Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: Genetic alterations in two c‐SCLC cases

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Amplification

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 expression is upregulated in the SCLC component of c‐SCLC tumors. (a) Examples of RNAscope in situ hybridization results of cores from a c‐SCLC tumor: two from the SCLC component (upper panel) and two from the NSCLC component (lower panel). Scale bar = 50 µm. RUNX1T1 mRNA signal is detected as red dots and MYC by the green dots. (b) Quantitation of the RUNX1T1 RNAscope signal of NSCLC and SCLC components (3 images/core for 2 cores of each histology) using CellProfiler. * P = 0.0292 from unpaired t‐test. Error bars represent mean ± SD. (c) INSM1 IHC stain of representative NSCLC and SCLC cores from c‐SCLC patient. Scale bar = 50 µm.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Expressing, RNAscope, In Situ Hybridization, Quantitation Assay, Staining

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 is highly expressed in SCLC. (A) RUNX1T1 mRNA levels in cancer cell lines based on Affymetrix data from the CCLE. SCLC is highlighted with a red arrow and NSCLC with a blue arrow. (B) Western blot of RUNX1T1 expression in eight SCLC cell lines (DMS79, H2171, H1694, SHP77, H82, H446, H69, and SW1271) and four NSCLC cell lines (A549, H1299, H1650, and PC9); beta‐actin was used as the loading control. (C) RUNX1T1 mRNA levels in tumor samples detected by RNA‐Seq comparing SCLC with other cancers (BRCA: breast cancer, CR: colorectal cancer, GBM: glioblastoma, LUAD: lung adenoma NSCLC, LUSC: lung squamous NSCLC, PRAD: prostate adenoma cancer, SKCM: skin melanoma cancer). (D) RUNX1T1 in situ hybridization examples using RNAscope on clinical tumor samples of ‘pure’ SCLC, lung adenocarcinoma and lung squamous cell carcinoma. Red dots identify RUNX1T1 mRNA signal, green dots MYC mRNA signal. Scale bar = 50 µm.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Western Blot, Expressing, Control, RNA Sequencing, In Situ Hybridization, RNAscope

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: Overexpression and knockout of RUNX1T1 lead to significant changes in hallmark pathways. (A) RUNX1T1 RT‐qPCR validating overexpression efficiency in cell lines ( n = 3 replicates of individual experiment/cell, error bars represent mean ± SEM). The house keep gene beta‐actin was used for normalization. No signal was detected in parental A549 and H1650 cells. (B) Western blot results validating RUNX1T1 overexpression (OE) and knockout (KO) in various cell lines. (C) Significantly changed hallmark pathways identified by GSEA after RUNX1T1 overexpression in NSCLC cell lines (H1299 and H1650) or RUNX1T1 knockout in SCLC cell lines (H82 and H2171). Blue and red bars show the overlapped pathways inversely affected by overexpression and knockout, respectively. NES: normalized enrichment score. (D) GSEA plots showing ‘E2F TARGETS’ genes depleted by RUNX1T1 overexpression in NSCLC (H1650 and H1299) and enriched by RUNX1T1 knockout in SCLC (H82 and H2171).

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, Knock-Out, Quantitative RT-PCR, Western Blot

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 overexpression increases E2F activity and decreases CDKN1A (p21) expression. (A) E2F luciferase reporter assays in RUNX1T1 overexpressing cell lines A549, H1299, H841, and SW1271 ( n = 4 replicates of individual experiment/cell, 2 experiments/cell). Error bars represent mean ± SD. Significance by unpaired multiple t‐test. (b) E2F luciferase activity in H841 and SW1271 cells with stable knockdown of RB1 by shRNA ( n = 4 replicates of individual experiment/cell). Error bars represent mean ± SD. Significance by unpaired t‐test (c) CDKN1A (p21) western blot and RT‐qPCR ( n = 3 replicates of individual experiment/cell, error bars represent mean ± SEM) in RUNX1T1 overexpressing (OE) cells compared with the corresponding control. Beta‐actin was used as the loading control in western blotting and for normalization in qPCR. Significance by unpaired t ‐test.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, Activity Assay, Expressing, Luciferase, Knockdown, shRNA, Western Blot, Quantitative RT-PCR, Control

