Journal: Cancer discovery

Article Title: Ontogeny Dictates Oncogenic Potential, Lineage Hierarchy, and Therapy Response in Pediatric Leukemia

doi: 10.1158/2159-8290.CD-25-0556

Figure Lengend Snippet: Single-cell profiling reveals developmentally regulated transcriptional and epigenetic programs in NUP98::NSD1-driven AML. A, UMAP of merged scRNA-seq reference, including normal and leukemic xenografts, categorized into 17 clusters. Control, n = 2 biological replicates; NUP98::NSD1, n = 4; WT1ko, n = 2; and NUP98::NSD1/WT1ko, n = 3. B, Contribution of each group to the merged scRNA-seq UMAP. The red dotted box highlights LMPP/early GMPs in the NUP98::NSD1 and NUP98::NSD1/WT1ko conditions. C, Beeswarm plot comparing log fold changes in nearest neighbor cells from different cell type clusters computed with Milo among NUP98::NSD1 to control, NUP98::NSD1/WT1ko to control, and NUP98::NSD1/WT1ko to NUP98::NSD1. Significant neighborhoods identified at FDR < 0.01 are indicated in color. D, Mapping of scRNA-seq for PDX #1 (NUP98::NSD1, FLT3-ITD) and PDX #2 (NUP98::NSD1, FLT3-ITD, WT1 mutation) onto the merged scRNA-seq reference, displaying relative cell abundance density. E, UMAP of FL NUP98::NSD1 and NUP98::NSD1/WT1ko samples, by AUC score for the LSPC quiescent gene signature. Higher AUC scores (red) indicate stronger enrichment of the quiescent signature, whereas lower scores (blue) represent weaker enrichment. F, Schematic experimental overview of the LDA experiment with CD34 + CD117 + -enriched LSCs and non-CD34 + CD117 + cells sorted from FL NUP98::NSD1 and NUP98::NSD1/WT1ko xenografts. G, Leukemia initiation stem cell frequency for CD34 + CD117 + enriched LSCs and non-CD34 + CD117 + cells based on the LDA approach described in F . ****, P < 0.0001 using Pearson’s χ 2 test, n = 14–15 mice per condition (4–5 mice per dosage). H, Mapping scRNA-seq for CB NUP98::NSD1/WT1ko onto the merged FL-derived scRNA-seq reference, contoured with relative abundance density of cells. I, Besswarm plot showing the log fold change comparing FL NUP98::NSD1/WT1ko xenografts and CB NUP98::NSD1/WT1ko xenografts for groups of nearest neighbor cells from different cell type clusters computed with Milo. Significant neighborhoods identified at an FDR < 0.01 are indicated in color. n = 3 for FL NUP98::NSD1/WT1ko and n = 2 for CB NUP98::NSD1/WT1ko. J, Bar plot showing significantly upregulated hallmark pathways in FL NUP98::NSD1/WT1ko cells relative to CB NUP98::NSD1/WT1ko in the LMPP/early GMP cluster. Pathways marked with an asterisk (*) are statistically significant based on the cutoff P value < 0.05. The x -axis represents −log 10 ( P value), with higher values indicating greater statistical significance. K, UMAP of CB NUP98::NSD1/WT1ko with cells colored by their AUC score for the LSPC quiescent signature using AUCell. L, Violin plots showing AUCell enrichment scores for LSPC quiescent (left) and LSPC cycling (right) in LMPP/early GMPs from FL NUP98::NSD1/WT1ko and CB NUP98::NSD1/WT1ko. Wilcoxon rank-sum test. M, Survival analysis of NSGS mice transplanted with FL- or CB-derived NUP98::NSD1/WT1ko cells. Survival was monitored over a 120-day observation period. ****, P < 0.0001 using the log-rank Mantel–Cox test, n = 4–5 mice per condition. (Created in BioRender. https://BioRender.com/r2dvel5 .)

Article Snippet: Antibodies used for these experiments are as follows: WT1 (Novus Biologicals, NB110–60011, 1:1,000), GAPDH (Abcam, ab8245, 1:20,000), and goat anti-mouse IgG horseradish peroxidase conjugate (Thermo Fisher Scientific, 32430, 1:20,000).

