1L depletion in HuH-7 cells. ( J ) Immunoblotting analysis of the DOT1L expression after overexpression of WT or catalytic inactive (CI) HA-DOT1L in DOT1L-depleted HuH-7 cells. ( K ) RT-qPCR analysis of immune-related genes after rescue of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). ( L ) Flow cytometry histograms of CD47 and PD-L1 after overexpression of WT or CI HA-DOT1L in DOT1L-depleted HuH-7 cells.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Western Blot, Quantitative RT-PCR, Flow Cytometry, RNA Sequencing, Expressing, Over Expression

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Volcano plot shows transcriptome analysis of transposon elements (TEs) expression after DOT1L KO in HuH-7 cells. ( B ) The expression changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( C ) RT-qPCR analyses of TEs in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=3). n.s., not significant, * P < 0.05, ** P < 0.01, unpaired Student’s t -test. ( D ) The ATAC-seq signal changes of four different categories of TEs in WT and DOT1L KO HuH-7 cells. ( E ) Binding density heatmap of ATAC-seq, H3K4me3, and H3K27ac ChIP-seq signals of up-regulated TEs caused by DOT1L KO in HuH-7 cells. ( F ) Flow cytometry histograms of dsRNA staining of WT or DOT1L KO HuH-7 cells. DAC, Decitabine served as a positive control. ( G and H ) RT-qPCR analysis of immune-related genes in DOT1L-depleted HuH-7 cells transduced with shRNA against scramble, MDA5 (G), or RIG-I (H). Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( I ) Heatmaps of IgG and DOT1L signals by fanChIP in HuH-7 cells. ( J ) Genomic annotations of DOT1L binding peaks by fanChIP in HuH-7 cells. ( K ) DOT1L fanChIP signals, ATAC-seq, and RNA-seq profiles in HuH-7 cells of LINEs (top) or LTR (bottom).

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Flow Cytometry, Staining, Positive Control, Transduction, shRNA, RNA Sequencing

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Venn diagrams of H3K79me2-associated genes and down-regulated genes in HuH-7 cells. ( B ) Expression levels of ZEB1 by RNA-seq in WT and DOT1L-depleted HuH-7 cells. Results are shown as mean ± SD (n=2). ** P < 0.01, unpaired Student’s t -test. RPKM, Reads Per Kilobase per Million mapped reads. ( C ) RT-qPCR analysis of ZEB1 upon DOT1L depletion in HuH-7 cells. Results are shown as mean ± SD (n=3). *** P < 0.001, unpaired Student’s t -test. ( D ) Immunoblotting analysis of ZEB1 protein levels using whole-cell lysates from WT and DOT1L KO HuH-7 cells. ( E ) DOT1L fanChIP tracks, H3K79me2, H3K4me3, and H3K27ac ChIP-seq signals, and ATAC-seq profiles of WT and DOT1L-depleted HuH-7 cells in the ZEB1 locus. ( F ) Enrichment of ZEB1 motifs in the accessible chromatin regions specific to DOT1L-deficient HuH-7 cells by HOMER analysis. ( G ) Cistrome toolkit analysis of ATAC-seq data revealed that DNA-binding sites of ZEB1 were more open in the DOT1L-deficient HuH-7 cells. ( H ) Binding density heatmap of ZEB1 ChIP-seq signals in the DOT1L-deficient HuH-7 cells. ( I ) ChIP-seq tracks of ZEB1 in WT and DOT1L-depleted HuH-7 cells in the JAK2 , STAT1 , and PD-L1 loci. ( J ) Immunoblotting analysis using whole-cell lysates from WT and ZEB1 KO HuH-7 cells. ( K ) Expression levels of immune-related genes by RT-qPCR analysis upon ZEB1 depletion in HuH-7 cells. Results are shown as mean ± SD (n=3).

