Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A ) Experimental design of the zebrafish tumor treatment assay. Tumor-bearing dbh:MYCN transgenic zebrafish, 3 wpf, were treated with DMSO or 13- cis RA (2 μM) for 6 days. Tumor size was quantified by the EGFP + surface area. ( B ) Representative images of 3-wpf dbh:MYCN transgenic zebrafish with EGFP + sympathoadrenal cell hyperplasia (white arrows) that were treated with DMSO or 2 μM 13- cis RA for 6 days. ( C ) Dot plot showing the distribution of posttreatment tumor size of DMSO ( n = 26) or 13- cis RA ( n = 16) treatment in zebrafish, as quantified by EGFP cross sectional area. Red bar indicates the median value. *** P < 0.001 by t test. ( D ) Representative images of 12-week-old dbh:MYCN transgenic zebrafish with EGFP + neuroblastomas (white arrows) that were treated with DMSO or 5 μM 13- cis RA for 6 days. ( E ) Dot plot showing the distribution of posttreatment tumor size of DMSO ( n = 6) or 13- cis RA ( n = 5) treatment in zebrafish, as quantified by EGFP cross-sectional area. Red bar indicates mean. ** P < 0.01 by t test. ( F ) Representative images of 12-wpf neuroblastoma tumor sections treated with DMSO or 5 μM 13- cis RA for 6 days and stained with hematoxylin and eosin (H&E) (left) or a PCNA-detecting antibody counterstained with hematoxylin (right). ( G ) Dot plot showing the percentage of PCNA + cells in neuroblastoma tumors treated with DMSO ( n = 4) or 13- cis RA ( n = 4). Red bar indicates mean. ** P < 0.01 by t test.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: Transgenic Assay, Staining

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A ) Cell growth time course for BE2C and NGP cells comparing treatment with DMSO or 5 μM ATRA for 6 days; *** P < 0.001 by ANOVA and t test at 6 days. Data show cell growth measurements with standard error bars for one representative experiment of three different independent experimental replicates. ( B ) Confocal images of BE2C and NGP neuroblastoma cells treated with DMSO or 5 μM ATRA for 6 days. Cells were stained with fibronectin (FN1), b3-tubulin (TUBB3), or vimentin (VIM) (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and phalloidin (red). Scale bar, 20 μm.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: Staining

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A ) Violin plot illustrating log 2 fold changes (ATRA/DMSO) among all highly expressed genes [base mean transcripts per million (TPM) > 10] when assayed by spike-in normalized RNA-seq in BE2C and NGP ( MYCN -amplified) cells. Changes are highlighted for MYCN (red), GATA3 (blue), and PHOX2B (green), all of which were reduced at the protein level as well. ( B ) Expression levels of adrenergic CRC transcription factor genes were determined by spike-in normalized mRNA-seq in BE2C and NGP cells treated with DMSO (gray) or 5 μM ATRA (red) for 6 days. ( C and D ) Normalized ChIP-seq tracks for H3K27ac and H3K27me3 depicting super-enhancers associated with the GATA3 (C) and PHOX2B (D) gene loci in BE2C cells. Cells were treated with 5 μM ATRA for 12 days. ChIP-seq read densities ( y axis) were normalized to reads per million (RPM) reads sequenced from each sample.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: RNA Sequencing, Amplification, Expressing, ChIP-sequencing

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A and B ) Super-enhancers were identified in the DMSO- and ATRA-treated BE2C (A) and NGP (B) cells and collapsed into one set of regions whose differential enrichment was assessed in an H3K27ac coverage scatterplot. Orange diagonal line indicates equivalent H3K27ac signal in control DMSO compared to ATRA-treated cells. Highlighted enhancers were associated with SOX4 (blue) and MEIS1 (red). ( C and D ) Normalized ChIP-seq tracks for H3K27ac showing acquired super-enhancers associated with SOX4 (C) and MEIS1 (D) in BE2C and NGP cells. Cells were treated with 5 μM ATRA for 12 days; shaded areas indicate super-enhancers identified by H3K27ac enrichment in the collapsed union list. ChIP-seq read densities ( y axis) were normalized to reads per million reads sequenced from each sample.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: Control, ChIP-sequencing

