Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Functional enrichment analysis on significantly downregulated genes in SU-DIPG13 cells treated with 10uM Sulfopin for 12 hours, compared to DMSO. Enrichr algorithm was used to compare downregulated genes against the Molecular Signatures Database (MSigDB) hallmark geneset . Dashed line denotes adjusted p-value = 0.05. MYC targets are significantly enriched among the Sulfopin downregulated genes. (B) RT-qPCR analysis of selected MYC target genes in SU-DIPG13 cells treated with 10uM Sulfopin for 12 hours, compared to DMSO. Fold change between Sulfopin and DMSO treated cells was calculated and the mean ± SD of two technical repeats is shown. (C) H3K27me3 Cut&Run read coverage over MYC target genes (‘MYC Targets V1’ hallmark geneset , n=200), in SU-DIPG13 cells treated with 10uM Sulfopin for 8 days compared to DMSO. Sulfopin treatment increases H3K27me3 levels on the TSS of MYC target genes. (D) Functional enrichment analysis of the genes associated with Sulfopin-unique H3K27me3 peaks, in SU-DIPG13 cells treated as in C. Dashed line denotes adjusted p-value = 0.05. MYC target geneset (‘MYC Targets V1’ hallmark geneset ) is mildly enriched among these genes, with adjusted p-value of 0.077. (E) Cell viability, as measured by CellTiterGlo, of eight DMG cultures (H3.3K27M: SU-DIPG13, SU-DIPG6, SU-DIPG17, SU-DIPG25 and SU-DIPG50. H3.1K27M: SU-DIPG36, SU-DIPG38 and SU-DIPG21), treated with Sulfopin for eight days with pulse at day four, compared to DMSO. Mean±SD of two technical replicates is shown. Logarithmic scale is used for the x-axis. Sulfopin treatment led to a mild reduction in cell viability in all H3-K27M glioma cultures. (F) Cell viability, as measured by CellTiterGlo, of two isogenic DMG cell lines (SU-DIPG13 and BT245) in which the mutant histone was knocked-out (KO), treated with the indicated concentration of Sulfopin for 8 days, compared to DMSO. For each cell line and concentration, the fold change in viability between Sulfopin and DMSO treated cells is shown. For SU-DIPG13-mean ± SE of at least two independent experiments is shown. For BT245-mean± SD of three technical replicates is shown. H3-K27M glioma cells show higher sensitivity to Sulfopin treatment compared to the KO cells. *P < 0.05; **P < 0.01 (two-sample t-test over all technical replicates). Significance adjusted after Bonferroni correction.

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Functional Assay, Quantitative RT-PCR, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Timeline demonstrating the treatment protocol for the combination of Sulfopin and Vorinostat. (B) Percentage of cell viability, as measured by CellTiterGlo of SU-DIPG13 cells treated with Sulfopin and Vorinostat at the indicated concentrations, compared to DMSO. (C) BLISS index measured as the ratio between the observed and the expected effect of the combination of Sulfopin and Vorinostat, for each pair of concentrations, in SU-DIPG13.Synergy: Bliss <1, Additive: Bliss=1, Antagonist: Bliss>1. (D) Cell viability as measured by CellTiterGlo, of eight DMG cultures treated with Sulfopin (0uM, 2.5uM, 5uM, 10uM, 20uM and 40uM) and Vorinostat (1uM), compared to DMSO. H3.3-K27M and H3.1-K27M cultures are indicated in blue and orange, respectively. Mean±SD of two technical replicates is shown. H3.3-K27M cells showed higher sensitivity to the combined treatment compared to H3.1-K27M cells. (E) The BLISS index of the combination of Sulfopin (10uM) and Vorinostat (1uM), in the indicated cultures. An additive effect was detected in all the H3.3-K27M cultures at this set of concentrations. (F) Pearson correlation coefficient matrix of BLISS index of the combined treatment (Sulfopin (10uM) and Vorinostat (1uM)) and mRNA levels of MYC and its target genes, in the eight DMG cultures tested. mRNA levels were measured by RT-qPCR (Fig. S2F-G). Blue and yellow colors indicate negative or positive correlation, respectively. Negative correlation was detected between the BLISS indexes and the expression levels of MYC and its target genes. (G) Unsupervised hierarchical clustering of expression levels of 620 significantly DE genes detected in SU-DIPG13 cells treated with either Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), the combination of Sulfopin and Vorinostat or DMSO. Gene expression rld values (log2 transformed and normalized) were standardized for each gene (row) across all samples. Color intensity corresponds to the standardized expression, low (blue) to high (red). Clusters 1 and 4 demonstrate additive transcriptional patterns associated with the combined treatment. (H) Top: Gene Set Enrichment Analysis (GSEA) on SU-DIPG13 treated with combination of 10uM Sulfopin and 1uM Vorinostat compared to DMSO, showing significant downregulation of mTORC1 signaling (‘HALLMARK_MTORC1_SIGNALING’ geneset ) in the combined treatment. NES: Normalized Enrichment Score. FDR: false discovery rate. Bottom: Expression levels of significantly DE genes detected in the combined treatment compared to DMSO that are part of the mTORC1 signaling geneset. MTOR gene was added manually to the heat-map. Heatmaps were generated as described in B. (I) Top: Gene Set Enrichment Analysis (GSEA) on SU-DIPG13 treated as in C, showing significant downregulation of the epigenetic BMI-1 pathway and the oncogenic cAMP pathway in the combined treatment (BMI1_DN.V1_UP; CAMP_UP.V1_UP; MSigDB C6 oncogenic signature , ). Bottom: Expression levels of significantly DE genes detected in the combined treatment compared to DMSO that are part of the BMI-1 and cAMP genesets. Heatmaps were generated as in described B. (J) Western blot of SU-DIPG13 treated either with Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), the combination of Sulfopin and Vorinostat or DMSO, using the indicated antibodies. β-tubulin is used as loading control.