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Immunoblotting analysis using whole-cell lysates from WT and Dot1l KO Hepa1-6 cells. ( B ) GO analysis of RNA-seq data showing top 10 pathways that are up-regulated in Hepa1-6 cells upon Dot1l deficiency. ( C ) GSEA of RNA-seq data showing IFN (top) and IL2_STAT5 (bottom) gene sets that are up-regulated upon DOT1L depletion in Hepa1-6 cells. ( D ) RT-qPCR analyses of TEs in WT and Dot1l-depleted Hepa1-6 cells. Results are shown as mean ± SD (n=3). n.s., not significant, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. ( E ) Flow cytometry histograms of dsRNA staining of WT or Dot1l KO Hepa1-6 cells. ( F ) Immunoblotting analysis using whole-cell lysates from Hepa1-6 cells with EPZ-5676 treatment for 7 days. ( G ) RT-qPCR analysis of immune-related genes of Hepa1-6 cells with EPZ-5676 treatment for 7 days. Results are shown as mean ± SD (n=3). ( H ) Schematic of ICB treatments in mice injected with Hepa1-6 WT or Dot1l KO cells. ( I ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 WT or Dot1l KO cells reconstituted as in (H) and subsequently treated with isotype control or anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( J ) Survival analysis conducted on the C57BL/6 mice described in (H). n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Logrank test. ( K ) Schematic of Dot1l inhibitor and ICB treatments in mice injected with Hepa1-6 cells. ( L ) Analysis of tumor volume in C57BL/6 mice subcutaneously injected with Hepa1-6 cells reconstituted as in (K) and subsequently treated with 20 mg/kg EPZ-5676 and anti-PD-L1 antibodies (n=6). n.s., not significant, *** P < 0.001, unpaired Student’s t -test. ( M ) Survival analysis conducted on the C57BL/6 mice described in (L). n.s., not significant, *** P < 0.001, Logrank test. ( N to R ), The intra-tumoral abundance of CD4+ T cells, CD8+ T cells, Tregs, NK, and Macrophages cells upon Dot1l inhibition alone or in combination (Combo) with anti-PD-L1 blockade. Results are shown as mean ± SD (n=5). P values were calculated by unpaired Student’s t -test.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Western Blot, RNA Sequencing, Quantitative RT-PCR, Flow Cytometry, Staining, Injection, Control, Inhibition

Journal: bioRxiv

Article Title: Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

doi: 10.1101/2025.05.01.651793

Figure Lengend Snippet: ( A ) Schematic of DOT1L inhibitor treatment in huHSC-NCG mice injected with HuH-7 cells. ( B ) Analysis of tumor volume in huHSC-NCG mice subcutaneously injected with HuH-7 cells reconstituted as in (A) and subsequently treated with 20 mg/kg EPZ-5676 (n=5). *** P < 0.001, unpaired Student’s t -test. ( C ) The intra-tumoral abundance of hCD8+ T cells upon DOT1L inhibition treatment in huHSC-NCG mice (n=5). * P < 0.05, unpaired Student’s t -test. ( D to G ) GSEA of published RNA-seq data showing immune-related gene sets that are up-regulated upon inhibition the methyltransferase activity of DOT1L in multiple cancer cell lines. ( H ) Analysis of DOT1L expression in tumors and normal tissues from TCGA patients with the indicated cancer types. n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, wilcox.test. ( I ) Correlation analysis between DOT1L expression levels and z-scores of the IFN gene sets in different tumor types of TCGA. ( J ) DOT1L mRNA expression levels of responder and non-responder in ICB treatment clinical trials with anti-PD-1 treatment in melanoma. P values were calculated by unpaired Student’s t -test. ( K ) A proposed model for the dual function of DOT1L in regulating the immune response of tumors.

Article Snippet: Transferred protein was immunoblotted using primary antibodies against DOT1L (Cell Signaling Technology, 90878), H3K79me2 (Abcam, ab3594), H3 (Proteintech, 17168-1-AP), HRP-conjugated-β-actin (Proteintech, HRP-60008), HA-tag (Cell Signaling Technology, 3724), NPM1 (Proteintech, 10306-1-AP), and ZEB1 (Proteintech, 21544-1-AP).