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 overexpression decreases histone acetylation at the CDKN1A (p21) promoter. (A) UCSC browser tracks showing CDKN1A gene annotation and precise transcription start site (TSS), as shown in GRO‐cap tracks of two different cell lines. DNaseI hyposensitivity and transcription factor ChIP‐seq tracks from ENCODE indicate tight regulation of the p21 gene. Enrichment of H3K27 ac (ENCODE ChIP‐seq data) and location of primer sets used in ChIP‐qPCR analysis are indicated. (B) RUNX1T1 ChIP‐qPCR results in overexpressing (OE) vs control H1299 and SW1271 cells targeting seven CDKN1A (p21) promoter and distal regions ( n = 3 replicates of biological duplicates/cell). Error bars represent mean ± SD (C) ChIP‐qPCR of pan‐histone 3 acetylation in OE vs control H1299 and SW1271 cells targeting the same seven CDKN1A (p21) promoter and distal regions ( n = 3 replicates of biological duplicates/cell). Error bars represent mean ± SD. (D) Western blot of CDKN1A (p21) in H1299 and SW1271 under Trichostatin A (TSA) treatment at 400nM and 2µM for six hours. Beta‐actin was used as the loading control. UT: untreated. DMSO: DMSO vehicle control.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, ChIP-sequencing, ChIP-qPCR, Control, Western Blot

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1‐RUNX1T1 (AML/ETO) knockdown increases global H3K9 acetylation. Heatmap (A) and metagene plot (B) showing the enrichment of H3K9ac near the TSS of 21,364 curated genes in Kasumi‐1 cells (H3K9ac ChIP‐seq dataset SRR203377 and SRR203378). (C) H3K9ac level at CDKN1A promoter proximal region is shown using IGV (integrated genome viewer).

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Knockdown, ChIP-sequencing

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Kaplan–Meier survival analysis of progeny of the ENU-treated Th-MYCN mouse #1590, that were either wild-type for Runx1t1 (red, unsuppressed) or had inherited the Y534H Runx1t1 mutation (black, suppressed); n = 11 per genotype, p < 0.0001 (log-rank (Mantel–Cox) test). b Scatter plot showing time to tumor development in progeny of the ENU-treated mouse #1590 that were wild-type for Runx1t1 (red), or had inherited the Y534H mutation (black), n = 11 per genotype. c Multiple protein sequence alignment of the RUNX1T1 NHR4 domain across a range of organisms. The suppressed tumor phenotype resulted from the substitution of histidine (H) for a highly conserved tyrosine residue (Y), denoted by the asterisk (upper panel). A schematic model of wild-type and mutant Runx1t1 NHR4 zinc-finger motif domain folding (bottom panel). d Kaplan–Meier survival analysis for homozygous Th-MYCN mice either wild type ( Th-MYCN +/+ Runx1t1 +/+) or with heterozygous knock-out ( Th-MYCN +/+ Runx1t1 +/−) of Runx1t1 . Wild-type Runx1t1 mice (red) demonstrated almost complete tumor penetrance, while Runx1t1 heterozygous knock-out mice (black) almost entirely lacked the ability to form tumors ( n = 101 for wild-type and n = 163 for heterozygous knock-out, log-rank (Mantel–Cox) test p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Mutagenesis, Sequencing, Residue, Knock-Out

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a The percentage neuroblast hyperplasia scored from homozygous Th-MYCN (+/+) mice or littermate mice lacking the MYCN transgene (−/−), with either wild-type (+/+) or heterozygous loss (+/−) of Runx1t1 . Scoring of N = 3–8 independent mice was performed for each genotype and timepoint. All data points were N = 3, except for +/+, +/+ week 1 and week 2 ( N = 4); +/+, +/− day 0 ( N = 8) and week 4 ( N = 4); −/−, +/− day 0 ( N = 6), week 1 ( N = 4) and week 4 ( N = 5). The graph is mean ± SEM. b Representative histology of RUNX1T1 staining in ganglia from mice homozygous for the Th-MYCN transgene, and either wild-type or heterozygous for Runx1t1 from day 0 and 4 weeks of age. Neuroblast hyperplasia is defined as ≥30 small round blue cells within a sympathetic ganglion . Photos were taken at 600X magnification, and the scale bars represent 20 microns. c βIII-tubulin staining of sympathetic ganglia isolated from Th-MYCN mice. The percent coverage of neurites was calculated and the area under the curve determined for each ganglia. N = 4 (+/+, +/−) or 5 (−/−, +/+ and +/+, +/+), Graphs are Mean ± SEM, * p = 0.0181, *** p = 0.0007. Two-tailed unpaired t -test. d Heatmap displaying gene clustering following RNA-Seq analysis of ganglia dissected from two-week-old Th-MYCN mice with either homozygous or heterozygous Runx1t1 , compared to fully developed murine neuroblastoma tumors. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Staining, Isolation, Two Tailed Test, RNA Sequencing

