DOI: 10.5061/dryad.2v6wwpzvs
title: Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic iron-sulfur assembly pathway

description: Glioblastoma (GBM), the most lethal primary brain tumor, harbors glioma
 stem cells (GSCs) that not only initiate and maintain malignant phenotypes
 but also enhance therapeutic resistance. Although frequently mutated in
 GBMs, the function and regulation of PTEN in PTEN-intact GSCs are unknown.
 Here we found that PTEN directly interacts with MMS19 and competitively
 disrupts MMS19-based cytosolic iron-sulfur (Fe-S) cluster assembly (CIA)
 machinery in the differentiated glioma cells (DGCs). Interrogation of
 GSCs, when compared with their matched DGCs, revealed that PTEN is
 specifically succinated at cysteine (C) 211 in GSCs. Isotope tracing
 coupled with mass spectrometry analysis confirmed that fumarate, generated
 by adenylosuccinate lyase (ADSL) in de novo purine synthesis pathway which
 is highly activated in GSCs, promotes PTEN C211 succination. This
 modification abrogates the interaction between PTEN and MMS19, thereby
 reactivating CIA machinery pathway in GSCs. Functionally, inhibiting PTEN
 C211 succination through re-expressing PTEN C211S mutant, depleting ADSL,
 or consuming fumarate by N-acetylcysteine (NAC), an FDA-approved
 prescription drug, impairs GSC maintenance. Importantly, re-expressing
 PTEN C211S or treating with NAC sensitizes GSC-derived brain tumors to
 temozolomide and irradiation, the standard-of-care treatments for GBM
 patients, by retarding CIA machinery-mediated DNA damage repair. These
 findings reveal an immediately practicable strategy to target GSCs for
 treating GBMs by combined therapy with repurposing NAC.
description: <strong>Mice</strong> To identify
 the potential tumor formation ability of GSCs and DGCs,
 luciferase-expressing cells (1 × 10<sup>4</sup>) were injected
 intracranially into 4-week-old female athymic old nude mice
 (BALB/cNj-Foxn1nu/Gpt, Strain NO. D000521, GemPharmatech) as previously
 described (<em>25</em>). Each group contained five mice. Mice
 were fed autoclaved food and water and maintained in a specific
 pathogen-free facility. Tumor volumes were monitored by detecting the flux
 activity using a bioluminescence imaging system at different time
 points. To teste whether inhibiting PTEN C211sc
 sensitizes GSCs to TMZ treatment, PTEN-depleted-MGG8 or MES28 GSCs
 expressing with or without WT Flag-PTEN or Flag-PTEN C211S were
 transfected with firefly luciferase. 1 × 10<sup>4 </sup>cells
 were injected intracranially into 4-week-old female athymic old nude mice.
 Each group contained five mice. 7 days after the injection, mice were
 treated with PBS or TMZ (20 mg/kg) by intraperitoneal injection. To teste
 whether inhibiting PTEN C211sc sensitizes GSCs to radiation treatment,
 PTEN-depleted-MGG8 or MES28 GSCs expressing with or without WT Flag-PTEN
 or Flag-PTEN C211S were transfected with firefly luciferase. 1 ×
 10<sup>4 </sup>cells were injected intracranially into
 4-week-old female athymic old nude mice. Each group contained five mice.
 On day 9 and 11, mice receive 5 Gy radiation treatment. Tumor volumes were
 monitored by detecting the flux activity using a bioluminescence imaging
 system at different time points. To explore the
 function of NAC sensitizing chemotherapy of brain tumors, 1 ×
 10<sup>4</sup> luciferase-expressing MGG8 and T3264 GSCs were
 injected intracranially into 4-week-old female athymic old nude mice. Each
 group contained five mice. 7 days after the injection, mice were treated
 with PBS, TMZ (20 mg/kg), NAC (200 mg/kg), or TMZ plus NAC by
 intraperitoneal injection. To explore the function of NAC sensitizing
 radiation therapy of brain tumors, 1 × 10<sup>4</sup>
 luciferase-expressing MGG8 and T3264 GSCs were injected intracranially
 into 4-week-old female athymic old nude mice. Each group contained five
 mice. 7 days after the injection, mice were treated with NAC (200 mg/kg)
 by intraperitoneal injection. On day 9 and 11, mice receive 5 Gy radiation
 treatment. Tumor volumes were monitored by detecting the flux activity
 using a bioluminescence imaging system at different time points.