Techniques: Injection, Inhibition, RNA Sequencing, Activity Assay, Expressing, Clinical Proteomics

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: Upregulated DOT1L expression correlates with PARPi resistance in OCA. The non-BRCA mutated cell lines OVCAR8 were subjected to a gradual increase in the concentration of Olaparib (from 0.5 to 20 µM) to allow for the development of acquired resistance. The IC50 values of Olaparib-resistant OVCAR8 (R8 OlaR) and original parent OVCAR8 (R8) cell lines were detected by CCK8 assay. B . Niraparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. C . Veliparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. D . Talazoparib IC50 curves of parent OVCAR8 and cells with acquired resistance to Olaparib. E . RNA-seq was performed on R8 OlaR (R) ( n = 3) and R8 (N) ( n = 3). Venn diagram illustrating the overlap between the RNA-seq data (R and N) and the classic epigenetic regulator genes. F . Volcano plot showing differential expression of mRNAs among overlap gene in E. Red dots represent differently expressed mRNAs with P < 0.05 and Log2FC > 1; blue dots represent mRNAs with P < 0.05 and Log2FC<-1; grey dots represent mRNAs with no significance. G . Heatmap showing differentially expressed epigenetic-related genes between R and N (F). H. DOT1L mRNA levels in R8 OlaR and its original parent OVCAR8 cells were analyzed by RT-qPCR. I . R8 OlaR and its original parent OVCAR8 cells were collected and subjected to western blotting to detect with the indicated antibodies. J - K . Analysis of DOT1L protein levels in PARP inhibitor-resistant ( n = 7) and sensitive fresh-frozen (FF) tissue tissues ( n = 7) ( J ). Quantified results are presented as the means ± SD ( n = 3), ** p < 0.01 ( K ). L . DOT1L mRNA levels in PARPi-resistant and sensitive OC tissues were analyzed by RT-qPCR. The data is presented as the means ± SD, * p < 0.05. M IHC staining of DOT1L in PARPi-resistant and sensitive OC tissues. Representative images are shown. Scale bars: 200 μm (upper); 100 μm (lower) (left). Quantification of DOT1L expression in PARPi-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01 (right)

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Expressing, Concentration Assay, CCK-8 Assay, RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L regulates OC sensitivity to Olaparib and contributes to PARPi resistance. A . DOT1L protein levels in a panel of OC cell lines were examined by western blotting. B . PLKO.1 and DOT1L shRNA (shDOT1L) plasmids were stably transfected into SKOV-3 cell line. Western blotting was used to determine DOT1L protein levels. C . PCMV, and PCMV DOT1L plasmids were stably transfected into OVCAR8 cell line. Western blotting was used to determine DOT1L protein levels. D . The CCK8 assay was performed to detect cell viability in SKOV-3 cells treated with Olaparib (Olap) for 96 h. E . The CCK8 assay was performed to detect cell viability in OVCAR8 cells treated with Olaparib for 96 h. F . Clonogenic assays were conducted to assess the colony formation efficiency of SKOV-3 cells in the presence of Olaparib for 7–14 days (left). The number of clones was quantified (right). G . Clonogenic assays were conducted to assess the colony formation efficiency of OVCAR8 cells in the presence of Olaparib for 7–14 days (left). The number of clones was quantified (right). H . A flow cytometry assay was performed to detect cell apoptosis in SKOV-3 cells treated with Olaparib (10 µM) for 48 h. I . A flow cytometry assay was performed to detect cell apoptosis in OVCAR8 cells treated with Olaparib (10 µM) for 48 h. (Data is presented as the mean ± SD; ns, p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001, n = 3). J-L. OVCAR8 and DOT1L stably overexpressed OVCAR8 cells (4 × 10 6 cells) were subcutaneously injected into the left armpit of each mouse. When the tumor volumes reached approximately 50 mm 3 , the mice were randomly divided into four groups (pc + PBS, pc + Olap, oeDOT1L + PBS, oeDOT1L + Olap) and received an intraperitoneal injection of Olaparib (Olap, 50 mg/kg) or PBS three times a week. Three weeks post-injection, the mice were sacrificed, and their body weights and tumor weight were quantified. Tumors from each group are shown in ( J ). Tumor growth curve (K) and tumor weights of each group ( L ) were quantified. M . The nude mice’s body weights of each group before and after administration. (Data are presented as the mean ± SD, ns, p > 0.05; * p < 0.05; ** p < 0.01, n = 5)

