Journal: Cell metabolism

Article Title: Transsulfuration activity can support cell growth upon extracellular cysteine limitation

doi: 10.1016/j.cmet.2019.09.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-MAT2A , Bethyl Laboratories , Cat# A304-279A-M; RRID: AB_2620475.

Techniques: Virus, Recombinant, SYBR Green Assay, Plasmid Preparation, Software, CRISPR

Journal: Molecular Cancer

Article Title: In vitro and in vivo effects of the PPAR-alpha agonists fenofibrate and retinoic acid in endometrial cancer

doi: 10.1186/1476-4598-5-13

Figure Lengend Snippet: Selected genes derived from ICA & GO analysis of gene array experiments. Genes are combined into ontologies. Average Fold Changes are calculated from normalised values over the number of spots shown for each gene. As can be seen, not all genes are differentially expressed according to t-test p-values. Genes are instead selected based on their ICA loading (see discussion)

Article Snippet: RT-PCR for three genes selected from microarray analysis was performed using the assays-on-demand primers below according to the manufacturers instructions (Applied Biosystems, USA). (a) Cyclin D1 (CCND1) (Cat. No. Hs00277039_m1) (b) Methionine adenosyltransferase II alpha (MAT2A) (Cat. No. Hs00428515_g1) (c) Phosphoenolpyruvate carboxykinase 2 (PCK2) (Cat. No. Hs00356436_m1)

Techniques: Derivative Assay, Membrane, Binding Assay, Protease Inhibitor, Activity Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Gene expression level of MAT2A in prostate cancer patients indicated subgroups (Primary, n = 714; CRPC, n = 316; NEPC, n = 19). p -values are indicated. B Heatmap of ERG and MAT2A expression values in the cohort of CRPC patients described in A . (Padj value 0.0014). C Gene expression level of MAT2A in CRPC described in A and divided in ERG positive ( n = 113) and negative ( n = 203). Adjusted p -value 0.0014. D Immunoblot of VCaP control (Ctrl) and MAT2Akd VCaP cells (Sh1, Sh2) with indicated Ab ( n = 3 independent experiments). E Sphere formation assay (SFA) in indicated cell lines ( n = 3 independent experiments with n = 4 (for sh1) and n = 3 (for ctrl and sh2) biological replicates. Bottom, representative images of tumor spheres. Scale bars, 100 µm. F Growth curve of VCaP xenograft from VCaP control (Ctrl) and VCaP with stable MAT2A knockdown (Sh1 and Sh2) were engrafted and growth was monitored by caliper every 2 days ( n = 5 mice /group). G Tumor weight of indicated tumor xenografts ( n = 5 mice/group). H Representative sections from the indicated tumor xenografts. Scale bars, 200 µm. Right, Immunoscore of Ki67 by Aperio tool ( n = 3 mice Ctrl, n = 5 mice Sh1, n = 5 mice Sh2). I Immunoblot of RWPE-1 cells with stable expression of ERG, MAT2A and ERG + MAT2A with indicated Ab ( n = 3 independent experiments). J SFA in indicated cell lines ( n = 3 independent experiments with 3 biological replicates). K Fold change of mRNA levels of NANOG and SOX2 in RWPE-1 cells with stable expression of ERG, MAT2A and ERG + MAT2A. ( n = 5-6 biological replicates). L Growth curve of RWPE-1 xenografts. Indicated cell lines were engrafted and growth was monitored by caliper every 2 days ( n = 6 mice/group). M Tumor weight of indicated tumor xenografts ( n = 6). N Representative sections from the indicated tumor xenografts. Scale bars are 200 µm. O Immunoscore of Ki67 using the Aperio tool ( n = 6 mice/group). Molecular weights are indicated in kilodaltons (kDa). All error bars, mean ± SD. For box-and-whisker plots in A and C , the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values. One-way-ANOVA was used to test significant differences between groups in all panels, except for ( K ) where 2-way-ANOVA was used. Data presented in F and L are independent replicates derived from individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Expressing, Western Blot, Control, Tube Formation Assay, Knockdown, Whisker Assay, Derivative Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Number of genes affected by knockdown of MAT2A (Sh1 and Sh2) in VCaP cells ( p -value ≤ 0.05). B Principal component analysis (PCA) plot of VCaP cell lines, colored according to the condition (Ctrl, Sh1, Sh2). C Heatmap of differentially expressed genes in MAT2Akd (VCaP Sh1 and VCaP Sh2) versus control VCaP cells (VCaP Ctrl). Replicates ( n = 3 biological replicates) are indicated. D Hallmark enrichment analysis of MAT2Akd VCaP cells (Sh1 and Sh2) compared to control cells, performed with cameraPR function. Down-regulated pathways are colored in blue, up-regulated pathways are colored in red. E Functional annotation of top-ranking features of MAT2Akd VCaP cells (Sh1 and Sh2) performed with enrichR (cell markers augmented). F Fold changes of selected oncogenic drivers in MAT2Akd cells (Sh1 and Sh2) versus VCaP control, evaluated by RNA-seq. G Cumulative gene expression level of down-regulated genes in MAT2Akd VCaP cells (Sh1 and Sh2) in the cohort of primary ( n = 714), CRPC ( n = 316) and NEPC ( n = 19) patients. H Fold changes of selected canonical androgen genes in MAT2Akd (Sh1 and Sh2) versus VCaP control cells evaluated by RNA-seq. I Cumulative gene expression level of up-regulated genes in MAT2Akd VCaP cells (Sh1 and Sh2) in the cohort of primary ( n = 714), CRPC ( n = 316) and NEPC ( n = 19) patients. J Venn diagram showing the overlap between down-regulated genes in VCaP MAT2Akd cells (Sh1 and Sh2) and gene set associated with NEPC. Test: Fisher exact test. Fold changes of selected NEPC ( K ) and cancer stem cell genes ( L ), repressed by MAT2Akd in VCaP cells (Sh1 and Sh2). M Hallmark enrichment analysis of ERG + MAT2A RWPE-1 cells compared to RWPE-1 MAT2A performed with cameraPR function. Down-regulated pathways are colored in blue, up-regulated pathways are colored in red. N Fold change of selected CSC- and EMT-related genes up-regulated in RWPE-1 ERG + MAT2A versus RWPE-1 MAT2A. For box-and-whisker plots in G and I , the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values. One-way-ANOVA was used to test significant differences between groups.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Knockdown, Control, Functional Assay, RNA Sequencing Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Schematic representation of ex-vivo SFA assay, starting from LuCaP 35. B Ex-vivo SFA from dissociated LuCaP 35 under treatment with the indicated concentration of PF-9366 Right, images of spheres at the indicated concentration ( n = 6 biological replicates). Scale bars are 100 µm. C Ex-vivo SFA from dissociated LuCaP 35 under treatment with the indicated concentration of AG-270. Scale bars are 100 µm. Right, images of spheres at the indicated concentration ( n = 3 biological replicates). D Schematic representation of systemic in vivo treatment of LuCaP 35 tumors with vehicle or AG-270. E Growth curve of LuCaP 35 xenografts. Mice ( n = 7/group) were treated by oral gavage with either vehicle or AG-270. Tumor growth was monitored every 2 days with caliper. F Ex-vivo SFA assay from explanted LuCaP 35 tumors ( n = 3 tumors/group) after treatment with either vehicle or AG-270. Right, images of spheres of indicated treatment groups. Scale bars are 100 µm. G Representative sections from the indicated LuCaP tumors. Scale bars are 200 µm. H Immunoscore of Ki67, ERG, EZH2, AR and CHGA of tumor xenografts shown in ( E ) (n = 7 mice/group/; n = 7 biological replicates) using the Aperio tool. I Representative immunohistochemical evaluation of MAT2A (left) and quantification (right) in prostate tissue from WT (Pb-Cre;ERGflox/flox; n = 4 mice, n = 8 technical replicates) and ERG + /PTEN-(Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG; n = 6 mice, n = 40 technical replicates). Scale bars, 200 µm. J Schematic representation of 3D organoids assay derived from ERG/PTEN prostates to evaluate the reversion of the organoid phenotype. K Organoids treated with the indicated doses of PF-9366 ( n = 8 biological replicates). Right, representative images are shown. Scale bars, 100 µm. L Organoids treated with the indicated doses of AG-270 ( n = 8 biological replicates). Right, representative images are shown. Scale bars are 100 µm. All error bars, mean ± s.d. Ordinary one-way-ANOVA was used to test significant differences between groups in all panels, except for ( K , L ) where was used two-way-ANOVA. P -value was determined using unpaired t -test in F – H and I . * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. Panels A , B and J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Ex Vivo, Concentration Assay, In Vivo, Immunohistochemical staining, Derivative Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Immunoblot of MAT2A in ERG positive and negative cell lines ( n = 2 independent experiments). B SFA from LNCaPabl cells under treatment with the indicated concentration of AG-270 (Left) ( n = 6 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. C SFA from LNCaP cells under treatment with the indicated concentration of AG-270 (Left) ( n = 6 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. D SFA from 22Rv1 cells under treatment with the indicated concentration of AG-270 (Left) ( n = 3 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. E Immunoblot of H3K4me2 in ERG positive and negative cell lines ( n = 2 independent experiments). F Immunoblot with indicated antibodies in LNCaPabl cells after treatment with the indicated dose of AG-270. Right, densitometry analysis of the indicated markers ( n = 2 independent experiments). G Schematic representation of ex-vivo SFA from dissociated patient-derived xenograft LuCaP 145.2. H Immunoblot of MAT2A in patient-derived xenograft LuCaP 145.2 ( n = 2 independent experiments). I Ex-vivo SFA from dissociated LuCaP 145.2 under treatment with the indicated concentration of AG-270 (Left) ( n = 3 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 6 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. J Schematic representation of organoid establishment from LuCaP 145.2 treatment with AG-270, and evaluation of NE markers. K Number and images of organoids at the indicated concentrations ( n = 7 biological replicates). L Immunoblot of NE markers in LuCaP 145.2 organoids treated with AG-270. Molecular weights are expressed in kDa ( n = 2 biological experiments). Scale bars indicated in all images are 50 µm except for B and I which are 100 µm. All error bars, mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. ns = no significant. One-way-ANOVA was used to test significant differences between groups in all panels, except for K where two-sided t -test was used. Panels G and J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Western Blot, Concentration Assay, Ex Vivo, Derivative Assay

Journal: Journal of the American Chemical Society

Article Title: The Transition-State Structure for Human MAT2A from Isotope Effects

doi: 10.1021/jacs.7b05803

Figure Lengend Snippet: Figure 1: MAT2A catalyzes the formation of SAM from ATP and methionine. KIE measurements were done using isotopically labeled substrates with the labels indicated. Each color corresponds to labels found on an individual substrate molecule.

Article Snippet: A synthetic plasmid encoding human MAT2A was designed in a modified pET vector (now deposited at AddGene).

Techniques: Labeling

Journal: Journal of the American Chemical Society

Article Title: The Transition-State Structure for Human MAT2A from Isotope Effects

doi: 10.1021/jacs.7b05803

Figure Lengend Snippet: Figure 4: (A) Geometric and electrostatic potential surface (EPS) maps for the MAT2A transition state. Red color designates partial negative charge while blue color designates partial positive charge. (B) Zoomed in EPS maps of the central sulfur atom for the MAT2A transition state (TS), S-adenosyl-L-methionine (SAM) and methionine (Met) are shown for comparison. The partial positive charge on the sulfonium group is significantly reduced in the transition state structure when compared to the SAM structure.

Article Snippet: A synthetic plasmid encoding human MAT2A was designed in a modified pET vector (now deposited at AddGene).

Techniques: Comparison

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: Expression and prognostic value of MAT2A in multiple myeloma. (A) Left side . MAT2A mRNA expression levels in normal bone marrow plasma cells (BMPC; N=22), cells from patients with monoclonal gammopathy of undetermined significance (MGUS; (N=44), or smoldering multiple myeloma (SMM; N=12), and multiple myeloma (MM) cells from newly diagnosed patients (N=345) from the UAMS TT2 cohort. Right side . MAT2A mRNA expression levels in normal bone marrow plasma cells (N=5), MGUS (N=5), primary MM cells from newly diagnosed patients (N=206), and human MM cell lines (N=42) from the Heidelberg-Montpellier cohort. ns, non-significant; * P <0.05; **** P <0.0001. (B) Prognostic value of MAT2A mRNA levels in terms of overall survival in newly diagnosed patients from the UAMS TT2 (N=256), and UAMS TT3 (N=158) cohort and in relapsed MM patients from the Mulligan cohort (N=264). Maxstat analysis was used to calculate the optimal separation of patients based on a cutoff value. (C) Overall survival and progression-free survival curves for newly diagnosed MM patients with high or low MAT2A RNA expression (MMRF CoMMpass trial, N=653). The Kaplan-Meier method was used to plot survival curves and group comparisons were made using the log-rank test. P <0.05 was considered as statistically different. (D, E) Basal mRNA (D) and protein (E) expression of MAT2A in nine human MM cell lines (ANBL6, AMO-1, JJN3, LP-1, OPM2, RPMI8226, U266, XG-2 and XG-7). HR: hazard ratio; OS: overall survival; PFS: progression-free survival.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, RNA Expression

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: MAT2A inhibition by siMAT2A reduces multiple myeloma cell growth and survival in vitro. Human myeloma cell lines, JJN3 and OPM-2, were transfected with 20 nM siRNA against MAT2A. (A) Cell viability was measured by the CellTiter-Glo Luminescent cell viability assay after 5 days. (B) Cell proliferation was measured by a bromodeoxyuridine incorporation assay followed by flow cytometric analysis after 4 days. (C) The cell cycle was analyzed using propidium iodide followed by flow cytometric analysis after 4 days. (D) Cell apoptosis was measured using flow cytometry by staining for annexin V-FITC/7-amino-actinomycin D after 5 days. (E) The levels of markers of apoptosis, i.e., PARP, caspase 9 and caspase 3 enzymes, were measured by western blotting. Statistical significance was determined by oneway analysis of variance. * P <0.05, ** P <0.01. si: short interfering; BrdU: bromodeoxyuridine; PARP: poly ADP ribose polymerase; cl: cleaved.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, In Vitro, Transfection, Cell Viability Assay, BrdU Incorporation Assay, Flow Cytometry, Staining, Western Blot

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: MAT2A interference inhibits protein synthesis by downregulating the mTOR pathway. (A) OPM2 cells were treated with 20 nM siMAT2A for 3 days and s-adenosyl-methionine concentration levels were measured by liquid chromatography-mass spectrometry. Significance was determined by one-way analysis of variance. (B) Western blot analysis of puromycin uptake after MAT2A knockdown for 4 days in JJN3 and OPM2 cells. (C) Western blot analysis of protein synthesis-related proteins, isolated after 4 days of MAT2A knockdown. *** P ≤0.001. si: short interfering; α-puro: α-puromycin.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Concentration Assay, Liquid Chromatography, Mass Spectrometry, Western Blot, Knockdown, Isolation

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: MAT2A inhibition by FIDAS-5 reduces multiple myeloma cell survival in vitro . (A, B) JJN3 and OPM2 cells were treated with increasing doses of FIDAS-5 for 48 h (A) or 5 days (B) and cell viability was measured by a CellTiter-Glo Luminescent cell viability assay. (C) Cell proliferation was measured using a bromodeoxyuridine incorporation assay followed by flow cytometric analysis after treatment with FIDAS-5 (250 nM) for 4 days. (D) Cell cycle was analyzed using propidium iodide staining followed by flow cytometry after treatment with FIDAS-5 (250 nM) for 4 days. (E) Western blot analysis of cell cycle proteins, isolated after 4 days of FIDAS-5 (250 nM) treatment. (F) Cell apoptosis was measured using flow cytometry by staining for annexin V-FITC/7-amino-actinomycin D after 5 days of treatment with increasing doses of FIDAS-5. (G) The protein levels of the apoptosis markers PARP, caspase 9 and caspase 3 and MCL-1 were measured by western blotting after treatment with FIDAS-5 (250 nM) for 5 days. All experiments were performed in conditioned medium. Statistical significance was determined by one-way analysis of variance or the Mann-Whitney U test. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. BrdU: bromodeoxyuridine; PARP: poly ADP ribose polymerase; cl: cleaved.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, In Vitro, Cell Viability Assay, BrdU Incorporation Assay, Staining, Flow Cytometry, Western Blot, Isolation, MANN-WHITNEY

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: Inhibition of MAT2A with FIDAS-5 impairs protein synthesis by downregulating the mTOR pathway. (A, B) siRNA-mediated knockdown was established using 20 nM siMAT2A in OPM2 cells for 3 days, after which the cells were treated with 0.5, 1, or 2 μM FIDAS-5 for 48 h. Cell viability was evaluated using a CellTiter-Glo Luminescent Cell viability assay (A) and cell apoptosis was measured using flow cytometry by staining for annexin V-FITC/7-amino-actinomycin D (B). (C) Western blot analysis of protein synthesis-related proteins, isolated after 4 days of FIDAS-5 (250 nM) treatment. (D) Western blot analysis of puromycin uptake after FIDAS-5 (250 nM) treatment for 4 days. All experiments were performed in conditioned medium. Statistical significance was determined by one-way analysis of variance. * P ≤0.05, ** P ≤0.01, *** P ≤0.001. Ctl: control; si: short interfering; α-puro: α-puromycin.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Knockdown, Cell Viability Assay, Flow Cytometry, Staining, Western Blot, Isolation, Control

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: Inhibition of MAT2A with FIDAS-5 blocks glycolysis and the tricarboxylic acid cycle. Media and cell samples were collected after 5 days of FIDAS-5 (250 nM) treatment, followed by liquid chromatography-mass spectrometry analysis of extracellular glucose and lactate and intracellular metabolites. For the analysis of extracellular metabolite, results represent % peak area ± standard deviation (SD) compared to that of cell-free media. For analysis of intracellular metabolites, data represent relative peak area of metabolite ± SD compared to that of the non-treated controls. Unpaired t tests were performed. ** P ≤0.01. Ctl: control; TCA: tricarboxylic acid.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Liquid Chromatography, Mass Spectrometry, Standard Deviation, Control

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: MAT2A inhibition by FIDAS-5 reduces tumor burden in vivo. (A, B) Murine multiple myeloma cells 5TGM1 were treated with increasing doses of FIDAS-5 for 5 days. Next, cell viability was measured by a CellTiter-Glo Luminescent cell viability assay (A), while cell apoptosis was measured using flow cytometry by staining for annexin V-FITC/7-amino-actinomycin D (B). (C) Tumor burden was analyzed by assessing the percentage of eGFP-positive cells using flow cytometry, bone marrow plasmacytosis on cytosmears and the amount of M-protein in serum. (D, E) Western blot analysis of protein synthesis-related proteins. Bar graphs represent the pixel intensity as measured by Image Studio Lite v5.2. Statistical significance was determined by one-way analysis of variance or the Mann-Whitney U test. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, In Vivo, Cell Viability Assay, Flow Cytometry, Staining, Western Blot, MANN-WHITNEY

Journal: Haematologica

Article Title: S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma

doi: 10.3324/haematol.2023.282866

Figure Lengend Snippet: Inhibition of MAT2A sensitizes multiple myeloma cells to bortezomib. (A) Cells were transfected with 20 nM siRNA against MAT2A for 5 days. On day 3, cells were treated with bortezomib (JJN3: 1.5 nM, OPM2: 2.5 nM) for an additional 2 days. Cell viability was measured by a CellTiter-Glo Luminescent cell viability assay. (B) Cells were treated with FIDAS-5 (JJN3: 2.5 μM, OPM2: 10 μM) and bortezomib (JJN3: 1.5 nM, OPM2: 2.5 nM) for 48 h in conditioned medium and cell viability was measured by a CellTiter-Glo Luminescent cell viability assay. (C, D) Cells were treated with FIDAS-5 (JJN3: 2.5 μM, OPM2: 10 μM) and bortezomib (JJN3: 1.5 nM, OPM2: 2.5 nM) for 24 h in conditioned medium. Next, cell proliferation was measured by a bromodeoxyuridine incorporation assay followed by flow cytometry (C) and cell cycle proteins were analyzed by western blot (D). (E, F) Cells were transfected with 20 nM siMAT2A (E) or treated with FIDAS-5 (JJN3: 2.5 μM, OPM2: 10 μM) in conditioned medium (F), combined with bortezomib (JJN3: 1.5 nM, OPM2: 2.5 nM). After 48 h, cell apoptosis was measured using flow cytometry by staining for annexin V-FITC/7-amino-actinomycin D. (G) The protein levels of the apoptosis markers PARP, caspase 9 and caspase 3 were measured by western blotting after treatment with FIDAS-5 (JJN3: 2.5 μM, OPM2: 10 μM) and bortezomib (JJN3: 1.5 nM, OPM2: 2.5 nM) for 48 h in conditioned medium. (H) CD138 + MM cells isolated from three patients’ samples were cultured for 24 h in conditioned medium and treated with bortezomib and FIDAS-5. Cell viability was measured by CellTiter-Glo assay. Statistical significance was determined by one-way analysis of variance. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. BZ: bortezomib; BrdU: bromodeoxyuridine; Combo: combination of bortezomib and FIDAS-5; PARP: poly ADP ribose polymerase; cl: cleaved.

Article Snippet: Bortezomib was purchased from Selleckchem (Munich, Germany), FIDAS-5 was purchased from Millipore Sigma (St. Louis, MO, USA) and MAT2A inhibitor 1 (Compound 196) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Transfection, Cell Viability Assay, BrdU Incorporation Assay, Flow Cytometry, Western Blot, Staining, Isolation, Cell Culture, Glo Assay

Journal: Cell Reports Medicine

Article Title: Methionine intervention induces PD-L1 expression to enhance the immune checkpoint therapy response in MTAP-deleted osteosarcoma

doi: 10.1016/j.xcrm.2025.101977

Figure Lengend Snippet: Reversing CD8A + T cell deficiency by methionine metabolism intervention enhancing immunotherapy response in MTAP-deleted osteosarcoma (A) Flowchart depicting methionine restriction diet combined with immune checkpoint therapy. (B and C) In vivo orthotopic tibia models of DuNN shMtap in C3H mice, depicting tumor growth curves (mean ± SD) and tumor weights at experimental endpoints, treated by methionine restriction diet combined with immune checkpoint therapy. ND n = 8; ICT n = 8; MR n = 8; Combined n = 8. Statistical analyses were performed using two-tailed Student’s t tests. (D) Metabolomics of the intestines in DuNN shMtap tumor-bearing C3H mice revealed downregulation of methionine-related metabolites following dietary methionine restriction. (E) Pathological characteristics and immunohistochemistry (CD8A) of tumor tissues from DuNN shMtap in C3H mice, treated by methionine restriction diet combined with immune checkpoint therapy. Arrows indicate T cells. Scale bar, 100 μm (F) Flowchart depicting MAT2A inhibition combined with immune checkpoint therapy. (G and H) In vivo orthotopic tibia models of DuNN shMtap in C3H mice, depicting tumor growth curves (mean ± SD) and survival curve at experimental endpoints, treated by MAT2A inhibition combined with immune checkpoint therapy. ND n = 6; ICT n = 6; MR n = 6; Combined n = 6. Statistical analyses were performed using two-tailed Student’s t tests. (I) Pathological characteristics and immunohistochemistry (CD8A, CD4, and FOXP3) of tumor tissues from DuNN shMtap in C3H mice, treated by MAT2A inhibition combined with immune checkpoint therapy. Arrows indicate T cells. Scale bar, 100 μm.

Article Snippet: MAT2A inhibitor screening assay kit , BPS bioscience , Cat#71402.

Techniques: In Vivo, Two Tailed Test, Immunohistochemistry, Inhibition

Journal: Cell Reports Medicine

Article Title: Methionine intervention induces PD-L1 expression to enhance the immune checkpoint therapy response in MTAP-deleted osteosarcoma

doi: 10.1016/j.xcrm.2025.101977

Figure Lengend Snippet: MAT2A inhibition triggering IKZF1-mediated PD-L1 expression in MTAP-deleted osteosarcoma (A, B, and C) Integrated analysis of transcription factors associated with PD-L1 expression in SGH-OS and TARGET-OS and genes upregulated by treating with MAT2A inhibitor rather than immune checkpoint therapy. Spearman correlation tests were performed, and data with p values < 0.05 were considered statistically significant. (D and E) Protein expression of MAT2A and PD-L1 in MTAP-deleted OS cells (143B) treated with MAT2A inhibitor PF-9366, and protein expression of IKZF1, MAT2A, and PD-L1 in 143B cells co-treated with siIKZF1, assessed by western blotting. (F) IKZF1 RNA expression in wild-type (WT) ( n = 36) and MTAP-deleted (DEL) ( n = 14) cases in SGH-OS cohort. Statistical analyses were performed using two-tailed Student’s t tests. (G) Transcriptomic pathway analysis of cells by treating with PF-9366, using GSEA analysis ( p value < 0.05) on Hallmark gene sets. ES refers to enrichment score, and NES refers to normalized enrichment score. (H and I) Survival curve of TARGET-OS and TCGA-SARC related with IKZF1 expression. Statistical significance was calculated using the log rank test. (J) Summary schematic illustrating the mechanism of methionine intervention enhancing immune checkpoint therapy in MTAP-deleted osteosarcoma (MTAP-del OS).

Article Snippet: MAT2A inhibitor screening assay kit , BPS bioscience , Cat#71402.

Techniques: Inhibition, Expressing, Western Blot, RNA Expression, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Methionine intervention induces PD-L1 expression to enhance the immune checkpoint therapy response in MTAP-deleted osteosarcoma

doi: 10.1016/j.xcrm.2025.101977

Figure Lengend Snippet:

Article Snippet: MAT2A inhibitor screening assay kit , BPS bioscience , Cat#71402.

Techniques: Control, Recombinant, Saline, Red Blood Cell Lysis, Lysis, Staining, Screening Assay, Transfection, Plasmid Preparation, In Vitro, CCK-8 Assay, Cell Isolation, Gene Expression, Sequencing, Methylation, In Vivo, Software, Microscopy, Refractive Index, Mass Spectrometry