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Transformation Assay, Generated, Western Blot, Control

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A) Scheme of the single-molecule imaging experimental setup : cell-derived mono-nucleosomes are anchored in a spatially distributed manner on polyethylene glycol (PEG)-coated surface. Captured nucleosomes are incubated with fluorescently labeled antibodies directed against the H3K27ac modification. Total internal reflection fluorescence (TIRF) microscopy is utilized to record the position and modification state of each nucleosome. Time series images are taken to allow detection of maximal binding events. (B) Single-molecule imaging quantification of the percentage of H3K27ac nucleosomes, in SU-DIPG13 cells treated with either Sulfopin (10uM, 8 days), Vorinostat (1uM, 72 hours), or the combination of Sulfopin and Vorinostat, normalized to DMSO. Mean fold ± SE of at least two independent experiments is shown. H3K27ac global levels are lower in the combined treatment compared to cells treated solely with Vorinostat. *P < 0.05 (two sample t-test). (C) SU-DIPG13 cells were treated as in B, and analyzed by western blot using the indicated antibodies. (D) Left panel: Heatmap shows H3K27ac read coverage around the TSS (+/-5Kb) of the significantly DE genes shown in , in SU-DIPG13 cells treated with the combination of 10uM Sulfopin and 1uM Vorinostat versus DMSO. Average coverage is shown on top. Color intensity corresponds to the standardized expression. Clusters 1-4 are indicated. Right panel: The log2 ratio of H3K27ac read coverage in SU-DIPG13 cells treated with the combination of 10uM Sulfopin and 1uM Vorinostat vs. DMSO was calculated. Heatmap shows the ratio around the TSS (+/-5Kb) of the significantly DE genes shown in , and average coverage is shown on top. Color intensity corresponds to the ratio between samples, low (red) to high (blue). Clusters 1-4 are indicated, with cluster 1 presenting the strongest local decrease in H3K27ac following the combined treatment compared to DMSO. (E) IGV tracks of MTOR and SLC7A5 gene promoters, showing H3K27ac coverage in SU-DIPG13 cells treated as indicated. (F) Functional enrichment analysis of the genes linked to enhancers (top targets of high confident enhancers) marked with H3K27ac exclusively in SU-DIPG13 cells treated with Vorinostat, and not in the combined treatment. gProfiler algorithm was used to calculate enrichment against the KEGG pathways DB . Dashed line denotes adjusted p-value = 0.05. Genes associated with Vorinostat-unique enhancers are enriched for oncogenic signaling pathways. (G) IGV track of AKT3 and SEC13 linked enhancers, showing H3K27ac coverage in SU-DIPG13 cells treated with 1uM Vorinostat or the combination of 10uM Sulfopin and 1uM Vorinostat. (H) Normalized expression levels of AKT3 and SEC13 genes in SU-DIPG13 cells treated as in G. Mean ± SD of three technical repeats is shown.*P < 0.05 (two-sample t-test).