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Western Blot, shRNA, Stable Transfection, Transfection, CCK-8 Assay, Clone Assay, Flow Cytometry, Injection

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-mediated transcription regulation directly influences DOT1L expression. A PLKO.1, PARP1 shRNA (shPARP1) plasmids were stably transfected into SKOV-3 cells. Whole cell lysate (WCL) and chromatin-binding protein (CHR) were extracted and analyzed by western blotting with the indicated antibodies. B . RT-qPCR was used to determine the DOT1L and PARP1 mRNA levels. C . PCMV, PCMV PARP1 plasmids were stably transfected into SKOV-3 cells. Whole cell lysate (WCL) and chromatin bind protein (CHR) were extracted and analyzed by western blotting with the indicated antibodies. D . Quantification of PARP1 and DOT1L mRNA levels in ( C ). E . SKOV-3 cells were transfected with control pcDNA, Flag-PARP1(WT), or enzymatically defective Flag-PARP1 (PARP1 E988K). Western blotting was performed to detect DOT1L protein expression levels. F . DOT1L mRNA levels in pcDNA-, PARP1(WT)-, or PARP1 E988K-transfected SKOV-3 cells were analyzed by RT-qPCR. The data represents the means ± SD ( n = 3). * p < 0.05. G - H . OVCAR8 and OVCAR8 OlaR cells were collected, and western blotting and RT-qPCR were performed to detect DOT1L protein expression(G) and mRNA ( H ) levels. I . ChIP–qPCR showing the level of the indicated proteins recruited to the DOT1L promoter regions. The data represents the means ± SD ( n = 3). * p < 0.05. Four independent sets of DOT1L primers were used. J . PARP1-ChIP assay was performed in pcDNA-, PARP1(WT)-, or PARP1 E988K- transfected SKOV-3 cells to examine PARP1 occupancy at DOT1L. K . STAT3-ChIP assay was performed in OVCAR8 and OVCAR8 OlaR cells to examine STAT3 occupancy at DOT1L. L. STAT3-ChIP assay was performed in pcDNA-, PARP1(WT)-, or PARP1 E988K- transfected SKOV-3 cells to examine STAT3 occupancy at DOT1L. M . OVCAR8 and OVCAR8 OlaR cells were transfected with the DOT1L promoter report gene. The luciferase activity was measured 36 h after transfection. N . SKOV-3 cells were transfected with the DOT1L promoter report gene, together with control pcDNA, wild-type Flag-PARP1, and mutant Flag-PARP1 E988K as indicated. The luciferase activity was measured 36 h after transfection. O . The luciferase reporter assays were performed in PARP1 stably knockdown SKOV-3 cells

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Binding Assay, Western Blot, Quantitative RT-PCR, Control, ChIP-qPCR, Luciferase, Activity Assay, Mutagenesis, Knockdown

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L facilitates PARPi resistance via H3K79 methylation. A . Heatmaps of H3K79me2 levels detected by CUT&Tag around gene body regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. 3 kb windows spanning the TSS to TES of all genes were plotted. Genes were arranged by their enrichment of H3K79me2 in shNC and shDOT1L cells. B . The distributions of H3K79me2-binding regions are shown in the pie charts. C . Venn diagram showing the overlap between RNA-seq data and CUT&Tag data. The KEEP analysis revealed the significantly enriched items based on H3K79me2 signature. D . IGV tracks showing the enrichment of H3K79me2 in ABCB1 and PLCG2 gene regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. E - F . ChIP–qPCR showing the level of the indicated proteins recruited to the PLCG2 ( E ) and ABCB1 ( F ) promoter regions in DOT1L-overexpressed OVCAR8 cells. The data represent the means ± SD ( n = 3). * p < 0.05. three independent sets of PLCG2 and ABCB1 primers were used. G . RT-qPCR was performed in DOT1L overexpressed OVCAR8 cells to determine PLCG2 and ABCB1 mRNA levels. H. PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L overexpressed OVCAR8 cells. I . An H3K79me2-ChIP assay was performed in DOT1L knockdown SKOV-3 cells to examine H3K79me2 occupancy at PLCG2 and ABCB1. J . RT-qPCR was performed in DOT1L knockdown SKOV-3 cells to determine PLCG2 and ABCB1 mRNA levels. K . PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L knockdown SKOV-3 cells