Techniques: Control, Mutagenesis, Derivative Assay

Journal: Cancer discovery

Article Title: Ontogeny Dictates Oncogenic Potential, Lineage Hierarchy, and Therapy Response in Pediatric Leukemia

doi: 10.1158/2159-8290.CD-25-0556

Figure Lengend Snippet: WT1 loss and ontogeny shape differential therapy responses in NUP98::NSD1-driven AML. A, Volcano plot of differentially expressed genes in LMPP/early GMPs between FL NUP98::NSD1/WT1ko and NUP98::NSD1. Genes with significant adjusted P values < 0.05 and log 2 fold change > 1 are colored; representative upregulated and downregulated genes are indicated. B, Bar plots showing over representation analysis (ORA) results of differentially expressed LMPP/early GMPs genes between NUP98::NSD1/WT1ko and NUP98::NSD1. Significant hallmark pathways are highlighted in bold and with an asterisk (*) based on P value < 0.05. C, UMAP of EGR1 expression in FL NUP98::NSD1 and NUP98::NSD1/WT1ko xenografts. D, UMAP of MYC expression in FL NUP98::NSD1 and NUP98::NSD1/WT1ko xenografts. E, Boxplots showing normalized expression of EGR1 (left) and MYC (right) in LMPP/early GMPs from FL NUP98::NSD1/WT1ko and CB NUP98::NSD1/WT1ko xenografts based on pseudobulk DESeq2 analysis. The solid lines in each box indicate median expression values, and the height of the box indicates the interquartile range (IQR). EGR1 : FC = 2.77, P = 3.42e–09; MYC : FC = −0.69, P = 0.002. F, Schematic experimental overview of in vivo studies to characterize the impact of cytarabine (Ara-C) and menin inhibitor (revumenib) treatment of FL NUP98::NSD1, FL NUP98::NSD1/WT1ko, and CB NUP98::NSD1/WT1ko xenografts. G, Engraftment of human CD45 + cells in control, cytarabine-treated, or revumenib-treated FL NUP98::NSD1, FL NUP98::NSD1/WT1ko, and CB NUP98::NSD1/WT1ko xenografts. Error bars represent SD. **, P < 0.01; ***, P < 0.001, unpaired t test; n = 4–19 mice per condition. H, Percentage of CD34 + CD117 + enriched LSCs in control, cytarabine-treated, and revumenib-treated NUP98::NSD1 and NUP98::NSD1/WT1ko xenografts. Error bars represent SD. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, unpaired t test. I, UMAP of scRNA-seq data from control, cytarabine- and revumenib-treated FL NUP98::NSD1/WT1ko xenografts, mapped to the merged scRNA-seq reference, contoured by relative abundance density of cells. J, Bar plots showing ORA results for pathways upregulated in NUP98::NSD1/WT1ko after treatment with cytarabine and revumenib. Significant hallmark pathways are highlighted in bold and marked with an asterisk (*) based on a 0.05 P value cutoff. (Created in BioRender. https://BioRender.com/2utsqm7 .)

Article Snippet: Antibodies used for these experiments are as follows: WT1 (Novus Biologicals, NB110–60011, 1:1,000), GAPDH (Abcam, ab8245, 1:20,000), and goat anti-mouse IgG horseradish peroxidase conjugate (Thermo Fisher Scientific, 32430, 1:20,000).

Techniques: Expressing, In Vivo, Control

Journal: Cancer discovery

Article Title: Ontogeny Dictates Oncogenic Potential, Lineage Hierarchy, and Therapy Response in Pediatric Leukemia

