Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) IUE-mediated H3K27M-tumor model. (B) PDGFRA MUT -p53 MUT -H3 WT (“PPW”) and PDGFRA MUT -p53 MUT -H3K27M (“PPK”) tumors are generated by IUE. Tumor growth is monitored by in vivo bioluminescence imaging and primary neurosphere cell cultures are generated by dissociation of tumor tissue. (C) Survival curve for PPW and PPK mice displays significantly reduced survival of PPK compared to PPW mice; **P=0.014, log-rank test. (D) IHC-stained images of PPK tumor show tumor-specific H3K27M expression and reduced H3K27me3 (representative of n=3 PPK tumors). Magnification = 10x (top row); 40x (bottom row). (E) Western blot (WB) of PPW and PPK primary neurospheres for assessment of H3K27M, H3K27ac and ID1 expression by H3 mutational status. (F) WB of murine PPK and PPK+ ACVR1 MUT (“PPK+A”) cells, and human DIPGXIIIp and DIPGXIIIp+ ACVR1 MUT cells, for assessment of ID1 and pSMAD expression. (G) ID1 expression of DIPG tissue (n=34) compared to matched normal brain tissue (n=18) from the SickKids cohort; ***P < .001, unpaired parametric t-test. (H) ID1 expression by scRNA-seq from the DFCI cohort, including brainstem (n=4), thalamus (n=2) and cortex (n=8); ****P<0.0001, one-way ANOVA t-test. (I) ID1 expression of DIPG tissue (n=68) compared to hemispheric pHGG tissue (n=130). Data from ICR cohort; ****P < 0.0001, unpaired t-test. (J) Kaplan-Meier survival curve of DIPG patients (n=66) grouped by high and low ID1 expression. *P =0.0282, Mantel-Cox test. (K) ID1 expression across Hist1H3B (H3C2) K27M (n=12), H3F3A (H3.3A) K27M (n=71), H3 WT (n=118) and H3F3A (H3.3A) G34R (n=19) DIPG tumors. Data from ICR cohort, presented in Mackay et al; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA t-test. (L) ID1 expression of pHGG tissue by ACVR1 mutational status (n=15 ACVR1 MUT ; n=205 ACVR1 WT ). Data from ICR cohort; **P<0.01, unpaired parametric t-test. (M) ID1 expression of pHGG tumors with ACVR1 mutation only (n=4), H3K27M only (n=72), H3K27M and ACVR1 mutations (n=11) and neither mutation (H3WT/ ACVR1 WT; n=114). Data from ICR cohort; *P<0.05, **P<0.01, one-way ANOVA t-test.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Generated, In Vivo, Imaging, Staining, Expressing, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) ID1 expression of DIPG tumor by cell malignancy from the Dana-Farber Cancer Institute (DFCI) DIPG cohort (n=4 patients). ID1 expression was compared between malignant DIPG cells (n=1841) and non-malignant tumor cells (n=189) from single-cell RNA-seq (scRNA-seq) data. Data represent mean +/- SEM; ****P<0.0001, unpaired parametric t test. (B) ID1 is frequently (35-69%) expressed in pontine DIPG cells and rarely (6-9%) expressed in thalamic DMGs.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Multifocal DIPG tumor samples were obtained at autopsy from n=2 patients with H3K27M mutation and wildtype ACVR1 (ACVR1 WT ), n=2 patients with H3K27M mutation and ACVR1 mutation (ACVR1 MUT ) and n=3 patients with wildtype H3 and ACVR1 . Circles over MRI images represent the approximate region of tumor. (B) ID1 expression (qPCR) for multifocal samples collected from patients in (A). Data represent mean+/-SEM; **P<0.01, ****P<0.0001, one-way ANOVA t-test. (C) ChIP-sequencing of H3K27ac and H3K27me3 deposition at the ID1 gene locus in normal human pontine tissue (n=1), H3 WT DIPG tumor tissue (n=1) and H3K27M DIPG tumor tissue (n=1). (D-E) ChIP-qPCR quantification of deposited (D) H3K27ac, and (E) H3K27me3 marks at gene body elements identified in part C for the ID1 gene. Data represent samples from patients in (A), mean+/-SEM; *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA t-test. (F) MRI image of H3K27M/ACVR1 MUT DIPG patient with circles representing regions where samples were obtained at autopsy. Color scale on right displays relative level of ID1 expression by qPCR (orange=higher ID1 expression; blue=lower ID1 expression. (G) ScRNA-seq data (DFCI, n=4 DIPGs) of malignant DIPG cells plotted to show ID1 expression across varying subtypes of cells [oligodendrocyte-like (OC-like); OPC-like; AC-like].

