Journal: Cell death discovery

Article Title: UM171 cooperates with PIM1 inhibitors to restrict HSC expansion markers and suppress leukemia progression.

doi: 10.1038/s41420-022-01244-6

Figure Lengend Snippet: Fig. 3 UM171 binds to PIM1 and activates its phosphorylation. A Q-RT-PCR analysis of HEL cells treated with UM171 (6 μM) for expression of the PIM1. B Q-RT-PCR analysis of HEL cells treated with UM171 (6 μM) for expression of the PIM2, and PIM3 genes. C Western blot of HEL cells treated with the indicated concentration of UM171 compound. GAPDH was used as a loading control. D Molecular docking of the compound UM171 and PIM1 (PDB:5O12). The phosphorylation site of PIM1 kinase was shown as insert. E The molecular binding energy between PIM1 and UM171 as well as pan-PIM inhibitors LGH447, TP3654, SGI1776 ad AZ1208. F Binding of PIM1 to UM171 in pull-down experiment using affinity ES6B beads. G Q-RT-PCR analysis of HEL cells treated with UM171 (6 uM), LGH447(5 μM), and UM171 + LGH447 for 24 hours for expression of PIM1. H Western blot of HEL cells treated with UM171 and DMSO for expression of LSD1.

Article Snippet: The antibodies used were as follow: Polyclonal rabbit PIM1 (ab54503), FLI1 Cell Death Discovery (2022) 8:448 (ab133485) and ERK (ab184699) were purchased from Abcam (UK); the GAPDH (AB-P-R001) antibody was obtained from Goodhere Biotech (CN); c-KIT (18696-1-AP), LSD1 (20813-1-AP) and P21CIP1 (10355-1-AP) were obtained from Protein Technology (CN); p-ERK (9101 S), stat3 (4904), p-stat3 (9145), goat anti-mouse IgG (H+ L) DyLight (TM) 680 (5470 s) and goat anti-rabbit IgG (H+ L) DyLight (TM) 680 (5151 s) antibodies were obtained from Cell Signaling Technology (US).

Techniques: Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Control, Binding Assay

Journal: ACS Chemical Biology

Article Title: Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C

doi: 10.1021/cb500374f

Figure Lengend Snippet: Peptide Sequences Targeting KDM1A, -B, and KDM4A–C Identified from Phage Display, As Well As the Apparent EC 50 Values of the Peptide-Phages

Article Snippet: KDM1A was purchased from BPSBioscience (Cat. No. 50097); KDM1B cloning, expression, and purification.

Techniques: Sequencing

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 1. LSD1 regulates phosphorylation of AKT in colorectal cancer. A, Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for LSD1 across different TCGA datasets. Boxlimitsare set at the third and first quartilerange with central line at the median, withwhiskers depicting 1.5 times the interquartile range. Data points outside this range are represented at outliers (black dots). Black and blue outline indicates data for WT and PIK3CA- mutant tumors, respectively. Red and purple fill represent significant increase and decrease in LSD1 expression, respectively, between PIK3CA-mutant and PIK3CA WT tumors. The numbers under the box plots are the number of samples used to generate the plots. Western blot analysis of empty vector (shEV) or LSD1 KD in SW480 (B) or HT29 (C) cells. Arrowhead indicates correct position of pT308-AKT band. Western blot quantified by densitometric analysis and normalized to b-actin and shEV. Results are represented as mean SD (n ¼ 3). Significance was determined by two-tailed Student t test. LSD1 CRISPR KO clones with or without LSD1 OE plasmid (HA-LSD1) in SW480 (D) or HT29 (E) cells analyzed by Western blot analysis. F, EV, LSD1 KD, or LSD1 OE cells treated with 250 mmol/L H2O2 for 1 hour. G, Brightfield and immunofluorescence images of EV or LSD1 KD HT29 cells under untreated or H2O2-treated conditions. A field was selected in the H2O2-treated shLSD1 cells to facilitate direct comparison of LSD1-deficient and LSD1-proficient cells. White arrow indicates cells with LSD1 expression and orange arrow indicates cells deficient in LSD1 (, P < 0.001; , P < 0.0001; ns, not significant).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Phospho-proteomics, Whisker Assay, Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Two Tailed Test, CRISPR, Clone Assay, Comparison