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A ) Transcript levels (TPM) of transcription factor genes up-regulated in BE2C cells by treatment with ATRA (5 μM) for 6 days. A subset of transcription factors retained from the adrenergic CRC is shown above, and transcription factors unique to the retino-sympathetic CRC are shown below. ( B and C ) Normalized ChIP-seq (H3K27ac and RARA) and CUT&RUN-seq (GATA3, PHOX2B, MEIS1, and SOX4) read coverage tracks depicting occupancy of transcription factors at the HAND2 gene locus in BE2C cells treated with DMSO (B) or ATRA (C) for 12 days. Rabbit IgG is shown as a control for the CUT&RUN-seq technique, and alignment read densities ( y axis) were normalized to RPM reads sequenced. IgG, immunoglobulin G. ( D ) Genome-wide co-occupancy heatmap for adrenergic and retino-sympathetic transcription factors in DMSO- and ATRA-treated BE2C cells as determined by ChIP-seq (H3K27ac and RARA) and CUT&RUN-seq (GATA3, PHOX2B, MEIS1, and SOX4). Genomic regions (rows) were defined as those enriched in sequencing reads for at least one target and are ranked by the integrated H3K27ac signal. ( E ) CRC transcription factors form an interconnected co-regulatory loop, and treatment of adrenergic neuroblastoma cells with ATRA suppressed the expression and activity of GATA3 , PHOX2B , and ASCL1 . Treatment with ATRA led to increased transcript levels and acquisition of new super-enhancers associated with MEIS1 and SOX4 in both BE2C and NGP cells. RARA had increased expression and were associated with super-enhancers under both DMSO and ATRA conditions. Regulatory elements and gene loci are denoted by rectangles, and proteins are denoted by oval symbols.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: ChIP-sequencing, Control, Genome Wide, Sequencing, Expressing, Activity Assay

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A and B ) Normalized ChIP-seq (H3K27ac) and CUT&RUN-seq (MEIS1, SOX4, and IgG) alignment tracks depicting occupancy of transcription factors at the SOX4 (A) and MEIS1 (B) gene loci in BE2C cells treated with DMSO (above) or ATRA (below) for 12 days. Super-enhancers were identified by H3K27ac signal and are shaded in ATRA-treated cells. Read densities ( y axis) were normalized to reads per million sequenced from each sample. ( C ) Western blot assay for SOX4 protein levels in wild-type control and SOX4 -knockout BE2C cells treated with 5 μM ATRA for 0, 1, 2, and 3 days. ACTB was used as a loading control. ( D and E ) Gene expression assayed by quantitative RT-PCR measuring the RNA levels of MEIS1 (D) and FN1 (E) in wild-type and SOX4 -knockout BE2C cells treated with 5 mM ATRA for 0, 1, 2, and 3 days and normalized to ACTB .

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: ChIP-sequencing, Western Blot, Control, Knock-Out, Gene Expression, Quantitative RT-PCR

Journal: Science Advances

Article Title: Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

doi: 10.1126/sciadv.abe0834

Figure Lengend Snippet: ( A ) H3K27ac ChIP-seq in NBL-S cells is treated with DMSO or ATRA (5 μM) for 12 days, showing the genomic region surrounding the t (2;4) structural variation involving the MYCN gene locus. The region downstream of FBXO8 is a super-enhancer that is translocated to within close proximity of the MYCN gene. ( B ) Western blot assay measuring MYCN or MYC protein levels in BE2C and NGP ( MYCN -amplified), along with NBL-S and SKNAS (enhancer hijacking) cells following treatment with ATRA (5 μM) for 0, 3, and 6 days. ( C ) Cell viability assay after 6 days of treatment with DMSO or ATRA (5 μM) in MYCN -amplified (BE2C and NGP) and MYC / MYCN enhancer–hijacked (NBL-S and SKNAS) cell lines. *** P < 0.001 by t test; N.S., not significant. ( D ) Bar graphs showing cell cycle distribution determined from hypotonic citrate propidium iodide (PI) staining of BE2C, NGP, NBL-S, and SKNAS cells treated with DMSO or ATRA (5 μM) for 6 days.

Article Snippet: Cell culture–grade DMSO was purchased from ATCC.