 <strong>Cell Culture</strong> GSCs
 (MGG8, T3264, CW839, T2907, GSC23, MES28, MES20, T3028 and CW738) were
 derived from human specimens as previously described (PMID: 30948495).
 Details of these patients are restricted by the institutional
 requirements. GSCs were maintained in Neurobasal medium
 (Life Technologies) supplemented with B27, L-glutamine, sodium pyruvate,
 10 ng/ml basic fibroblast growth factor and 10 ng/ml epidermal growth
 factor (R&D Systems). For inducing DGCs, Fetal Bovine Serum (FBS)
 was added into culture medium for 7 days, and culture medium was changed
 every other day. The GSC phenotype was validated by stem cell marker
 expression (SOX2, Olig2 and GFAP) and tumor propagation <em>in
 vivo</em>. <strong>Plasmids and lentiviral
 transduction</strong> Polymerase chain reaction
 (PCR)-amplified full-length or truncated human FH, Flag-PTEN, and HA-MMS19
 were cloned into the pLVX vector. Human CIAO1 and CIAO2B were cloned into
 pColdI (His) vector. PTEN C211S was generated using the QuikChange
 site-directed mutagenesis kit (Stratagene, La Jolla, CA). shRNA-resistant
 (r) PTEN was constructed by introducing nonsense mutations in
 shRNA-targeting sites as previously described (<em>10</em>).
 shRNA targeting PTEN (5’-GCCAGCTAAAGGTGAAGATATAT-3’) was inserted into the
 pGIPZ vector as previously described (<em>10</em>). Lentiviral
 clones expressing shRNAs against human MMS19 (TRCN0000096384), human ADSL
 (TRCN0000078271), human PRPS1 (TRCN0000010123), human PPAT
 (TRCN0000031614) or a control shRNA were purchased from Sigma-Aldrich (St.
 Louis, MO, USA). 293FT cells were used to generate lentiviral particles
 through co-transfection of the packaging vectors pCMV-dR8.2 and pCI-VSVG
 using a standard calcium phosphate transfection method in Neurobasal
 complete medium as previously described
 (<em>25</em>).
 <strong>Immunoprecipitation and Immunoblotting
 Analysis</strong> Protein samples were extracted
 from transfected GSCs using IP cell lysis buffer (50 mM Tris-HCl, pH 7.5,
 0.01% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA,
 100 μM PMSF, 100 μM leupeptin, 1 μM aprotinin, 100 μM sodium
 orthovanadate, 100 μM sodium pyrophosphate, and 1 mM sodium fluoride).
 Immunoblot and immunoprecipitation analyses were performed using indicated
 antibodies as previously described (<em>58</em>).
 <strong>Purification of Recombinant
 Proteins</strong> Expression of His-CIAO1 and
 CIAO2B were induced in bacteria, and protein purification was performed as
 previously described (<em>58</em>). Briefly, 6xHis-tagged
 recombinant proteins were cultured in 250 ml of lysogeny broth (LB) medium
 until the OD reached 0.6. 0.5 mM. Isopropyl β-D-1-thiogalactopyranoside
 (IPTG) was used to induce protein expression overnight at 16 ℃. Then,
 bacteria were collected and lysed. The cell lysates were loaded onto a
 Ni-NTA column, washed with five column volumes of 20 mM imidazole, and
 eluted with 250 mM imidazole. Purified proteins were desalted using 10-kDa
 cut-through spin columns by washing with PBS.