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 2. LSD1 catalytic activity is dispensable for regulation of gene expression and activation of AKT. A, Metageneplot and heatmap depicting ChIP-seq of LSD1 in WT (n ¼ 2) or LSD1 KO (n ¼ 1) SW480 cells at gene enrichment sites genome-wide. Average plots and heatmaps depicting: LSD1 enrichment peak overlap with DNase-seq peaks in SW480 (B) and H3K4me2 ChIP-seq signal at TSS enrichment sites genome-wide in shEV and shLSD1 SW480 cells (N ¼ 3; C). A–C, Values are derived from CPM (counts per million) normalized reads. E, Differentially expressed genes (DEG) from RNA-seq (log2FC 1 and FDR 0.05 ¼ purple) after 40 nmol/L GSK-LSD1 for 48 hours versus DMSO or shLSD1 versus shEV in SW480 cells (N ¼ 3). D, ChIP-seq gene tracks of representative DEGs in LSD1 versus EV KD SW480 cells with or without LSD1 promoter enrichment. F, Cells pretreated with DMSO or 40 nmol/L GSK-LSD1 for 48 hours then treated with 250 mmol/L H2O2 for 1 hour. Western blots were quantified by densitometric analysis and normalized to loading control and DMSO. Graph represents mean SD; ns, not significant. Significance determined using one-way ANOVA with Tukey multiple comparisons test (N ¼ 3). G, Mixed population LSD1 KO cells were transfected with vector control, HA-LSD1, or HA-LSD1 (K661A) for 48 hours. Whole-cell extract from untreated and cells treated with 250 mmol/L H2O2 for 1 hour were analyzed by Western blot analysis.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Activity Assay, Gene Expression, Activation Assay, ChIP-sequencing, Genome Wide, Derivative Assay, RNA Sequencing, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 3. LSD1 regulates AKT activation via scaffolding of the CoREST complex on chromatin. A, Chromatin affinity assay performed in shEV or shLSD1 with whole-cell extract (WCE) or chromatin-bound fraction. Western blots were quantified by densitometric analysis and normalized to loading control and shEV fraction. Significance determined by two-way ANOVA with Sidak multiple comparisons test (n ¼ 3). Graphs depict mean SD (, Padj < 0.01; , Padj < 0.0001; ns, not significant). B, shEV or shRCOR1 cells treated with 250 mmol/L H2O2 for 1 hour and analyzed by Western blot analysis. C, shEV or shHDAC1 cells treated as in B. D, Model for corin inhibitor mode of action. E, Cells treated with DMSO or 3, 5, or 7 mmol/L corin over time course and analyzed by Western blot analysis. F, Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for RCOR1 across different TCGA datasets. Box limits are set at the third and first quartile range with central line at the median, with whiskers depicting 1.5 times the interquartile range. Data points outside this range are represented as outliers (black dots). Black and blue outline indicates data for WT and PIK3CA-mutant tumors, respectively. Red fill represent significant increase in LSD1 expression, respectively, between PIK3CA-mutant and PIK3CA WT tumors. G, Fraction of patients with PIK3CA mutation separated by high expression of LSD1 or RCOR1 versus low expression of both LSD1 and RCOR1. Plotted below is bootstrapped 90% confidence interval for the mean difference in fraction for patients with the PIK3CA mutation between the two groups. Significance determined by permutation test (, P < 0.001).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Activation Assay, Scaffolding, Western Blot, Control, Whisker Assay, Expressing, Mutagenesis

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 4. Gastrointestinal cell lines with mutant PIK3CA are sensitive to LSD1 KD. A, SW480, LoVo, HT29, AGS, HCT116, and RKO cell growth over a 5-day time course determined by the CellTiter-Glo Luminescent Cell Viability Assay (n ¼ 4). Graph depicts mean þ SD. Statistical analyses are performed using two-way ANOVA and Sidak multiple comparisons test with all statistically significant comparisons shown. , Padj < 0.01; , Padj < 0.0001. B, Correlation plot of RNA-seq data from LSD1 versus EV KD in SW480 and HT29 cells. Significant data points are defined as abs(Log2FC) 1 and FDR 0.05 for LSD1 compared with EV KD for each cell line, including those unique to HT29 (purple), unique to SW480 (red), and shared between HT29 and SW480 (black; n ¼ 3). C, Ridge plot depicts LSD1 versus EV KD expression changes of genes contributing to max enrichment score of Hallmark and curated gene sets accessed from the molecular Signatures Database (v6.2). GSEA Ratio shows the ratio of genes contributing to max enrichment score, tothe total number of genes in the gene set. Heatmap on right shows Log2FC value for each gene enriched in HALLMARK_PI3K_AKT_MTOR_SIGNALING in either HT29 or SW480 cells sorted by HT29 Log2FC. D, Network analysis of uniquely upregulated genes in HT29 after LSD1 KD with significantly enriched processes from the Reactome database. Similar terms were manually grouped and size and color of circles were set to indicate number of genes and the P value, respectively. E, Clonogenic growth assay. Significance determined by two-way ANOVA with Sidak multiple comparisons test (n ¼ 3). Results are represented as mean SD (, Padj < 0.01; ns, not significant).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Mutagenesis, Cell Viability Assay, RNA Sequencing, Expressing, Growth Assay

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 5. LSD1 regulates Snail stability via AKT in PIK3CA C2 domain–mutant cancer cells. Western blot analysis of HT29 (A) or AGS cells treated with DMSO or 10 mmol/L GSK690693 for 48 hours (B). C, Real-time PCR analysis of SNAI1 RNA expression levels after 48-hour DMSO or 10 mmol/L GSK690693 treatment in HT29 and AGS cells. Expression was normalized to Gapdh and DMSO. Results are represented as mean SD. Significance determined by two-way ANOVA with Sidak multiple comparisons test. ns, not significant. D, Proportion of total PIK3CA mutations occurring in the different domains indicated across various cancer types in the TCGA pancancer datasets. E, shEV and shLSD1 HT29 cells treated with DMSO or 10 mmol/L MG-132 for 4 hours and analyzed by Western blot analysis. F, Real-time PCR analysis of LSD1 and SNAI1 RNA expression levels in shEV and shLSD1 HT29 cells as in C. , Padj < 0.0001. G, HT29 shLSD1 cells were transfected with HA-LSD1 alone or in combination with 10 mmol/L GSK690693.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Transfection

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 6. LSD1 is required for EGF-induced migration of cells with an active AKT–GSK3b–Snail axis. A, 10 Brightfield images of crystal violet–stained HT29 cells after 48-hour migration through transwell insert. WT, LSD1 KO, 10 mmol/L GSK690693, or 3 mmol/L corin cells were cotreated with 100 ng/mL EGF for 48 hours. B, Quantification of migration normalized to migration counts for untreated cells. Results are represented as mean SD. Significance was determined by one-way ANOVA with Tukey multiple comparisons test. All significant comparisons are shown. , Padj < 0.0001. C, shEV or shLSD1 cells were treated with 100 ng/mL EGF for 24 or 48 hours and analyzed by Western blot. D, Model depicting CoREST complex in the regulation of AKT.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Migration, Staining, Western Blot