Techniques: ChIP-sequencing, Western Blot, Amplification, Viability Assay, Staining

Journal: bioRxiv

Article Title: Modeling platinum resistance in a stem-like patient-derived ovarian cancer sample

doi: 10.1101/2024.01.30.577975

Figure Lengend Snippet: Cisplatin and olaparib resistant patient-derived cell line expresses markers of epithelial-mesenchymal transition (EMT) and stemness. A. PDX4 was obtained from a patient diagnosed with stage IVB high grade serous ovarian cancer (HGSOC). After sample dissociation and processing, cells were grown in vitro in increasing concentrations of cisplatin. B. While growing at 10 µM of cisplatin, PDX4 CR (cisplatin resistant) had cisplatin and olaparib IC50 of 12 µM (n=4) and 5.8 µM (n=3), respectively, as measured by thiazolyl blue tetrazolium bromide (methylthiazolyldiphenyl-tetrazolium bromide; MTT) viability assay. C. RNA sequencing revealed a total of 7,506 differentially expressed genes (DEGs), with 3,890 genes downregulated and 3,616 genes upregulated in PDX4 CR (n=3) relative to PDX4 SE (cisplatin sensitive; n=3). Each dot represents a single gene. The highlighted genes are associated with cisplatin resistance, cancer stemness, and/or EMT. Adjusted p-value < 0.1 and absolute log2(FC) > 0.58. D. PROGENy pathway signatures identified in cisplatin sensitive and resistant samples. E. Clustering heatmap displaying the top 50 differentially expressed genes (DEG) within the RNA-seq data. The logFC column indicates the expression relative to the cisplatin sensitive sample and the “AvgExp” column displays the average normalized count values across all samples. Above the logFC column, the box and whiskers plot summarizes the spread of the logFC expression values. F. Gene expression profiles of putative EMT (CDH2, VIM, TGFB1, TGFB2, and ZEB2) and stemness genes (BMI1, ENG, MYC, NOTCH1, and POU5F1B) in the cisplatin sensitive and cisplatin resistant samples as identified by RNA-seq. * p < 0.05, **p < 0.01, *** p < 0.001. Created with Biorender.com .

Article Snippet: Cell viability was measured using thiazolyl blue tetrazolium bromide (MTT; 00697; Chem-Impex, Wood Dale, IL, USA) assays.

Techniques: Derivative Assay, In Vitro, MTT Viability Assay, RNA Sequencing Assay, Expressing

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: (A) Interaction map for BH3 peptides and BH3 mimetics with BCL-2 family proteins. Red, Kd < 100nM, determined by fluorescence polarization. Ven, venetoclax; Nav, navitoclax. (B) % hCD45+ circulating blasts in AML PDXs on venetoclax treatment (100 mg/kg, PO, 5 days/week) for 2 weeks. Mean ± SEM, n=5 mice; *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t-test.

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques: Fluorescence, Two Tailed Test

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: (A) Schematic of in vivo venetoclax-resistant models generation.

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques: In Vivo

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: (A-B) Heat map of delta priming response in AML cell lines after treatment with (A) 5nM venetoclax and (B) 50nM S63845 for 16 h, determined by dynamic BH3 profiling (n=3).

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques:

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: (A) Cell viability of parental and venetoclax-resistant Molm-13 cells treated with MIK665 and +/− 1.3 μM venetoclax at 72 h. Mean±SEM, n=2.

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques:

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: (A-C) Parental or venetoclax-resistant myeloblasts from indicated PDXs were serially transplanted into NSG mice and assigned into treatment arms after 4 weeks post-transplant. (A and C) % hCD45+ peripheral blast count and corresponding survival curves in response to quizartinib. (B) % hCD45+ blast reduction across different compartments upon quizartinib treatment. Mean±SEM, n=5 mice; *P<0.05, **P<0.01, ***P<0.001 one-way ANOVA. Survival curve analysis; log-rank test. In C, VEN-R+quizartinib arm study was stopped before endpoint reached due to COVID.

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques:

Journal: Cancer cell

Article Title: Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia

doi: 10.1016/j.ccell.2020.10.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Clinical grade venetoclax (Medchem express) was formulated in a mixture of 60% phosal 50 PG, 30% PEG 400, and 10% EtOH.

Techniques: Blocking Assay, Derivative Assay, Recombinant, Red Blood Cell Lysis, Protease Inhibitor, Immunoprecipitation, Isolation, Sample Prep, Reverse Transcription, Sequencing, Software