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Runx1t1 and MYCN mRNA expression in liver, brain, and tumor samples from homozygous Th-MYCN mice with intact Runx1t1 relative to the control, Gusb . Means ± SEM ( n = 3) are indicated. Data were analysed using one-way-ANOVA and corrected for the multiple comparison using the Tukey test. *** p = 0.0042, ns p = 0.1385 for Runx1t1 ; *** p = 0.0006 for MYCN . Three independent mice were used for each tissue. b Western blot for RUNX1T1 and MYCN in liver, brain, and tumor from homozygous Th-MYCN mice; n = 3 independent mice. c RUNX1T1 mRNA expression relative to the control GUSB in MYCN -non-amplified (non-AMP) and amplified (AMP) neuroblastoma cell lines. Means ± SEM ( n = 5 data points representing five different cell lines in each group, and the value of each data point is the mean of three biological repeats) are indicated, two-tailed Mann–Whitney test, P = 0.3095. d Western blot for RUNX1T1 and MYCN across a panel of MYCN non-amplified (non-AMP) and MYCN amplified (AMP) neuroblastoma cell lines. This experiment has been repeated once with similar results. e Scatter plot of RUNX1T1 mRNA expression in MYCN non-amplified (non-AMP) and amplified (AMP) tumors from a publicly available dataset (SEQC) of tumor samples from the neuroblastoma R2 database, n = 410 non-AMP and n = 92 AMP samples; two-tailed unpaired t -test, **** p < 0.0001. f A tissue microarray (TMA) of human neuroblastoma tumor samples ( n = 66) was stained with antibodies to either MYCN or RUNX1T1. Photos from three representative MYCN -non-amplified samples and three MYCN -amplified samples are shown. Numbers in panels indicate staining intensity. Photos were taken at 600× magnification, and the scale bars represent 20 microns. H&E, hematoxylin and eosin. g RUNX1T1 staining intensity was scored for all samples in the TMA. Neuroblastoma samples were split into non-amplified ( n = 52) and amplified ( n = 14) and compared to benign diseases of ganglioneuroblastoma (GNB) ( n = 5) and ganglioneuroma (GN) ( n = 12). Violin plot describes distribution of intensity, width describes frequency of score in tumors. The thick line represents the median, thin line represents the 25th and 75th percentiles. The bounds of the box show the range of scores. Unpaired t -test, two-tailed * p = 0.0403; ** p = 0.0064; *** p = 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Expressing, Control, Comparison, Western Blot, Amplification, Two Tailed Test, MANN-WHITNEY, Microarray, Staining

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Western blot of SH-EP TET-21/N cells after 72 h of treatment with 1 µg/mL doxycycline (left panel). Quantitation from three independent experiments demonstrated significantly decreased MYCN (**** p = 0.0001) and RUNX1T1 expression (*** p < 0.0001) (right panel). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. b MYCN and RUNX1T1 mRNA expression after 72 h doxycycline treatment, showing significant downregulation of MYCN (*** p = 0.0009) but no significant change in RUNX1T1 ( p = 0.9559). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. c Western blot of SH-EP neuroblastoma cells overexpressing MYCN and empty vector (EV) control (left panel). Quantitation of western blot, from three independent experiments (right panel) demonstrated significantly increased RUNX1T1 expression (* p = 0.0339). Values represent means from three independent experiments ±SD, two-tailed unpaired t -test. d RUNX1T1 and MYCN mRNA expression in SH-EP cells overexpressing MYCN, relative to the control gene ( GUSB ). Values represent means from three independent experiments ±SD. Two-tailed unpaired t -test, **** p < 0.0001; ns: not significant ( p = 0.5679). e Cyclohexamide chase e xperiment showing RUNX1T1 protein stability in three neuroblastoma cell lines (BE(2)-C, KELLY, SH-EP) over a 12 h time course. Values represent means from three independent experiments ±SD. f Luciferase activity following transfection of murine Runx1t1 5’UTR into MYCN overexpressing or EV SH-EP cells. Values represent means from three independent experiments ±SD. Groups were analysed using two-way ANOVA and Bonferroni multiple comparison. *** p = 0.0007; ns not significant ( p = 0.9335). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Western Blot, Quantitation Assay, Expressing, Two Tailed Test, Plasmid Preparation, Control, Luciferase, Activity Assay, Transfection, Comparison

