Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: Sp1 is deacetylated by HDAC1/2/6 in TMZ-resistant GBM cells. (A) The wild-type (Wt) and TMZ-resistant (TMZ-R) GBM cells,12 as well as GBM spheroids formed in serum-free medium/suspension (S/S) culture and the control attached (Adh) cells13 were used for the immunoprecipitation (IP) assay with rabbit IgG, anti-Sp1 (Sp1, panel a), and anti–acetyl-lysine (ac-K, panels b and c) antibodies, and analyzed using immunoblotting (IB) as indicated. In panel c, the protein level of acetylated Sp1 was normalized to its total protein and quantified. (B) Gene expression profiles of HDACs in brain tumors were analyzed using the Oncomine database. HDAC1/3/6/9, shown by the arrows, were upregulated more in cancer tissues than in normal samples. Red indicates upregulation; blue indicates downregulation. The number in the cell represents the number of datasets that pass the filter criteria (threshold: P < 0.05). (C to F) Cells were harvested and analyzed using IB. The Wt (C and E) and TMZ-R (E) A172 cells were treated with the indicated concentrations of TMZ for 3 days. (D) The protein expression of HDACs in Wt and TMZ-R P11 GBM cells was normalized to the loading control and quantified. (F) The levels of HDAC6 and tubulin acetylation in attached GBM cells and tumorspheres. (G) Wt and TMZ-R P11 cells were used for IP assay with anti-Sp1 antibodies and rabbit IgG, and analyzed using IB as indicated. (H) TMZ-R U87MG cells were transfected with a nontargeting control siRNA or HDAC1/2/6-specific siRNAs as indicated. After knockdown, the cells were used for IP assay. (t-test: *P < 0.05, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Control, Immunoprecipitation, Western Blot, Gene Expression, Expressing, Transfection, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: HDAC1/2/6 inhibition significantly reduces the growth rates of TMZ-resistant GBM cells. (A) U87MG cells, as well as primary cultures of neurons and glial cells, were treated with 1 μM SAHA (SA), 1 μM azaindolyl sulfonamide compound 12 (MPT0B291, MP), or dimethyl sulfoxide (DMSO) (DM) for 4 days. After treatment, cell viability was assessed using colorimetric MTT assay. (B) In the focus formation assay, parental and TMZ-resistant (TMZ-R) U87MG cells were seeded at low density onto 60-mm plates, and treated with TMZ or MP alone or in combination at different doses every 3 days. Following a 2-week incubation period, the forming foci were stained using crystal violet. Representative images are shown. (C) TMZ-R GBM cell lines, including U87MG-R, A172-R, and P11-R cells, were treated with DMSO or different doses of MP (1, 3, 6 μM) for various time intervals (1 to 4 days). Cell viability was assessed using the MTT assay. (D) TMZ-R U87MG inoculated orthotopic mice were treated with 25 mg/kg TMZ to maintain a TMZ-resistant phenotype, and co-treated with or without 25 mg/kg MP every 2 days for 3 weeks. The brain tumors were observed using serial histology sections along the tumor using hematoxylin and eosin staining. (E) TMZ-R P3 inoculated orthotopic mice were randomly grouped and treated with DMSO, 10 mg/kg TMZ (T), or TMZ plus 10 mg/kg MP (T+M) every 2 days. Survival was plotted using a Kaplan–Meier curve. (F) Cells were transfected with HDAC1-, HDAC2-, and/or HDAC6-specific siRNAs or a nontargeting control siRNA as indicated for 2 days. After knockdown, cell viability was assessed using the MTT assay. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Inhibition, MTT Assay, Tube Formation Assay, Incubation, Staining, Transfection, Control, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: The HDAC/Sp1 pathway plays an important role in regulating cell cycle progression and proliferation. (A) Venn diagram illustrating overlaps between number of genes that were altered by more than 1.5–fold in TMZ-resistant (TMZ-R) cells and in spheroids (serum-free medium/suspension culture, S/S) following MP treatment and were also targeted by Sp1 in U87MG cells. (B) The IPA software program was applied on 139 potential HDACs/Sp1-regulated genes (the intersection genes of Sp1 ChIP-seq data and gene expression microarray data from MP-treated TMZ-R and S/S in [A]) to identify top 10 scoring canonical pathways. (C) Heat map representing the expression levels of 41 cell cycle–related genes, obtained from IPA analysis in (B), following MP treatment. (D) Relationships between MP-treated microarray data (in horizontal) and TCGA-GBM NGS data (in vertical) using Pearson’s correlation coefficient (PCC, r). Each dot represents the expression value of a cell cycle-related gene. (E) Forest plots showing hazard ratios for risk of death in low-grade and high-grade glioma patients with higher expression of the indicated gene(s). The lines on both sides denote 95% confidence intervals. All the original data (Kaplan–Meier curve) were obtained from PROGgeneV2 (Supplementary Figure 15A). Hazard ratios above 1 indicate a worse outcome. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Software, ChIP-sequencing, Gene Expression, Microarray, Expressing