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Methylation, Control, Knockdown, Binding Assay, RNA Sequencing, ChIP-qPCR, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-DOT1L-PLCG2/ABCB1 axis contributes to PARPi resistance. A . H3K79me2-ChIP assay was performed with OVCAR8 Olaparib-resistant and parent OVCAR8 cell lines to determine H3K79me2 occupancy at PLCG2 and ABCB1. B . PLCG2 and ABCB1 mRNA levels were determined in R8 OlaR and parent OVCAR8 cells by RT-qPCR. C . Western blotting was performed in Olaparib-resistant OVCAR8 and parent OVCAR8 cell lines to examine PLCG2 and ABCB1 (P-gly) protein expression levels. D . Western blotting was performed in R8 OlaR and parent OVCAR8 cells which were transfected with shNC and DOT1L shRNA respectively with the indicated antibodies. E . R8 OlaR and parent OVCAR8 cells were transfected with shNC, ABCB1 shRNA and PLCG2 shRNA. After 72 h of transfection, cells were collected and analyzed by western blotting with the indicated antibodies. F – G . Colony formation ( F ) and cell apoptosis assay ( G ) were performed in R8 OlaR and parent OVCAR8 stably transfected cell lines. H . Immunohistochemistry (IHC) staining of DOT1L, PARP1, ABCB1 (P-gly), and PLCG2 in PARP inhibitor-resistant human ovarian carcinomas (OC) tissues and sensitive tissues. Representative images are shown. Scale bars: 400 μm (upper); 160 μm (lower). I – K . Correlation analysis between PARP1 and DOT1L(I), DOT1L and P-gly ( J ), and DOT1L and PLCG2 ( K ) were analyzed. L . Quantification of P-gly (right) and PLCG2 (left) expression in PARP inhibitor-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Apoptosis Assay, Stable Transfection, Immunohistochemistry

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: Targeted inhibition of DOT1L sensitizes OC to PARPi in vitro and in vivo. A – B . Bliss synergy analysis of SGC0946 and Olaparib treatment in R8 OlaR ( A ) and SV3 OlaR ( B ) cell lines. Synergy and antagonism degrees between the drugs were determined using SynergyFinder. A positive score represents a synergistic effect. C . A colony formation assay was conducted to detect the effect of combination therapy with SGC0946 (20µM) and Olaparib(10µM) on R8 OlaR and SV3 OlaR proliferation (upper). Quantification of the relative survival rate of clones (lower). D . A flow cytometry assay was performed to detect cell apoptosis in R8 OlaR cells treated with Olaparib (10 µM), SGC0946 (20µM), or Olaparib (10 µM) + SGC0946 (20µM) for 48 h. E . R8 OlaR cells (4 × 10 6 cells) were subcutaneously injected into the left armpit of each mouse. When the tumor volumes reached approximately 50mm 3 , the mice were randomly divided into four groups (ctrl, Olap, SGC, Olap + SGC) and they received an intraperitoneal injection of Olaparib (Ola, 50 mg/kg), SGC0946(SGC, 50 mg/kg), Ola (50 mg/kg) + SGC (SGC, 50 mg/kg) or PBS three times a week. Three weeks post-injection, the mice were sacrificed, and mouse body weights and tumor weights were quantified. Tumors from each group are shown. F – G . The tumor volume curve ( F ) and weight of each group ( G ) were shown. H . The difference in nude mice’s body weights of each group before and after administration. (Data are presented as the mean ± SD, ns, p > 0.05; * p < 0.05, n = 5). I – K . The synergistic effects of SGC0946 and Olaparib on the viability of the indicated PDOs. Organoids were exposed for 5 days to combine treatments with suboptimal doses of SGC0946 (10 and 20 µM), and Olaparib (5 and 10 µM) ( I ). CI values less than 1, which suggest synergism, were calculated for drug combinations relative to the individual drugs and are indicated in the above graphs ( J – K ) (Data is presented as the mean ± SD, *** < 0.001, n = 3). L . Schematic diagram of molecular mechanism. Working model of the role of DOT1L in PARPi resistance