 <strong>Neurosphere Formation
 Assay</strong> <em>In vitro</em>
 limiting dilution was performed to measure neurosphere formation as
 previously described. Briefly, decreasing numbers of GSCs (100, 50, 25,
 10, 2) per well were plated into 96-well plates. The presentation and
 numbers of neurospheres in each well were recorded. Extreme limiting
 dilution analysis was performed using software available at
 http://bioinf.wehi.edu.au/software/elda as previously described
 (<em>5</em>). <strong>Quantitative
 RT-PCR</strong> Total cellular RNA was isolated
 using Trizol reagent (Sigma Aldrich). The qScript cDNA Synthesis Kit
 (Quanta BioSciences) was used for reverse transcription into cDNA. Applied
 Biosystems 7900HT cycler using SYBR-Green PCR Master Mix (Thermo Fisher
 Scientific) was employed to perform quantitative real-time PCR. The
 primers used in this study were described in Table S4.
 <strong>Cell Proliferation
 Analysis</strong> A total of<strong>
 </strong>1000 GSCs suspended in 200 μL Neurobasal medium were plated
 in a 96-well plate. CellTiter-Glo (Promega, Madison, WI, USA) was used to
 measure cell proliferation according to the manufacturer’s instructions.
 All data were normalized to those of day 1 and presented as mean ± SD from
 three independent experiments.
 <strong><sup>55</sup></strong><strong>Fe Incorporation Assay</strong> <sup>55</sup>Fe incorporation assays were performed as previously described (<em>21</em>). Briefly, 16 mM <sup>55</sup>FeCl3 (Perkin Elmer) was incubated in 100mM HCl, 63 μM nitrilotriacetic acid (NTA), and 20 mM HEPES pH to 6.0 with Tris followed by titration to pH 7.0 with 100 mM NaOH to obtain <sup>55</sup>Fe-NTA. 1 × 10<sup>5</sup> cells were seeded into 6-well plates followed by 2 mCi/mL <sup>55</sup>Fe-NTA treatment for 18 hours. Then, cells were washed, collected, and lysed using RIPA buffer (150 mM NaCl, 5 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0, 1% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% SDS (w/v), 1 mM DTT, and 1X protease inhibitor cocktail (Roche)). MMS19 targeting proteins were Immunoprecipitated and <sup>55</sup>Fe incorporation was measured by scintillation counting. Data were normalized to cellular protein concentrations. <strong>Fumarate Quantification</strong> Intracellular fumarate levels were quantified using Fumarate Assay Kit (MAK060, Sigma-Aldrich) according to manufacturer’s instructions. For intracellular fumarate measurement, 1 × 10<sup>6</sup> cells were lysed using fumarate assay buffer from the kit, followed by centrifuging at 13,000 g for 10 min to remove insoluble material. For intracranial fumarate measurement, mice were perfused with PBS. The tumor samples in mouse brains were collected and chopped into pieces. 4 mg tissues were rapidly homogenized in 10 mL of Fumarate Assay Buffer, followed by centrifuging at 13,000 g for 10 min to remove insoluble material. Set up the Master Reaction Mix using fumarate assay buffer, fumarate developer, and fumarate enzyme mix from the kit. Add 100 μL Master Reaction Mix to each well of plates from the kit, followed by incubation for 30 min at room temperature. Measure the absorbance at 450 nm and calculate fumarate levels. Data were normalized to cellular protein concentrations. <strong>Mass Spectrometry Analysis</strong> PTEN succination site was determined as previously described (<em>10</em>). Briefly, Flag-PTEN protein was immunoprecipitated from two GSC cultures (MGG8 and T3264) and digested by dithiothreitol (5 mM) for 30 min at 56 ℃ followed by alkylation with iodoacetamide (11 mM) for 15 min at room temperature in a dark environment. Then, protein was diluted to concentration less than 2 M using TEAB (100 mM). Finally, chymotrypsin (chymotrypsin : protein = 1 : 50) was used to digest protein