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 induces senescence and diminished the expression stemness-related markers in both GBM spheroids and TMZ-resistant (TMZ-R) cells. (A) Mitotic P3 cells were released into the cell cycle by removing nocodazole from the culture, and then harvested at different time points as indicated. Cells were then fixed for cell cycle progression assay using flow cytometry. The percentages of cells in G1 phase and G2/M phase are shown in right panel. (B) U87MG spheroids (S/S) were treated with DMSO or MP. At 4 days posttreatment, senescence was examined using SA β-gal staining. Photomicrographs of spheroids were randomly selected in microscopic fields, and SA-β-gal positive cells were counted. (C) Cells dissociated from U87MG spheroids via trypsinization were grown in soft agar, followed by treatment with different doses of MP every 4 days. After 3 weeks of incubation at 37°C, the colonies that arose from these single cells were photographed randomly, and a histogram of average colony numbers was plotted after performing the experiment in triplicate. (D and E) Cells, as indicated, after 2 days of MP treatment were harvested, and the cell lysates were analyzed using IB with the indicated antibodies. (t-test: **P < 0.01)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Expressing, Flow Cytometry, Staining, Incubation

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 decreases Sp1 binding to BMI1 and hTERT promoters. (A) The Sp1 ChIP-seq reads mapped to the promoter region of BMI1 and hTERT. Forward reads are shown in green and reverse reads are shown in red. The significance of ChIP peaks, generated using the CLC Genomics Workbench 10.1.1 software, indicated Sp1 binding loci. (B and C) U87MG spheroids were treated with DMSO or MP for 6 h, and the level of Sp1 binding to the promoter regions of BMI1 and hTERT was assessed using a ChIP assay with rabbit IgG or anti-Sp1 antibodies. DNA was then extracted from the sample for PCR with the primers as indicated. Rabbit IgG acted as a negative control for nonspecific precipitation, and E-box was used as a negative control for nonspecific binding. (D to F) U87MG spheroids were treated with different doses of MP for 2 days. After treatment, the mRNA (D) and protein (E) levels of hTERT in cells were analyzed using real-time PCR and IB, respectively. Furthermore, relative telomerase activity (F) was also detected using the TRAP assay and normalized to the value of the internal PCR control in each reaction. The cell lysis buffer was used as a negative control. The arrow points to the 36-bp internal control. (t-test: *P < 0.05, **P < 0.01)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Binding Assay, ChIP-sequencing, Generated, Software, Negative Control, Real-time Polymerase Chain Reaction, Activity Assay, TRAP Assay, Control, Lysis

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) Schematic overview of workflow for identifying TIFAB-interacting proteins in a human del(5q) AML cell line. (B) Silver-stained gel for vector-transduced protein eluate and TIFAB-transduced protein eluate from a tandem-affinity purification procedure. (C) Interaction network for TIFAB-interacting proteins with the top 100 precursor intensity values. STRING interaction data were plotted in Cytoscape , which calculates degree, a measure of connectivity (red, low; blue, high). (D) Dot plot representing p values of statistically over-represented pathways, generated by analyzing TIFAB-interacting proteins in Reactome. (E) Venn diagram of TIFAB-interacting proteins identified in two independent biological replicates of the proteomics screen (1A) and analyzed using two mass spectrometry (MS) approaches with varying sensitivities. (F) Immunoblotting of endogenous USP15 on lysates immunoprecipitated for FLAG-TIFAB that were isolated from HL60 cells transduced with retrovirus encoding FLAG-TIFAB and subsequently used for MS analysis.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Staining, Plasmid Preparation, Affinity Purification, Generated, Mass Spectrometry, Western Blot, Immunoprecipitation, Isolation, Transduction