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Mutagenesis, Expressing, ChIP-sequencing, ChIP-qPCR

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Violin plots depicting ID1 expression in three subtypes of H3K27M-DIPG malignant cells [Data from pontine DIPG patients in ]. (B) Violin plots depicting ID1 expression in cycling vs non-cycling malignant H3K27M-DIPG cells; P=1.6e -12 , Mann-Whitney U test. [OPC-Oligodendrocyte precursor cell; OC-Oligodendrocyte; AC-Astrocyte]. Primary data for parts (A) and (B) from Filbin et al., Science , 2018.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Heat map showing relative ID1 expression by in situ hybridization (ISH) in murine brain across development. Available from: http://developingmouse.brain-map.org/ . (B) ISH of sagittal developing murine brain sections showing higher ID1 RNA in embryonic hindbrain than forebrain, and minimal ID1 RNA in all post-natal brain [Allen Developing Mouse Brain Atlas. Available from: http://developingmouse.brain-map.org/ ]. (C) Heatmap of ID1 expression across varying cell types during normal human pontine development [data from Fan et al. ]. Circle size indicates the percentage of cells that express ID1 and color indicates the expression level in ID1 + cells (red=high expression; blue=low expression). (D) ID1 IHC staining of normal human pontine tissue displays higher ID1 expression in cells lining the 4 th ventricle at 20.5 weeks gestation and minimal expression in brain tissue at 3.5 years of age. (E) ID1 IHC of normal murine pontine tissue at embryonic day 18 (E18) displays higher ID1 expression compared to postnatal day 7 (P7). Magnification = 10x (top row); 40x (bottom row). (F) Overlap of genes expressed by cell types in the developing human pons Fu et al. in DIPG tumor cell subsets. (Red=cell type marker genes enriched in DIPG cells; blue=cell type marker genes not enriched in DIPG cells). (G) Immunostaining of SPARCL1 (green) and ID1 (red) in human DIPG tissue showing co-localization of ID1 and SPARCL1 in a subset of cells (white arrow). Scale bar, 20 µm. Tumor nuclei were stained with DAPI (blue). [For (A), from left to right (row headings), RSP: rostral secondary prosencephalon, Tel: telencephalic vesicle, PedHy: peduncular (caudal) hypothalamus, P3: prosomere 1, P2: prosomere 2, P1: prosomere 3, M: midbrain, PPH: prepontine hindbrain, PH: pontine hindbrain, PMH: pontomedullary hindbrain, MH: medullary hindbrain (medulla); from top to bottom (column headings), E11.5/15.5: embryonic day 11.5/15.5, P4: postnatal day 4].

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Marker, Immunostaining, Staining

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: ID1 RNA is high in the developing embryonic murine brain, and drastically reduced in the post-natal brain. Image credit: Allen Institute. © 2014 Allen Developing Mouse Brain Atlas. Available from: http://developingmouse.brain-map.org/

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques:

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) H3K27ac peaks at the ID1 locus in E15.5 mouse brain and predicted ID1 enhancer regions [Image generated using IGV browser]. (B) Relative enrichment of H3K27ac at predicted ID1 enhancers in E15.5 murine brain regions. Data retrieved from ENCODE Consortium; highlighted regions quantified using EaSeq ( http://easeq.net ).

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Generated

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A-B) Violin plots from analysis of Fan et al., Science Advances , 2020, depicting that AC-like cells show maximum ID1 expression during normal murine pontine development. Data points from all gestational weeks are combined for each cell type and sorted by median. [Astro-Astrocyte].