twice. After digestion, the protein peptides were analyzed by MALDI-TOF/TOF MS (MALDI-7090, Shimadzu Kratos). The total MS/MS data was compared against SwissProt Database by the following parameters: chymotrypsin digestion allowing up to 1 missed cleavage, fixed modifications of cysteine (carbamidomethylation), variable modifications of methionine (oxidation) and cysteine (succination), precursor peptide tolerance of 0.05 Da, and MS/MS tolerance of 0.2 Da. Analysis results with e values less than 0.01 was considered as positive identifications. To confirm the results that purine synthesis fuels PTEN C211sc through ADSL, MGG8 GSCs expressing with control shRNA or ADSL shRNA were washed with aspartate-free medium and incubated in fresh medium containing <sup>13</sup>C-aspartate (1 mM) for 24 h. <sup>13</sup>C-labelled C211sc of PTEN was analyzed by MS as described above. To determine the abundance of intracellular IMP, AMP, GMP, and GSH, approximately 1 × 10<sup>5</sup> GSCs were seeded in 10 cm dishes in triplicate. Cells were lysed, extracted in 90/9/1 (v/v/v) acetonitrile/water/formic acid and subjected to high-resolution mass spectrometry. Pure samples of IMP, GMP, AMP and GSH were purchased from Sigma-Aldrich. Samples were centrifuged and supernatants were dried using Termovap Sample Concentrator. Samples were then resolved in ammonium acetate (10 mM) containing 0.2% ammonium hydroxide. Samples were injected into a Luna NH2 column (P/N 00B-4378-B0; 5 μM, 50 × 2.0 mm; Phenomenex, Torrance, CA) heated to 35°C with mobile phase A (0.77 g NH4OAc, 1.25 mL NH4OH, 25 mL ACN, and 300 µL acetic acid [HAc] dissolved in 500 mL water) and mobile phase B (acetonitrile). Using a flowrate of 0.3ml/min, the elution program was: 0.1 min, 85% B; 3 min, 30% B; 12 min, 2% B; 15 min, 2% B; and 16–28 min, 85% B. Data were acquired using a Thermo Orbitrap Fusion Tribrid Mass Spectrometer via Selected Ion Mode (SIM) electrospray positive mode. <strong>EC<sub>50</sub> Measurement</strong> GSCs were exposed to TMZ with increasing concentrations from 2.9 to 1500 μM. Cell viability was measured at 24 h after treatment. The EC<sub>50</sub> of TMZ for GSCs was calculated using GraphPad software. <strong>Combination Effect Analysis</strong> The synergistic effect of TMZ and NAC combination was evaluated by a calculation of CI according to the Chou-Talalay method. Data were analyzed using CompuSyn software (CompuSyn Inc.): CI = 0.85 to 0.9, slight synergism; CI = 0.7 to 0.85, moderate synergism; CI = 0.3 to 0.7, synergism; CI = 0.1 to 0.3, strong synergism; CI &lt; 0.1, very strong synergism. <strong>Immunohistochemical (IHC) Analysis</strong> Sections of paraffin-embedded xenografted tumors were stained with Ki-67, γ-H2AX, PTEN, and PTEN C211sc, respectively. The percentage of Ki-67 and γ-H2AX positive cells was quantified in five randomly selected fields using Image Pro Plus software (Media Cybernetics). The staining of PTEN and PTEN C211sc were quantified according to the percentage of positive cells and staining intensity as described previously. The staining intensity was scored on a scale of 0-3: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion scores were assigned to the sections: 0 if 0% of tumor cells exhibited positive staining, 1 for 0 to 1% positive cells, 2 for 2% to 10% positive cells, 3 for 11% to 30% positive cells, 4 for 31% to 70% positive cells, and 5 for 71% to 100% positive cells. The intensity and proportion scores were then added to obtain a total score ranging from 0 to 8 as described before. <strong>TUNEL Analysis</strong> GSCs-derived tumors were cut into 4 mm slices. The rate of apoptotic cells in tumors was analyzed using the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme) according to the manufacturer’s instructions.