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) Top: immunoblotting of HA-TIFAB on lysates immunoprecipitated for FLAG-USP15 deletion mutants that were isolated from HEK293T cells transfected with HA-TIFAB and the indicated FLAG-USP15 mutants. Bottom: schematic of human USP15 with key functional domains. DUSP, domain present in ubiquitin-specific peptidases; UBL, ubiquitin-like domain; UCH, ubiquitin C-terminal hydrolase. (B) Immunoprecipitation of FLAG-TIFAB and immunoblotting for wild-type or catalytically inactive USP15 (C269A) from HEK293T cells transfected with FLAG-TIFAB alone, HA-USP15 constructs alone, and FLAG-TIFAB combined with HA-USP15 constructs (lanes 4 and 5). (C) Cell-free deubiquitination assay utilizing internally quenched fluorescent (IQF) diubiquitin as a substrate for USP15. 500 nM K48-linked diubiquitins was incubated with 50 nM USP15 plus mock buffer (black) or USP15 plus 50 nM recombinant TIFAB (red). Relative fluorescent units (RFUs) measured over time by spectrometry are a readout of USP15 activity. Four replicates were analyzed per group for two independent experimental assays. (D) The reaction rate (RFU/s) of the linear phase of the reaction (~500 s) plotted as a function of diubiquitin substrate concentration using an allosteric sigmoidal model to calculate Vmax. Increasing amounts of diubiquitins were added to the reaction with 50 nM USP15 and 50 nM TIFAB until the rate of ubiquitin hydrolysis by USP15 began to saturate. The USP15 rate of reaction is increased by adding TIFAB to the reaction (red versus black). Four replicates were analyzed per group for two independent experimental assays. (E) Schematic of the orthogonal deubiquitination assay. (F) Coomassie-stained gels containing diubiquitin incubated with USP15 alone or USP15 with TIFAB for the indicated times. Diubiquitins for which USP15 has a high affinity (K11, K6, and K48) are in the top row (USP15 dependent). Diubiquitins for which USP15 has low affinity (K29, linear, and K27) are in the bottom row (USP15 independent). Panels were constructed by assembling images from the respective sections of gels containing the proteins indicated.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Western Blot, Immunoprecipitation, Isolation, Transfection, Functional Assay, Ubiquitin Proteomics, Construct, Incubation, Recombinant, Activity Assay, Concentration Assay, Staining

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) Schematic overview of TIFAB and USP15 substrates exerting homeostatic control of p53, left, and de-repression of p53 by deletion of TIFAB (middle) or by deletion of USP15 (right). (B) Immunoblotting of HA-K48-Ub on lysates immunoprecipitated for MDM2 that were isolated from HEK293T cells transfected with TIFAB, USP15, and/or MDM2. Transfected cells were incubated with the proteasome inhibitor (MG-132) to enrich for ubiquitinated MDM2. (C) Immunoblotting of Usp15, Keap1, and vinculin in Tifab +/+ and Tifab −/− BM mononuclear cells. (D) Immunoblotting of Mdm2, p53, and vinculin in Tifab +/+ and Tifab −/− Lin − BM cells. (E) Immunoblotting of USP15, Keap1, p53, and vinculin in Usp15 +/+ and Usp15 −/− BM mononuclear cells. (F) Immunoblotting of Mdm2 and vinculin in Usp15 +/+ and Usp15 −/− BM mononuclear cells.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Control, Western Blot, Immunoprecipitation, Isolation, Transfection, Incubation

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) Overview of experimental scheme. (B) Kaplan-Meier survival curves for nontransplanted and transplanted mice reconstituted with Tifab +/+ or Tifab −/− BM cells (four groups total). All groups except transplanted Tifab −/− exhibited 100% survival. Summary from two independent transplants analyzed for up to 16 months. A subset of the transplanted mice were previously characterized at early time points . (C) Table displaying top 25 enriched pathways in TIFAB-deficient LSK cells, identified by RNA microarray and gene set enrichment analysis, with false discovery rate (FDR) <0.5. Colored pathways denote shared functions. (D) Heatmap representing Z score values for established p53 target genes in Tifab +/+ and Tifab −/− BM LK cells . (E) Immunoblotting of caspase-3 and actin on lysates isolated from BM mononuclear cells of mice reconstituted with Tifab +/+ and Tifab −/− BM cells. (F) Peripheral blood count recovery after 5-FU injection (150 mg/kg) of mice reconstituted with BM from Tifab +/+ (n = 16), Tifab −/− (n = 16), and Tifab −/− ;p53 −/− (n = 5) mice. When comparing Tifab −/− and Tifab −/− ;p53 −/− mice, *p < 0.05. (G) Immunoblotting of p53 in lineage-negative (Lin − ) BM cells isolated from Tifab +/+ and Tifab −/− mice treated with 0, 2, or 6 Gy irradiation in vitro . (H) Colony formation in methylcellulose of Lin − BM cells isolated from Tifab +/+ or Tifab −/− mice treated in vitro with 0, 2, or 6 Gy irradiation (n = 3). *p < 0.05. (I) Colony formation in methylcellulose of Lin − BM cells of Tifab +/+ or Tifab −/− after infection with mCherry-expressing lentivirus (LV; pReciever-mCherry) (n = 2). A representative image is shown on the right as an image section of the entire well. *p = 0.09. (J) Relative number of colonies of Tifab −/− Lin − BM cells transduced with vectors encoding shp53 or control shRNA (shCtl) (n = 3). Data are normalized to Tifab +/+ expressing shControl. A representative image is shown on the right as an image section of the entire well. *p = 0.08.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Microarray, Western Blot, Isolation, Injection, Irradiation, In Vitro, Infection, Expressing, Transduction, Control, shRNA