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Single-cell ID1 expression in varying cell types in normal murine pontine development. (B) Heatmap of ID1 expression during normal murine pontine development [E15.5-Embryonic day 15.5; P0-Postnatal day 0]. Red arrow indicates increased ID1 expression in astrocytes from P0-P6. Primary data for parts (A) and (B) from Jessa et al., Nature Genetics , 2019.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Western blot (WB) confirming ID1 knockdown in DIPG007 cells. (B) WB depicting reduction in SPARCL1 expression along with decreased ID1 expression in ID1 -knockdown DIPG007 cells. (C) Effect of ID1 knockdown on invasion as measured by Matrigel-coated Boyden chamber assay. Images show invading cells stained with crystal violet. Each data point represents an individual image; **P < 0.01, unpaired parametric t-test. (D-E) Effect of ID1 knockdown on DIPG007 migration as measured by scratch assay, quantified as percent wound closure. Images show representative scratch at 0 and 24 hours outlined in dotted red line. Experiment was completed in triplicate and data points represent mean+/-SEM, **P < 0.01; images taken with Incucyte; area measured by ImageJ. (F) WB for ID1 and ACTB expression in DIPG007 and PPK cells treated with increasing concentrations of CBD or DMSO control. (G) Viability of DIPG007 and PPK cells treated with increasing concentrations of CBD (0.5-20µM) relative to DMSO-treated control. Experiment was completed in triplicate and data points represent mean+/-SEM. (H) DIPG007 cells were treated for 2 days with DMSO (control), 2.5µM or 5µM CBD and invasion was measured by Matrigel-coated Boyden chamber. Each data point represents an individual image, mean+/-SEM; ****P < 0.0001, unpaired parametric t-test. (I) Effect of CBD treatment (5-10µM) on DIPG007 migration as measured by scratch assay, quantified as percent wound closure. Experiment was completed in triplicate and data points represent mean+/-SEM, **P < 0.005, two-way ANOVA t-test. (J) Histogram showing increase in DCF (ROS) with increasing doses of CBD. (K) Production of ROS mediates the inhibitory activity of CBD through ID1. DIPG007 cells were treated for 72 hours with vehicle (DMSO) or different concentrations of CBD (10, 8, 6, 4, 2, 1 µM) in the presence and absence of 50 µM TOC. IC 50 was 2.3µM for CBD treatment alone and 10.34µM for CBD + TOC; ****P < 0.0001, two-way ANOVA t-test. Cell proliferation was measured using XTT assay.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Western Blot, Knockdown, Expressing, Boyden Chamber Assay, Staining, Migration, Wound Healing Assay, Control, Activity Assay, XTT Assay

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Western blot confirming ID1 knockdown in HEK293 cells. (B) Effect of ID1 knockdown on cell invasion, as measured by Matrigel-coated Boyden chamber assays. Each data point represents an individual image (4 random images were taken per well). NS, P > 0.05, unpaired t test. (C) Effect of ID1 knockdown on migration as measured by scratch assay. NS, P > 0.05, unpaired t test.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Western Blot, Knockdown, Migration, Wound Healing Assay

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) ID1 western blot of human DIPGXIIp and PBT-29 cells treated with increasing concentrations of CBD, or DMSO control (UT-untreated). Expression levels of ID1 and ACTB were measured. (B) Viability of DIPGXIIIp and PBT-029 cells treated with increasing concentrations of CBD (0.5 µM to 20 µM) relative to DMSO-treated control. Experiment was completed in triplicate and data points represent mean +/- SEM. (C) Western blot of ID1 and SPARCL1 expression in PBT-29 cells treated with increasing concentrations of CBD or DMSO control (UT). Experiments for all western blots were completed in triplicate.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Western Blot, Control, Expressing

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Standard Kaplan-Meier survival plot reveals notable increase in survival for PPK-Sh-ID1 [ PDGFRA- , TP53- and H3K27M-mutant with ID1 knockout (n=8)] mice with median survival 58 days post-IUE injection compared to PPK-Sh-control (n=8) mice with median survival of 49 days; P=0.01, Log-rank test. (B) Representative bioluminescence images of PPK-Sh-control tumors and PPK-Sh-ID1 (representative from n=8), 49 days after IUE injection, displaying lower average luminescence in the PPK-Sh-ID1 group than in the PPK-Sh-control. (C) IUE PPK bioluminescence tumor monitor growth data with statistical significance between PPK-Sh-control and PPK-Sh-ID1 groups 49 days after IUE injection. *P<0.05, one-way ANOVA t-test. ( D ) IHC analysis of ID1 and Ki67 expression in tumors from PPK-Sh-ID1 and PPK-Sh-control mice. Images representative of each experimental cohort. Magnification=40x. (E) IHC quantification for PPK-Sh-control and PPK-Sh-ID1 mice for ID1 and Ki67 expression levels. **P=0.0065 and ****P ≤ 0.0001, one-way ANOVA t-test. Data points include 3 animals per treatment group and 4 images per animal. Data represent the mean+/-SEM. (F) Images of IUE-generated PPK-Sh-Control and PPK-Sh-ID1 tumor borders for assessment of tumor cell invasiveness. Magnification = 10x (top row); 40x (bottom row).