description: # Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic
 iron-sulfur assembly pathway [https://doi.org/10.5061/dryad.2v6wwpzvs
 ](https://doi.org/10.5061/dryad.2v6wwpzvs) In this study, we have shown
 that PTEN to be a substrate of fumarate, it is possible that other
 substrates could be succinated contributing to glioma progression. In
 addition, to evaluate the clinical applicability of NAC supplementation in
 glioma treatment, the GSCs-derived xenograft transplantation mouse model
 was used to evaluate tumor growth in vivo. While more mouse models, like
 PDX mouse model, will make the results more solid. Moreover, PTEN C211sc
 expression in clinical samples are needed to further evaluate the
 correlation between PTEN C211sc expression and TMZ or radiation therapy
 sensitivity of glioma patients. ### Description of the data and file
 structure Datasets included: 1\. Uncropped blots Each blots contains the
 information of cell line, treatment and the antibodies, and is named by
 figure location. For example, “Fig.1C” image refers to the immunoblots in
 Fig.1C. The blot on the top left indicates that Flag-PTEN expressed T3264
 GSCs and DGCs was collected for immunoprecipitation with antibody against
 Flag, the 2SC levels were measured. 2\. IHC a. Each folder contains the
 representative IHC images displayed in figures, and is name by figure
 location. For example, “Fig.7H” folder refers to the images displayed in
 Fig.7H. b. Each image is named by the group. For example, in “Fig.7H”
 folder, “TMZ+NAC” indicates that this IHC image is from tumors received
 TMZ and NAC treatment. 3\. TUNEL a. Each folder contains the
 representative TUNEL images displayed in figures, and is name by figure
 location. For example, “Fig.S7G” folder refers to the images displayed in
 Fig.S7G. b. Each image is named by the group. For example, in “Fig.S10J”
 folder, “TMZ+NAC” indicates that this TUNEL image is from tumors received
 TMZ and NAC treatment. 4\. GSCs spheres a. Each folder is name by figure
 location. For example, “Fig.1I” folder refers to the results of Fig.1I. b.
 Each image is named by the cell line and group. For example, in “Fig.1I”
 folder, “MES28 C211S” indicates that this image refers to the formed
 spheres of PTEN C211S mutated MES28 GSCs. 5\. Data file S1 a. Sheet “ELDA”
 contains the data for ELDA results. “cell numbers” means plated cell
 numbers in each well; “wells” means well numbers of cell plated; “spheres
 positive wells” means the number of wells in which spheres were formed;
 “group” means the treatment for cells. b. Sheet “Cell number” contains the
 data for GSCs growth. For example, in table Fig.1J, “MGG8 GSC” indicates
 the cells used for experiment; “WT” and “C211S” indicate the cell
 transfection; cell numbers were measured on day1, 3, 5. c. Sheet “BLI”
 contains the data for the quantification results of BLI analyses. For
 example, in table Fig.3I, “MGG8” and “MES28” indicate the cells for
 intracranial injection; “ctrl, TMZ, shMMS19, shMMS19+TMZ” indicate the
 treatment for tumors; the flux activity was measured on day 7, 14, 21, and
 28. d. Sheet “55Fe incorporation” contains the data for 55Fe incorporation
 into CIA target proteins. For example, in table Fig.3E, “MGG8” and “MES28”
 indicate the cells used for experiment; “POLA1, POLD1 et.al” are the CIA
 targets. e. Sheet “Fumarate levels” contains the data of cellular fumarate
 levels measurement. For example, in table Fig.4A, “MGG8, T3264, MES28,
 MES20” indicate the cells used for experiment; “ctrl, shADSL” indicate the
 cell transfection. f. Sheet “Cell viability” contains the data for EC50
 measurement. For example, in table Fig.5B, “MES28” is the cell used in
 this experiment; cells were treatment with increased dosage (2.929688,
 5.859375 et. al) of TMZ; “Ctrl, Flag-PTEN WT, Flag-PTEN C211S” indicate
 the cell transfection. g. Sheet “Sphere number” contains the data of
 spheres formation. For example, table Fig.1H shows that MGG8, T3264, MES28
 GSCs were transfected with Flag-PTEN WT or C211S, formed spheres were
 measured. h. Sheet “IHC score” contains the quantification of IHC results.
 For example, in table Fig.7H, MGG8 derived tumors were treated with CTRL,
 NAC, TMZ, TMZ+NAC, 2SC levels in tumors were scored. i. Sheet “TUNEL”
 contains the quantification of TUNEL results. For example, in table
 Fig.S10J, MGG8 derived tumors were treated with CTRL, NAC, TMZ, TMZ+NAC,
 TUNEL positive rates in tumors were scored.