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a, b RUNX1T1 knock down after 72hrs of doxycycline (1 µg/mL) treatment in KELLY and BE(2)-C cells respectively, with two independent shRNA constructs. Quantitation from four independent experiments demonstrated significantly decreased RUNX1T1 levels following shRNA-mediated knockdown. Values represent means from three independent experiments ±SD. Ordinary one-way ANOVA with Tukey’s multiple comparisons test; KELLY sh#1 **** p < 0.0001, sh #2 ** p = 0.0018, ns p = 0. 6962; BE(2)-C sh #1 *** p = 0.0006, sh #2 * p = 0.0295, ns p = 0.8753. c , d Colony formation after doxycycline-induced knockdown of RUNX1T1 in KELLY and BE(2)-C cells respectively, with two independent shRNA constructs. Colonies are represented as a percentage relative to the untreated control in each experiment. Values represent means from three independent experiments ±SD. RUNX1T1 shRNA-mediated knockdown resulted in significantly decreased colony numbers compared to controls. Ordinary 1-way ANOVA with Tukey’s multiple comparisons test; KELLY sh #1 **** p < 0.0001, sh #2 **** p < 0.0001, ns p = 0. 3589; BE(2)-C sh #1 *** p = 0.0005, sh #2 **** p < 0.0001, ns p = 0.8549. e , f Kaplan–Meier survival analysis of NSG mice xenografted with doxycycline-inducible RUNX1T1 shRNA#1 in KELLY and BE(2)-C cells, respectively. Mice were split into two groups ( n = 10 mice per group for KELLY and n = 6 mice per group for BE(2)-C) and fed doxycycline or control food once a 50 mm 3 tumor was measurable. P values were determined using the Log-Rank (Mantel–Cox) test, p < 0.0001 for KELLY and p = 0.0035 for BE(2)-C. Growth curves (lower panels) plot size over time, post-doxycycline treatment. Graphs are Mean ± SEM. g Representative images of KELLY cells at 7 days and endpoint ± doxycycline. Tumor samples were stained with H&E or immunohistochemically for RUNX1T1 and Ki67. Photos were taken at 600× magnification, and the scale bars represent 20 microns. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Knockdown, shRNA, Construct, Quantitation Assay, Control, Staining