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Inhibition, In Vitro, In Vivo, Colony Assay, Clone Assay, Flow Cytometry, Injection

Journal: Experimental Hematology & Oncology

Article Title: Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins

doi: 10.1186/s40164-024-00488-5

Figure Lengend Snippet: LAMP5-AS1 regulates LAMP5 expression through the binding of DOT1L to the LAMP5 locus. ( a ) Western blotting show that DOT1L was enriched by LAMP5-AS1 pull-down assays. Antisense of LAMP5-AS1 sequence indicates the negative control. FT: flow through. ( b ) Western blotting for the protein levels of H3K79me2, H3K79me3, and LAMP5 in MLL leukemia cells transduced by LAMP5-AS1 siRNA and control. H3 and beta-actin as the internal control. The H3K79me2 or H3K79me3/ H3, LAMP5/Actin densitometric ratio were recorded by ImageJ, respectively. ( c ) ChIP-seq profiles (shown in GSE150483) of H3K79me2 and H3K79me3 at the LAMP5 genomic loci in LAMP5-AS1 knockdown (red) compared with control (blue) MOLM-13 cells. The y-axis scales represent read density per million sequenced reads. ( d, e ) ChIP Quantitative RT-PCR assays show H3K79me2 ( d ) and H3K79me3 ( e ) on the LAMP5 gene locus clearly declined upon LAMP5-AS1 knockdown in MLL leukemia cells. Error bars reflect ± SEM (**, p < 0.01; ***, p < 0.001) from three independent experiments. ( f ) Diagram depicts the procedure of ChIRP. ( g ) Quantitative RT-PCR for the LAMP5-AS1(upper, left) and LAMP5 locus (upper, right) enrichment in ChIRP experiment captured by LAMP5-AS1 probes. GAPDH and NC-probe were used to be the negative control. Western blotting shows the DOT1L and H3K79me2 protein levels that captured by LAMP5-AS1 probes in ChIRP experiment. GAPDH protein as the negative control. ( h ) RNA and DNA FISH and IF experiments showed that LAMP5-AS1 and DOT1L co-localized at LAMP5 locus in the cell nucleus. ( i ) Proposed model for LAMP5-AS1 regulate LAMP5 in MLL leukemia. LAMP5-AS1 regulates LAMP5 by directly interacting with DOT1L

Article Snippet: For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).

Techniques: Expressing, Binding Assay, Western Blot, Sequencing, Negative Control, Control, ChIP-sequencing, Knockdown, Quantitative RT-PCR

Journal: Experimental Hematology & Oncology

Article Title: Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins

doi: 10.1186/s40164-024-00488-5

Figure Lengend Snippet: The LAMP5-AS1-DOT1L-LAMP5 axis suppresses autophagy and maintains MLL fusion protein. ( a ) The resulting decline in MLL-AF9 levels by knocking down either LAMP5-AS1 was reversed upon treatment with the autophagy inhibitor bafilomycin A1 (25 nM, 12 h). MLL-AF9, LC3B-II, and LAMP5 were assayed by western blotting, and beta-tubulin as a loading control. The MLL-AF9, LC3B-II, or LAMP5 /Tubulin densitometric ratio were recorded by ImageJ. ( b ) Proposed model for the LAMP5-AS1-DOT1L-LAMP5 axis regulates autophagy and maintains MLL fusion protein

Article Snippet: For co-localization studies, after RNA and DNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with DOT1L primary antibody (CST, 90,878 S) and followed by DIG-FITC (Abcam, ab119349) and Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).

Techniques: Western Blot, Control