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Mutagenesis, Knock-Out, Injection, Control, Expressing, Generated

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Representative images of bioluminescent tumors from intracranial injection of scrambled-control or ID1-shRNA DIPG007 cells at DPI-97. (B) Bioluminescence of intracranially-injected scrambled or ID1-shRNA DIPG007 cells over days-post-injection . (C) Example images of IHC staining for Ki67 (left) and ID1 (right) in a sagittal tissue section (tumors generated from implantation of DIPG007 cells). Magnification is 20x.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Injection, Control, shRNA, Immunohistochemistry, Generated

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Survival curve for PPK mice shows that median survival for control condition was 45 days post-IUE injection (n=8) and 55 days for CBD condition (15 mg/kg, n=8). **P<0.005, Log-rank test. (B-C) IHC analysis and quantification of tumor images reveals that CBD treatment reduced expression of ID1 and Ki67 compared to vehicle-treated tumors (representative of n=3 tumors); **P=0.0065 and ****P ≤ 0.0001, Dunnett’s multiple comparisons test. N=3 animals per treatment group and 4 images per animal. Data represent the mean+/-SEM. Magnification=10x. (D) Analysis of tumor invasion in tumor-bearing mice (n=3 mice per group) with genetic (sh-ID1) or pharmacologic (CBD) ID1 knockdown. Invasion was defined as tumor infiltration into the contralateral hippocampus (Hip). (E) Timeline for pharmacokinetic (PK) liquid chromatography (LC)/mass spectrometry (MS) analysis of CBD treatment by intraperitoneal (IP) injection in normal mouse plasma and brainstem. (F) Timeline for PK mass spec analysis of CBD treatment by IP injection in PPK mouse plasma, normal brain and tumor. (G) PK analysis results for normal (non-tumor-bearing) mice treated with 45 mg/kg CBD (n=3 mice per time point). Data represent CBD concentrations as determined by LC/MS for the plasma and brainstem; mean+/-SEM. Blue dashed line represents estimated IC 50 of CBD for DIPG007 cells. (H) PK analysis results for n=3 PPK mice treated with 45 mg/kg CBD. Data represent CBD concentrations as determined by LC/MS for the plasma, normal brain and tumor; mean+/-SEM.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Control, Injection, Expressing, Knockdown, Liquid Chromatography, Mass Spectrometry, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Plot representing the CBD dosing range (mg/kg/day) in human patients, including one high (UMPED83) and one low (UMPED86) dose of CBD, as indicated by red and blue lines. (B-C) IHC-stained tumor tissue from DIPG patients (B) UMPED83 treated with CBD (25 mg/kg) and (C) UMPED86 treated with CBD (0.4 mg/kg/day) during treatment course for assessment of ID1 expression. IHC images representative of n=3 images taken using Aperio ImageScope, magnification=40x. (D) ID1 IHC analysis and quantification of human DIPG tumor samples with low (n=6) and high dose (n=4) of CBD; *P=0.0388, Mann-Whitney U test. (E) Survival of H3K27M-mutant tumor patients treated with CBD from higher than 3mg/kg (n=4) and lower than <3mg/kg (n=6) with historical control (n=98).

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Staining, Expressing, MANN-WHITNEY, Mutagenesis, Control

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: The proposed model is made up of the following sub-sections: (a) H3K27M inhibits PRC2, leading to global decreases in H3K27me3 and subsequently allowing for increased H3K27ac. (b) Regional or tissue-specific factors and/ or (c) constitutively activating ACVR1 mutations increase ID1 expression via SMAD protein signaling. We propose that ID1 expression replicates the developing cell subtype OAPC transcriptional program, which promotes migration. (d) ID1 expression is reduced by CBD treatment, which partially acts through increasing intracellular levels of reactive oxygen species (ROS). Image created with BioRender.

Article Snippet: Western blotting was performed using antibodies against ID1 (1:1000, Cat#133104, Santa Cruz Biotechnology), Vinculin (1:10000, Cat#700062, Invitrogen), H3K27M (1:500, EMD, Cat#ABE419), H3K27me3 (1:500, EMD, Cat#07-449) and ACTB (1:10000, Cat#A2228, Sigma-Aldrich), Secondary antibodies biotinylated horse anti-mouse IgG (Cat#BA2000, Vector Laboratories), HRP goat anti-rabbit IgG (Cat#PI1000, Vector Laboratories), and m-IgG k BP-HRP (Cat#sc516102, Santa Cruz Biotechnology) were used.

Techniques: Expressing, Migration