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) TIFAB mRNA expression in AML patients. Original TIFAB expression values (from TCGA) are normalized using the RNA-Seq by Expectation Maximization (RSEM) software. The mean values of all samples were subtracted from each sample’s RSEM count, which is depicted as “TIFAB mRNA.” (B) Toppgene pathway analysis was performed on genes expressed in TIFAB-“high”-expressing AML patients (top quartile). (C) Relative TIFAB mRNA expression in a panel of human leukemic cell lines normalized to HL60 cells, which are haploinsufficient for TIFAB. Red bars denote cell lines with MLL rearrangements. (D) Colony formation in methylcellulose of CD34 + cells (left) and MV4;11 cells (right) expressing control shRNA (shControl) or shRNA targeting TIFAB (shTIFAB). *p < 0.005. (E) Representative images of MV4;11 colonies from (D) (n = 3) as image sections of the entire well. (F) Colony formation in methylcellulose of CD34 + cells (left) and MV4;11 cells (right) expressing control shRNA (shControl) or shRNA targeting USP15 (shUSP15). *p < 0.05. (G) Representative images of MV4;11 colonies from (F) (n = 3) as image sections of the entire well.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Expressing, RNA Sequencing, Software, Control, shRNA

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: (A) Generation of Tifab +/+ and Tifab −/− MLL-AF9 leukemia model and schematic overview of the experimental setup. (B) Colony formation in methylcellulose of Tifab +/+ ;MLL-AF9 or Tifab −/− ;MLL-AF9. Two biological experiments were performed in triplicate. Representative images are shown. ***p < 0.0005. (C) Kaplan-Meier plot for overall survival of recipient mice transplanted with Tifab +/+ ;MLL-AF9 or Tifab −/− ;MLL-AF9 cells. (D) Relative mRNA expression of the indicated genes in Tifab +/+ ;MA9 or Tifab −/− ;MA9 cells. Three independent experiments were performed in triplicate. Error bars represent SD. *p < 0.05, ***p < 0.0005. (E) Cell growth analysis in liquid culture of Tifab +/+ ;MLL-AF9 or Tifab −/− ;MLL-AF9 cells expressed as live cell number per day, determined by trypan blue exclusion. *p < 0.05. (F) Relative cell growth of Tifab −/− ;MLL-AF9 (GFP + ) cells transduced with lentivirus encoding empty vector (Vector) or USP15 and mCherry was determined by flow cytometry (GFP + mCherry + ) at the indicated time points (n = 6 replicates). *p < 0.05. (G) Relative colony formation in methylcellulose of Tifab +/+ ;MLL-AF9 or Tifab −/− ;MLL-AF9 cells treated with vehicle (DMSO) or doxorubicin (Dox; 5 nM). The number of colonies was normalized to DMSO controls for each group (n = 2 biological experiments, performed in triplicate). ***p < 0.0005. (H) Experimental setup for generation of Tifab −/− ;MLL-AF9 knockin (KI) mice. (I) Colony formation in methylcellulose of cKit + MLL-AF9 KI cells expressing FLAG-TIFAB (MSCV-IRES-GFP) or empty vector. Representative images are shown to the right as image sections of the entire well (n = 3). *p < 0.005. (J) Immunoblotting of p53 and FLAG-TIFAB on lysate isolated from MLL-AF9 KI cKit + BM cells transduced with FLAG-TIFAB (MSCV-IRES-GFP) or empty vector, with actin as a loading control.

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Knock-In, Western Blot, Isolation, Control

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Taqman Gene-expression probe, mouse Tifab , Applied Biosystems , Mm04210261_m1.

Techniques: Ubiquitin Proteomics, Virus, Plasmid Preparation, Recombinant, Silver Staining, Transfection, Purification, Lysis, Microarray, Negative Control, Software