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a One-dimensional 1 H NMR spectra of the Runx1t1 MYND domain showed that the wild-type spectrum (top) has sharp peaks and good dispersion whereas the spectrum of the Y534H mutant (bottom) indicates a protein not well-ordered and/or aggregated—consistent with the mutation disrupting proper folding of the domain. b Co-IP between RUNX1T1_WT/YH-3xFLAG and LSD1-HA (top left), CoREST3-HA (top right), HDAC3-HA (middle left), HDAC1-HA (middle right), HDAC2-HA (bottom left), and HAND2-HA (bottom right). HEK-293T cells were transiently co-transfected with pCMV14 RUNX1T1_WT- or Y534H-3xFLAG and HA-tagged constructs. Nuclear fractions were immunoprecipitated with anti-FLAG antibody and immunoblotted with both anti-FLAG antibody (IP) and anti-HA (Co-IP). An experiment was performed twice. The input sample represents 5% of the non-immunoprecipitated sample and the control sample (EV) resulted from the co-transfection with the HA-tagged construct and the pCMV14 empty vector. c Summary of the Co-IP results. d Binding curves and calculated affinities for peptides RCOR3_1-3 and NCOR2 at pH 6 and RCOR3_3 and NCOR2 at pH 7.5. Binding curves were derived by tracking the combined 1 H and 15 N Chemical Shift Perturbations of three signals in the MYND domain 15 N-HSQC following titration of each peptide in increments of 0.5 molar equivalents into samples of 15 N-HNR4 MYND, each of which was fitted to a simple 1:1 binding model. The affinities reported for each peptide are the average (±SD) of three sets of measurements. We estimate the uncertainty for the KD’s to be ~25%. The affinity of NCOR2 at pH 7.5 was calculated by ref. and is listed for comparison. e Schema of CoREST3/RCOR3 deletion mutants (left). HEK-293T cells were transiently co-transfected with pCMV14 RUNX1T1_WT-3xFLAG and HA-tagged CoREST3 constructs (right, n = 1 experiment). Nuclear fractions were immunoprecipitated with anti-FLAG antibody and samples immunoblotted and probed with anti-FLAG antibody (IP, top boxes), or anti-HA (Co-IP, bottom boxes). Input represents 5% of non-immunoprecipitated sample; −/+ indicates co-transfection of pCMV14 EV and HA-CoREST3 constructs (−) or co-transfection of pCMV14 RUNX1T1_WT-3xFLAG and HA-CoREST3 constructs (+). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: Dispersion, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Control, Cotransfection, Plasmid Preparation, Binding Assay, Derivative Assay, Titration, Comparison

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a ChIP-seq analysis of RUNX1T1 binding across three replicates showing the number of peaks and their distance in base-pairs (bp) to the transcription start site (TSS). b Proportion of the peaks occurring at different binding site locations from the RUNX1T1 three ChIP-seq replicates. RUNX1T1 binding occurring at either a promoter, gene body, distal intergenic or downstream in relation to a gene (left), binding in an exon, intron or intergenic (middle), and binding within exons at either a coding region (CDS), 5’ untranslated region (UTR) or 3’ UTR (right). c Motif discovery using homer analysis showing significantly enriched motifs from HAND2, PHOX2A, and TCF4. d UpSet plot of the intersection of ChIP-seq peaks of RUNX1T1, HAND2, LSD1, and RCOR3. The number of peaks identified are indicated for each gene and the intersection of gene peaks. RUNX1T1 peaks that co-localized with HAND2, LSD1, and CoREST3 are shown by the blue bar. e GREAT analysis (using Binomial test) showing enriched gene ontology (GO) biological process of the common peaks between RUNX1T1, HAND2, LSD1, and RCOR3. f Number of active, poised, and primed enhancer sites where RUNX1T1 binds in the presence (wildtype; WT) and absence of RUNX1T1 (knock out; KO).

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: ChIP-sequencing, Binding Assay, Knock-Out