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Imaging, Derivative Assay, Incubation, Labeling, Modification, Fluorescence, Microscopy, Binding Assay, Western Blot, Expressing, Functional Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Dual Targeting of Histone Deacetylases and MYC as Potential Treatment Strategy for H3-K27M Pediatric Gliomas

doi: 10.1101/2024.02.05.578974

Figure Lengend Snippet: (A-B) SU-DIPG13P* cells were injected to the pons of immunodeficient mice to form tumors. Ten days post injection, mice were treated for 18 days with either DMSO, Sulfopin, Vorinostat, or the combination of Sulfopin and Vorinostat. (A) In-vivo bioluminescent imaging of DMG xenografts following 18 days of treatment. The heat map superimposed over the mouse head represents the degree of photon emission by DMG cells expressing firefly luciferase. (B) DMG xenograft tumor growth as measured by change in bioluminescent photon emission following 15 days of treatment with either DMSO (n=8), Sulfopin (n=4), Vorinostat (n=5) or the combination of Sulfopin and Vorinostat (n=6). Data points represent the fold-change in maximum photon flux between day 3 and day 18 under treatment for each mouse. *P < 0.05 (two-tailed Mann-Whitney U-test). (C-D) Immunofluorescent staining of brain sections from mice treated with DMSO (n=4) or the combination of Sulfopin and Vorinostat (n=4). (C) Representative fluorescence images of H3-K27M (red) and mTOR (green). (D) Percentage of mTOR positive cells out of the total H3-K27M-positive cells. H3-K27M positive cells show lower levels of mTOR following the combined treatment compared to DMSO. *P < 0.05 (two-tailed t-test).

Article Snippet: Treatment was given daily for 18 days, by intraperitoneal injection according to the following groups: Vorinostat 200mg/kg daily (LC, V-8477-SU-DIPG13P*; MCE, HY-10221-SU-DIPG13), Sulfopin 40 mg/kg (gift from Dr. Nir London’s Lab), combination 200 mg/kg Vorinostst and 40 mg/kg Sulfopin, control mice received solvent 15% DMSO in 50% PEG400 (Sigma) in 0.15M NaCl.

Techniques: Injection, In Vivo, Imaging, Expressing, Luciferase, Two Tailed Test, MANN-WHITNEY, Staining, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 2. Pin1 binding to the AF-1 is enhanced by ERK2 phosphorylation. Overlays of 2D [1H,15N]-HSQC NMR spectra of (A) 15N-labeled AF-1, (B) 15N-labeled pAF-1, and (C) 15N-labeled pAF-1 P76A mutant without or with 1 molar equivalent of human Pin1 reveal larger chemical shift perturbations (CSPs) in the phosphorylated state indicating stronger binding of Pin1 to phosphorylated AF-1. Peaks belonging to regions of particular interest noted by blue and green labeling are discussed further in the results. (D) AlphaFold3 model of Pin1 interaction with pAF-1 containing phosphorylated S112 (pS112) and T75 (pT75) residues with NMR CSPs from 15N-labeled pAF-1 ± 1x Pin1 (2B) shows the Pin1-binding epitope on pAF-1 extends to regions beyond the pS112 region. Spheres represent residue alpha carbons colored by CSP magnitude [white (CSP = 0) to magenta (CSP = 0.03)] or noting peaks that disappear (black). CSP plots comparing addition of Pin1 full-length, WW domain, or PPIase domain at 1 molar equivalent to (E) 15N-labeled AF-1, (F) 15N-labeled pAF-1, and (G) 15N-labeled pAF-1 P76A mutant reveal that each Pin1 domain binds weakly to AF-1, but binding of full-length Pin1 elicits larger CSPs in targeted regions of pAF-1 and pAF-1 P76A mutant.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Phospho-proteomics, Labeling, Mutagenesis, Residue

