Journal: Cell reports

Article Title: Menin-MLL1 complex cooperates with NF-Y to promote hepatocellular carcinoma survival

doi: 10.1016/j.celrep.2025.116619

Figure Lengend Snippet: (A) Scheme of epigenome-focused CRISPR-Cas9 screening in 2D/3D settings. (B) Summary of the most common mutations in HCC cell lines used for CRISPR-Cas9 screening. (C and D) Comparison of 2D/3D CRISPR screens in HLF (C) and PLC/PRF/5 (D) HCC cells using robust rank aggregation (RRA) scores. Subunits of menin-MLL1 are shown in red. (E) Heatmap of HCC-specific common negative hits among epigenetic regulators detected by CRISPR-Cas9 screens in HCC cell lines in 2D/3D settings. (F) Heatmap of hits showing opposite enrichment in 2D and 3D CRISPR-Cas9 screens in HCC cell lines. (G) Logarithmic counts of sequenced sgRNAs targeting ASH2L , MEN1 , or KMT2A genes in 2D/3D screens in HLF (left) and PLC/PRF/5 (right) cells, comparing initial and final time points. (H) Immunoblotting against Cas9 and β-Actin proteins in HLF and PLC/PRF/5 parental and knockin cells. (I and J) Summary of negative-selection CRISPR-Cas9 screen with sgRNAs targeting the MEN1 , ASH2L , or KMT2A gene and PCNA or Rosa26 serving as the positive or negative controls, respectively, in 2D (I) and 3D (J) depicted at days 3, 14, 21, and 28. Bar graphs represent the mean of measurements of 3 independently transduced sets of cells. Error bars represent the standard deviation (SD).

Article Snippet: Menin-MLL1 inhibitior (SNDX-5613) was purchased from MedChemExpress (HY-136175, CID: 132212657).

Techniques: CRISPR, Comparison, Western Blot, Knock-In, Selection, Standard Deviation

Journal: Cell reports

Article Title: Menin-MLL1 complex cooperates with NF-Y to promote hepatocellular carcinoma survival

doi: 10.1016/j.celrep.2025.116619

Figure Lengend Snippet: (A) HCC and liver cell viability following SNDX-5613 treatment assessed by CellTiter-Glo (CTG) assay after 14 days in 2D. Error bars represent the SD of 3 independent experiments, each performed in 4 technical replicates. (B and C) HCC and murine liver cell viability and growth after 28 days of SNDX-5613 treatment in 3D media. Cell viability was measured using the CTG assay (B), and cell growth and colony formation ability were assessed by microscopy (C). Micrographs were taken at ×1.25 amplification, with the bar indicating 1.3 mm. (D–F) Immunoblotting against menin (D), MLL1 (E), and H3K4me3 (F) with corresponding controls, such as β-Actin or total H3, in HLF cells following treatment with 5 μM SNDX-5613 for 4 days. (G) Distribution of binding sites relative to genomic features for H3K4me3, menin, and MLL1. (H and I) Venn diagrams of detected independent (H) or overlapping (I) menin, MLL1, and H3K4me3 peaks in HLF cells following treatment with DMSO or 5 μM SNDX-5613 (SNDX) for 4 days. Occupancy peaks for all the groups are selected based on menin binding sites. (J) Heatmaps showing the correlation of promoter peaks in a ±2-kb window with occupancy of H3K4me3, menin, and MLL1 across CUT&RUN data from HLF cells treated with 5 μM SNDX-5613 for 4 days. The bottom metaplots show the mean of overall peak signals detected at the regions comparing DMSO and SNDX-5613 treatments in HLF cells. (K) Examples of CUT&RUN tracks showing distribution of H3K4me3, menin, MLL1, and immunoglobulin (Ig)G binding to the promoter regions in HLF cells treated with 5 μM SNDX-5613 for 4 days. (L) Logarithmic fold changes (FCs) of the H3K4me3 mark, menin, and MLL1 protein binding to the menin sites comparing conditions of HLF cells treated with 5 μM SNDX-5613 to those treated with DMSO for 4 days. (M) GREAT analysis of the menin-MLL1 complex targets using the hallmark gene set as a reference, comparing HLF cells treated with SNDX-5613 to those treated with DMSO.

Article Snippet: Menin-MLL1 inhibitior (SNDX-5613) was purchased from MedChemExpress (HY-136175, CID: 132212657).

Techniques: CTG Assay, Microscopy, Amplification, Western Blot, Binding Assay, Protein Binding

Journal: Cell reports

Article Title: Menin-MLL1 complex cooperates with NF-Y to promote hepatocellular carcinoma survival

doi: 10.1016/j.celrep.2025.116619

Figure Lengend Snippet: (A) Immunoblotting against menin and β-Actin proteins in HLF and PLC/PRF/5 knockout (KO) and NTC cells. (B) Logarithmic fold changes between MEN1 KO relative to NTC and SNDX-5613 treatment relative to DMSO from RNA-seq of HLF or PLC/PRF/5 cells. (C) Heatmap of differentially expressed genes in HLF and PLC/PRF/5 cells with MEN1 or NTC KO or treated with SNDX-5613 or DMSO. (D) UpSet diagrams showing commonly up- and downregulated genes in HLF and PLC/PRF/5 cells treated with SNDX-5613 or upon MEN1 KO. (E) GSEA ontologies with hallmark gene set as a reference affected by SNDX-5613 treatment and MEN1 KO in HLF and PLC/PRF/5 cells. (F) GSEA analysis of gene expression changes in HLF or PLC/PRF/5 cells treated with SNDX-5613 with PI3K/AKT/mTOR signaling pathway from the hallmark gene set. (G) Logarithmic fold changes in gene expression and in menin and MLL1 binding to these genes in HLF cells treated with SNDX-5613. (H) Logarithmic fold changes in gene expression depending on menin-MLL1 binding in HLF cells treated with SNDX-5613.