Journal: Nature Communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: a Gene set enrichment analysis (GSEA) for Hallmark gene sets performed on RNAseq data obtained following RUNX1T1 shRNA knockdown in KELLY cells. A bubble plot is shown with the size of the bubble representing the significance based on the nominal p value. NES: normalized enrichment score showing the strength and enrichment direction. The column labeled ganglia represents the same analysis performed on ganglia (Th-MYCN +/+ ; Runx1t1 +/− ) at 2 weeks versus tumor obtained from Th-MYCN +/+ ; Runx1t1 +/+ mice. b GSEA plot showing Hallmark MYC_target_V1 genes following RUNX1T1 shRNA downregulation in KELLY cells. c GSEA showed significant enrichment of PRC2 target genes (BenPorath_PRC2_targets) following RUNX1T1 shRNA downregulation in KELLY cells. d RUNX1T1 shRNA downregulation in aRMS cell lines (Rh41 and Rh3) significantly decreased cell proliferation, n = 3 independent experiments; graph is mean ± SEM, two-way ANOVA with Dunnett’s multiple comparison. Rh41 sh #1 ± Doxy 6d *** p = 0.0003, 7d and 8d **** p < 0.0001; Rh3 sh #1 ± Doxy 6d * p = 0.0149, 7d and 8d **** p < 0.0001. The immunoblots show decreased RUNX1T1 levels following shRNA-mediated knockdown. Each experiment has been repeated three times with similar results. e RUNX1T1 shRNA downr e gulation in SCLC cell lines (DMS-273 and DMS-53) significantly decreased clonogenic capacity, n = 3 independent experiments, graph is mean ± SEM, unpaired two-tailed t -test. DMS-273 EV ns p = 0.6682, sh#1 * p = 0.0127, sh#2 * p = 0.0465; DMS-53 EV ns p = 0.2210, sh#1 * p = 0.0325, sh#2 ** p = 0.0075. The immunoblots show decreased RUNX1T1 levels following shRNA-mediated knockdown. Each experiment has been repeated three times with similar results. f Proposed mechanism of action of RUNX1T1 in MYCN -amplified neuroblastoma. High levels of MYCN resulting from gene amplification drive increased protein translation which includes increased RUNX1T1 protein levels. High-level RUNX1T1 expression is necessary to maintain an ESC-like phenotype as well as generate enhancer-mediated repression of HAND2 targets genes otherwise required for neuronal differentiation. Loss of RUNX1T1 allows upregulation of HAND2-driven pro-differentiation genes while at the same time inhibiting MYCN activity by decreasing the level of the obligate MYCN dimerization partner MAX. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies for RUNX1T1 (rabbit polyclonal antibody, 15494-1-AP, Proteintech, 1:1000 in 4% skim milk in TBST), MYCN (mouse monoclonal B8.4.B, sc-53993, Santa Cruz, 1:1000 in 4% skim milk in TBST), GAPDH (mouse monoclonal G-9, sc-365062, Santa Cruz, 1:5000), HAND2 (Abcam, RabmAb Clone: EPR19451, 1:1000 in 4% skim milk in TBST), anti-FLAG M2 (Sigma Aldrich clone M2, affinity isolated antibody, 1:3000 in 4% skim milk in TBST), anti-HA (AbCAM ab9110, Rabbit Polyclonal HA tag antibody, 1:3000 in 3.5% skim milk in TBST), and ACTIN (rabbit polyclonal antibody, A2066, Sigma-Aldrich, 1:5000).

Techniques: shRNA, Knockdown, Labeling, Comparison, Western Blot, Two Tailed Test, Amplification, Expressing, Activity Assay

Journal: Journal of proteome research

Article Title: Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics

doi: 10.1021/pr200834f

Figure Lengend Snippet: Mass spectrometry-based quantification of SUMO conjugation via SILAC approach. . (A) Representative SILAC peptide pairs for an upregulated protein (RUNX1) and a downregulated protein (TIF1β). Peptide identifications are included in the figure, and associated m/z and intensity information in Table S1. (B) Non-normalized ratio plot including all quantified peptides in the experiment, with 2 peptides from RUNX1 and 31 peptides from TIF1β in black and peptides from the other 186 proteins in grey.

Article Snippet: Antibodies used in this study include anti-SUMO2/3 polyclonal antibody (Covance), anti-HA polyclonal antibody (Cell Signaling), anti-ubiquitin monoclonal antibody (Cell Signaling), anti-TIF1β polyclonal antibody (Cell Signaling), and anti-RUNX1 polyclonal antibody (Novus Biologicals).

Techniques: Mass Spectrometry, Conjugation Assay

Journal: Journal of proteome research

Article Title: Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics

doi: 10.1021/pr200834f

Figure Lengend Snippet: Verification of proteomic analysis. Proteomic analysis data were verified for RUNX1 (A) and TIF1β (B). HA-tagged SUMO3 conjugated proteins of extracts from control and OGD cultures were immunoprecipitated using anti-HA agarose and eluted with 100 μg/ml HA peptide. Eluates were left intact or incubated with SUMO protease to release SUMO from target proteins, loaded onto SDS-PAGE gels and immunoblotted using antibodies against RUNX1 (A) and TIF1β (B). WB, Western blotting; IP, immunoprecipitation.

Article Snippet: Antibodies used in this study include anti-SUMO2/3 polyclonal antibody (Covance), anti-HA polyclonal antibody (Cell Signaling), anti-ubiquitin monoclonal antibody (Cell Signaling), anti-TIF1β polyclonal antibody (Cell Signaling), and anti-RUNX1 polyclonal antibody (Novus Biologicals).

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot