PMC

Biological effects of TET2 downregulation are comparable to GHB treatment or ALDH5A1 downregulation. a TET2 downregulation using siRNA (siTET2) without changes in TET1 or TET3 mRNA levels. Q-PCR assay. Mean ± SD, n = 3 independent biological samples. siC, control siRNA. b–d TET2 downregulation translates into decreased 5-hmC levels (b), decreased Nanog immunofluorescent (IF) nuclear signal (c), and decreased proliferation (d). TET2 siRNA vs control siRNA, mean ± SD, n = 4 independent biological samples. e Schematic representation of GHB mechanism of action

GHB accumulates in GBM and DIPG stem-like cells and decreases levels of the 5-hydroxymethylcytosine epigenetic mark. a Increased intracellular GHB levels detected by mass spectrometry following GHB supplementation (n = 3 independent biological samples). b Chemical structure of α-KG and GHB. c Example of 5-hmC and 5-mC detection by dot immunoblotting of DNA extracts from control (C) and GHB-treated GBM stem-like cells (TG1). Methylene blue (MB) was used as a loading control. d GHB decreases 5-hmC levels in GBM (TG1) and DIPG (TP54) stem-like cells without changing 5-mC levels. Densitometric analysis of dot immunoblotting of DNA extracts (10 mM GHB, 1 week). Mean ± SD, n = 3 (TG1) and n = 4 (TP54) independent biological samples. e ALDH5A1 downregulation in GBM (TG1) or DIPG (TP54) stem-like cells results in decreased 5-hmC levels in face of unchanged 5-mC levels. Mean ± SD, n = 4 (TG1) and n = 3 (TP54) independent biological samples

GHB inhibits GBM and DIPG stem-like cell proliferation and self-renewal. a Immunocytochemical detection of Olig2. 1-week GHB 10 mM. Scale bars 10 µm. b Quantification of Olig2 nuclear immunofluorescent (IF) signal. n = 4 independent biological samples. c Upregulated expression of p21/CDKN1A in GBM and DIPG stem-like cells (10 mM GHB, 1 week, Western blot assay, mean ± SD, n = 3 independent biological samples). p21 MW, 21 kDa; Actin MW, 42 kDa. RDU relative densitometry units. d GHB inhibits self-renewal of GBM (TG1) and DIPG (TP54) stem-like cells. Mean ± SD, n = 3 independent biological samples. e GHB inhibits cell growth of stem-like cells isolated from adult GBM and pediatric DIPG. Total numbers of viable cell evaluated after 1-week 10 mM GHB treatment. Mean ± SD, n = 3 independent biological samples. f, g GHB arrests cell cycle. f Example of cell cycle FACS analysis of GHB-treated TG1 GBM stem-like cells. BrdU labels cells having undergone DNA replication. DAPI is an index of DNA content. g Quantification of the cell cycle analyses. Mean ± SD, n = 3 independent biological samples. h ALDH5A1 downregulation in GBM (TG1) and DIPG (TP54) stem-like cells inhibits cell proliferation. siA (ALDH5A1 siRNA) versus siC (control siRNA), mean ± SD, n = 3 independent biological samples

GHB decreases 5-hydroxymethylcytosine levels via inhibition of TET2 activity. a GHB does not reduce expression of TET isoforms. Q-PCR assay. Mean ± SD, n = 4 (TG1) and 3 (TP54) independent biological samples. b Doxycycline-dependent TET2 expression in the leukemic UT7 cell line. UT7 cells were treated for 12 h with doxycycline (Dox) at a final concentration of 1 µg/mL. TET2 mRNA levels were assayed using Q-PCR. Dox-treated versus -untreated UT7, mean ± SD, n = 3 independent biological samples. c GHB inhibits TET2 activity in the leukemic UT7 cell line expressing TET2 in a doxycycline-dependent manner. 12 h 10 mM GHB. 5-hmC levels determined by DNA dot immunoblotting. GHB-treated vs control UT7, mean ± SD, n = 3 independent biological samples. d Doxycycline-dependent TET2 expression in GBM stem-like cells (TG1) stably overexpressing TET2 in a doxycycline-dependent manner following lentiviral transduction. Q-PCR assays, mean ± SD, n = 5 biological samples. e Inhibitory GHB effects on TET2-mediated 5-hmC formation in GMB stem-like TG1 cells overexpressing TET2 in a doxycycline-dependent manner, mean ± SD, n = 4 biological samples. f–h In silico analysis of α-KG (f) and GHB (g) binding pockets within the TET2 protein. α-KG and GHB are colored in white and their oxygens in red. Ribbon representation of TET2 C-terminal domain. Progression from the N- to C- parts of the C-terminal domain are colored from blue to orange. h TET2 amino acid residues in contact with α-KG or GHB

Loss of stem and tumorigenic properties by GBM stem-like cells is accompanied with GABA metabolism deregulation characterized by enhanced GHB levels. a Increased GABA by-products to α-KG ratios in TG1-miR compared to TG1. The “+” sign represents the mean value in the whisker box. Mean ± SD, n = 6 independent biological samples. b Schematic reconstruction of metabolic pathways with green and red boxes signaling metabolites decreased or increased in TG1-miR compared to TG1, respectively. Enzyme names are within grey boxes. When relevant, the corresponding gene designation is indicated below the enzyme name. SSADH: succinic semialdehyde dehydrogenase. SSAR succinic semialdehyde reductase. c Downregulation of the ALDH5A1 protein product SSADH in TG1-miR. Western blot analysis. SSADH MW, 57 kDa; Actin MW, 42 kDa. Mean ± SD, n = 3 independent biological samples. d Decreased ALDH5A1 mRNA levels in TG1-miR compared to TG1. Q-PCR assays. Mean ± SD, n = 3 independent biological samples. e Targeting of the ALDH5A1 transcript by miR-302. Expression of Renilla Luciferase mRNA containing the wild-type form of ALDH5A1-3′UTR is strongly reduced in TG1-miR compared to TG1. Deletion of miR-302 putative target sequence in the 3′UTR of ALDH5A1 mRNA (ALDH5A1-3′UTR-DEL) prevents the binding of the miR, and rescues luciferase activity. n = 3 independent biological samples. f Decreased SSADH levels in GBM and DIPG stem-like cells (TG1, TP54) upon ALDH5A1 downregulation with siRNAs (siA). siC (control siRNA). Mean ± SD, n = 3 independent biological samples. RDU, relative densitometry units. g ALDH5A1 down regulation results in enhanced GHB intra-cellular levels. Mean ± SD, n = 3 independent biological samples. h SSADH immunoreactive cells are enriched in proliferative/non-differentiated GBM territories (P ^HIGH/D ⁻) and rare in non-proliferative/differentiated (P ^LOW/D ⁺) tumor territories of patients’ GBM, as revealed by immunohistochemical staining of Ki67, Olig2, and GFAP. HES: hematoxylin and eosin staining. Scale bar 100 µm. i GHB/α-KG ratios in proliferative/non-differentiated (P ^HIGH/D ⁻) and weakly proliferative/differentiated territories (P ^LOW/D ⁺) of patient GBM. GC–MS/MS analysis. Mean ± SD, n = 5 independent patient’s GBM neurosurgical samples

GHB promotes GBM and DIPG stem-like cell differentiation and decreases tumor burden. a Positive and negative correlation of ALDH5A1 and AKR7A2 expression with stemness genes (p < 0.01 after correction for multiple testing with false discovery rate). Gene ontology (GO) analysis using transcriptomes of 484 untreated primary human GBM of the TCGA dataset. Of the 142 listed in KEGG pathway category «Signaling pathways regulating pluripotency of stem cells» , 122 were detected in the dataset. 61 were significantly correlated with ALDH5A1 (48 positive/13 negative) and 38 with AKR7A2 (3 positive/35 negative). b Opposite regulation of ALDH5A1 and AKR7A2 transcript levels in cells with and without self-renewing and tumor-initiating properties (SR/TI) isolated from 4 human GBM []. Mean ± SD. c–h GHB inhibits Nanog expression. c Examples of immunocytochemical detection of Nanog. 1-week GHB 10 mM. Scale bars 10 µm. (d) Quantification of nuclear Nanog immunofluorescent (IF) signal. Mean ± SD, n = 3 independent biological samples. Microphotographs illustrate 240 nm single optical sections of TG1 cells immunostained for Nanog. e Decreased Nanog expression in response to ALDH5A1 downregulation. siA, ALDH5A1 siRNA. siC, control siRNA. Mean ± SD, n = 4 independent biological samples. f Nanog Western blot assay with protein extracts from control (Cont) and 10 mM GHB-treated TG1 GBM stem-like cells (1-week treatment). Nanog MW, 47 kDa; Actin MW, 42 kDa. n = 3 independent biological samples. RDU, relative densitometry units. g FACS analysis of Nanog immunofluorescent signal. Grey and red lines delineate unstained TG1 cells in control and GHB-treated conditions, respectively. h Fold change of mean fluorescent intensity of Nanog signal per cell (control vs 1 week-GHB 10 mM), as determined by FACS. TG1 and R633 GBM stem-like cells. TP83 DIPG stem-like cells. Mean ± SD, n = 4 independent biological samples. i GHB promotes cell adherence and membrane extension. Microphotograph of TG1 GBM stem-like treated with 10 mM GHB for 24 h and quantification of the numbers of adhering cells. Mean ± SD, n = 6 independent biological samples (TG1), n = 3 (5706**), n = 3 (TP83). Scale bar 100 µm. j Bioluminescent analyses of tumor growth initiated by grafting 6240** GBM stem-like cells transduced with a luciferase construct and a control (shC) or ALDH5A1 (shA) shRNA construct. 49 days post-graft. Quantification of the bioluminescent signals. Mean ± SD, n = 15 mice per group. Photographs correspond to examples of bioluminescent in vivo images of tumors in mice. k Quantification of tumor cells numbers following xenografts of GFP-expressing 5706** GBM or TP54 DIPG stem-like cells transduced with a control or ALDH5A1 shRNA construct. Mice were killed at 64 (5706**) or 71 (TP54) days post-graft, and the numbers of GFP-expressing cells determined. Mean ± SD, n = 3 (5706**) and 4 mice (TP54) per group