Article Snippet: Menin-MLL1 inhibitior (SNDX-5613) was purchased from MedChemExpress (HY-136175, CID: 132212657).

Techniques: Western Blot, Knock-Out, RNA Sequencing, Gene Expression, Binding Assay

Journal: Cell reports

Article Title: Menin-MLL1 complex cooperates with NF-Y to promote hepatocellular carcinoma survival

doi: 10.1016/j.celrep.2025.116619

Figure Lengend Snippet: (A and B) Venn diagram of the overlap between NF-YB and menin peaks (A) or NF-YB peaks alone (B) in HLF cells treated with DMSO or SNDX-5613. (C) Volcano plot of logarithmic differential binding of NF-YB in HLF cells upon SNDX-5613 treatment. (D) Distribution of binding sites relative to genomic features for NF-YB based on its binding status (lost, stable, or gained). (E) Heatmaps showing the CUT&RUN or ATAC-seq signal in a ±2-kb window for NF-YB, menin, MLL1, H3K4me3, H3K27ac, and H3K27me3 from HLF cells treated with SNDX-5613 at NF-YB gained or lost peaks. (F and G) Logarithmic fold changes between NF-YB binding and chromatin accessibility (F) or gene expression (G) upon SNDX-5613 treatment relative to DMSO in HLF cells from CUT&RUN and ATAC-seq data (F) or RNA-seq data (G). (H and I) Logarithmic fold change of chromatin accessibility (H) or gene expression (I) based on the NF-YB binding status (lost, stable, or gained). (J) Metaplots showing the normalized signal in a ±2-kb window of NF-YB sites for ATAC-seq chromatin accessibility, menin, H3K27ac, and H3K27me3 across CUT&RUN-seq data from HLF cells treated with SNDX-5613. (K) Motif enrichment of sites that either gain or lose NF-YB and are found in open or closed chromatin. (L) Expression of the nearest gene to NF-YB peaks from (K) in HLF cells treated with SNDX-5613.

Article Snippet: Menin-MLL1 inhibitior (SNDX-5613) was purchased from MedChemExpress (HY-136175, CID: 132212657).

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The histone methyltransferase MLL1 complex inhibits expression of fetal hemoglobin

doi: 10.1016/j.jbc.2025.110863

Figure Lengend Snippet: BCL11A is a direct transcriptional target of the MLL1 complex. A , heatmap showing differential gene expression at 72 h after MEN1 knockdown in HUDEP-2 cells. B , Volcano plot showing differential gene expression upon MEN1 knockdown in HUDEP-2 cells. C , left panel , real-time RT–PCR analysis of BCL11A mRNA levels in HUDEP-2 cells at 72 h after infection with lentiviral shRNAs targeting MEN1 (MEN1-sh1 and -sh2) or nontargeting control lentiviral shRNA (NC-sh). Relative expression levels were calculated by normalizing to RPL4 mRNA levels in the same sample and also to cells infected with NC-sh virus. Data are represented as mean ± SD (n = 3, technical replicates). Right panel, representative Western blotting analysis of protein levels of BCL11A and control β-ACTIN in HUDEP-2 cells at 72 h after infection with the indicated lentiviral shRNAs. D , left panel , real-time RT–PCR analysis of BCL11A mRNA levels in HUDEP-2 cells at 72 h after infection with lentiviral shRNAs targeting KMT2A (KMT2A-sh1 and -sh2) or NC-sh. Data are represented as mean ± SD (n = 3, technical replicates). Right panel, representative Western blotting analysis of protein levels of BCL11A and control β-ACTIN in HUDEP-2 cells at 72 h after infection with the indicated lentiviral shRNAs. E , quantitative ChIP analysis of indicated regions of BCL11A promoter (−0.6 kb), enhancer (+58 kb), and control (−16.8 kb) region in HUDEP-2 cells at 72 h after transduction with lentiviral shRNA NC-sh or MEN1-sh2 using a MEN1-specific antibody. Data are represented as mean ± SD (n = 3, technical replicates). F , similar ChIP analysis as in ( E ) using HUDEP-2 cells at 72 h after transduction with lentiviral shRNA NC-sh or KMT2A-sh2 and a KMT2A-N-specific antibody. G , real-time RT–PCR analysis of γ-, ε-, and β-globin mRNA levels in HUDEP-2 cells at 72 h after infection with the indicated combination of lentiviruses, including the NC-sh, MEN1-targeting MEN1-sh1 (Sh1), a BCL11A-expressing virus (BCL11A), and its control empty virus (Vector). Relative expression levels were calculated by normalizing to RPL4 mRNA levels in the same sample and also to cells infected with NC-sh virus. Data are represented as mean ± SD (n = 3, technical replicates). H , representative Western blotting analysis of protein levels of BCL11A, MEN1, and control β-ACTIN in the same cells as in G . ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001 (two-tailed Student’s t test). ChIP, chromatin immunoprecipitation.

Article Snippet: Immunoprecipitations were performed using anti-MLL1 antibody (Bethyl Laboratories), anti-H3K4me3 antibody (Abcam), and control rabbit immunoglobulin G (Cell Signaling).

Techniques: Gene Expression, Knockdown, Quantitative RT-PCR, Infection, Control, shRNA, Expressing, Virus, Western Blot, Transduction, Plasmid Preparation, Two Tailed Test, Chromatin Immunoprecipitation