doi: 10.1158/2159-8290.CD-25-0556

Figure Lengend Snippet: Targeting BCL2 dependency in fetal-origin LSCs with venetoclax and combination therapies diminished AML. A, OS of patients with NUP98::NSD1 AML stratified by fetal-oncogenic gene signature. The light blue and red curves show the survival of patients with low and high fetal-oncogenic gene signatures, respectively. Kaplan–Meier method and log-rank test; n = 104 patients. B, EFS of patients with NUP98::NSD1 AML stratified by age. The light blue and red curves show patients above and below 10 years of age, respectively. Kaplan–Meier method and log-rank test; n = 104 patients. C, ChIP-seq tracks at BCL2 , BCL2A1 , and BCL2L1 loci from NUP98::NSD1/WT1ko leukemia cells for PRDM16 and H3K27ac; n = 3 biological replicates per condition. D, UMAP of BCL2 expression in FL NUP98::NSD1 and NUP98::NSD1/WT1ko secondary xenografts. E, BCL2 mRNA expression in NUP98::NSD1/WT1ko leukemia cells with control or PRDM16ko detected by qRT-PCR. Error bars represent SD; ****, P < 0.0001 using an unpaired t test; n = 3 biological replicates per condition. F, OCR in NUP98::NSD1/WT1ko leukemia cells with venetoclax treatment using Seahorse assays. Analysis of basal respiration, ATP-linked respiration, maximal respiration, and spare respiration. Error bars represent SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 using a two-way ANOVA; n = 5 biological replicates per condition. G, Schematic experimental overview of in vivo assessment of venetoclax in FL and CB NUP98::NSD1/WT1ko xenotransplanted mice. H, Engraftment of human CD45 + cells and percentage of CD34 + CD117 + enriched LSCs. Error bars represent SD; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 using an unpaired t test, n = 5–8 mice per condition. I, Experimental in vivo strategy to assess the effects of cytarabine (Ara-C), revumenib, venetoclax, and combinations of (a) cytarabine + venetoclax and (b) revumenib + venetoclax on FL NUP98::NSD1/WT1ko cells. J, Engraftment of human CD45 + cells. Error bars represent SD. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 using an unpaired t test, n = 8–13 mice per condition. K, Percentage of CD34 + CD117 + -enriched LSCs. Error bars represent SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 using an unpaired t test, n = 8–13 mice per condition. L, UMAP visualization of scRNA-seq data from NUP98::NSD1/WT1ko: control vs. venetoclax-treated conditions. Data are mapped on the merged scRNA-seq reference, contoured with the relative abundance density of cells in each sample. M, Experimental in vivo strategy to assess the effects of cytarabine (Ara-C), revumenib, venetoclax, and combinations of cytarabine + venetoclax and revumenib + venetoclax in a relapsed NUP98::NSD1/FLT3-ITD/WT1-mutant AML PDX model. N, Engraftment of human CD45 + cells. Error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 using an unpaired t test, n = 7–10 mice per condition. O, Oligomycin; F, FCCP(carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone); R/A, Rotenone/Antimycin A. (Created in BioRender. https://BioRender.com/6ezoyjb .)

Article Snippet: Antibodies used for these experiments are as follows: WT1 (Novus Biologicals, NB110–60011, 1:1,000), GAPDH (Abcam, ab8245, 1:20,000), and goat anti-mouse IgG horseradish peroxidase conjugate (Thermo Fisher Scientific, 32430, 1:20,000).

Techniques: ChIP-sequencing, Expressing, Control, Quantitative RT-PCR, In Vivo, Mutagenesis

Journal: Pakistan journal of medical sciences

Article Title: Differentiation of CD117 + Amniotic Fluid Stem Cells towards Nephron Progenitors.

doi: 10.12669/pjms.38.6.4887

Figure Lengend Snippet: Fig.3A: Characterization of NPCs colonies for WT1 by immunofluorescence staining as performed on three different amniotic fluid derived NPCs. This figure shows immunofluorescence analysis for positive nuclear marker WT1 (a) Bright field image of NPCs at 40x showed irregular with ill-defined borders having tightly packed cells for the three different amniotic fluid derived NPCs. (b) Counter DAPI nuclear stain showed blue color of Nucleus. (c) Characteristic of NPCs, WT1 is shown in nuclear compartment of cells.

Article Snippet: Immunocytochemistry: The amniotic fluid samples were washed thoroughly with Phosphate Buffered Saline then fixed at 1:1 acetone: methanol for twenty minutes at 25°C, after washing with Buffer Saline containing tween 20 (PBST) three times Triton X-100 was use for permeabilization of cell membrane then again washed with PBST, it was clogged by bovine serum albumin (BSA) 1% plus PBS (without Calcium &Magnesium) for 30 minutes at Room Temperature and incubated with primary monoclonal unconjugated antibody WT1 (WT1/857 + 6F-H2 clone, Novus Biologicals, Biotechne, USA) as positive and CD117 (CD117 APC, Thermofisher, 104D2) as a negative control for an hour.

Techniques: Immunofluorescence, Staining, Derivative Assay, Marker