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 4. AF-1 peptides delineating contributions of specific AF-1 regions in binding to Pin1. (A) Sequence and relative locations of peptides corresponding to AF-1 regions that include the S112 phosphorylation site with unphosphorylated S112 (green) or phosphorylated S112 (teal); T75 phosphorylation site with unphosphorylated T75 (purple) or phosphorylated T75 (coral); and the PFWP motif (mustard). Overlays of 2D [1H,15N]-TROSY HSQC NMR spectra of 15N-labeled Pin1 with (B) S112, (C) T75, (D) pS112, (E) pT75, and (F) W39 peptides titrated up to 5 molar equivalents. Shown alongside spectra are AlphaFold3 models of Pin1 colored by magnitude of CSP [pale blue/white (CSP = 0) to magenta (CSP = 0.1)] elicited by 2X titration of respective peptide shown in complex with Pin1. The WW domain is colored pale blue, and the PPIase domain is white. Peptides in models are colored corresponding to their identity as indicated in 5A. (G) CSP plots comparing addition of unphosphorylated or phosphorylated peptides at 5 molar equivalents to 15N-labeled Pin1. CSP profiles reveal Pin1 interaction patterns unique to each AF-1 peptide, with phosphorylated AF-1 peptides driving enhanced interaction to the WW domain.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Sequencing, Phospho-proteomics, Labeling, Titration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site.

doi: 10.1073/pnas.2414606122

Figure Lengend Snippet: Fig. 6. Approaches to studying Pin1 binding and catalysis. Previous structural studies of Pin1 isomerization depended heavily on the use of short peptide sequences (~20 amino acids). In addition to short peptides, our studies using a larger protein domain with multiple potential canonical and noncanonical Pin1 sites uncovered a multivalent binding interaction that catalyzes a noncanonical W-P motif with functional cellular relevance.

Article Snippet: Full- length human Pin1 and the Pin1 WW domain (1–39) protein were expressed from a pMCSG7 plasmid vector, purchased through Addgene (#40773) as TEV- cleavable N- terminal 6xHis fusion proteins.

Techniques: Binding Assay, Functional Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data *

doi: 10.1074/mcp.M115.050773

Figure Lengend Snippet: RRP analysis of Pin1 interactome. (A) Graphical presentation of analyzed Pin1 interactome based on: Protein Interaction Network analysis (2). (B) Phase contrast microscopy images of PC-3 cells transfected with indicated siRNAs on 384-well plate. (C) Graphical presentation of correlation of cell viability effects of individual siRNAs in PC-3 and PNT2 cells. (D) RRP ranking of top ten proteins with most similar function to Pin1. FunCoup similarity index (min. 0, max. 1) indicates for very high similarity in function with Pin1 for most RRP top ranked proteins. NA, not applicable due to lack of sufficient database information. Validation indicates either physical or functional validation for indicated interaction. N.d., not determined.

Article Snippet: The antibodies against PIN1 (H-123:SC-15340), c-Jun, MYC, FTSJ1 (B-2:SC-390355), DDX24 (C-12:SC-104863), FNBP3 ( d -20:SC-68080), PME-1 (B-12: SC-25278), Lamin A/C, (H-110 SC-20681; H-110), rabbit anti goat IgG-HRP (SC-2922), and normal rabbit IgG (SC-2027) were purchased from Santa Cruz Biotechnology.

Techniques: Microscopy, Transfection, Functional Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein–Protein Interaction Data *

doi: 10.1074/mcp.M115.050773

Figure Lengend Snippet: Validation of CSKN2B and PTOV1 as functional Pin1 interacting proteins. (A) Western blot analysis of phosphorylated c-Jun expression in PC-3 cells depleted of either Pin1, PTOV, or CSKN2B. (B) Western blot analysis of Pin1 expression in PC-3 cells transfected either with Pin1, PTOV, or CSKN2B siRNAs.

Article Snippet: The antibodies against PIN1 (H-123:SC-15340), c-Jun, MYC, FTSJ1 (B-2:SC-390355), DDX24 (C-12:SC-104863), FNBP3 ( d -20:SC-68080), PME-1 (B-12: SC-25278), Lamin A/C, (H-110 SC-20681; H-110), rabbit anti goat IgG-HRP (SC-2922), and normal rabbit IgG (SC-2027) were purchased from Santa Cruz Biotechnology.

Techniques: Functional Assay, Western Blot, Expressing, Transfection