cancers Article Glioblastoma Cells Counteract PARP Inhibition through Pro-Survival Induction of Lipid Droplets Synthesis and Utilization Jara Majuelos-Melguizo 1,2,† , José Manuel Rodríguez-Vargas 1, *,† , Nuria Martínez-López 3,‡,§ , Daniel Delgado-Bellido 1 , Ángel García-Díaz 1 , Víctor J. Yuste 4 , Marina García-Macía 5 , Laura M. López 1 , Rajat Singh 6,7 and F. J. Oliver 1, * 1 Department of Immunology and Cell Biology, Institute of Parasitology and Biomedicine “López-Neyra”, IPBLN, CSIC, CIBERONC, 18016 Granada, Spain; jara_mm@hotmail.com (J.M.-M.); ddelgado@ipb.csic.es (D.D.-B.); agdiaz@ipb.csic.es (Á.G.-D.); laural@ipb.csic.es (L.M.L.) 2 Department of Biochemistry, Autonomous University of Madrid (UAM), ‘Alberto Sols’ Biomedical Research Institute (CSIC-UAM), 28029 Madrid, Spain 3 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; nuria.martinez-lopez@einsteinmed.org 4 Cell Death, Senescence and Survival Group, Department of Biochemistry and Molecular Biology and Institute of Neurosciences, Faculty of Medicine, Autonomous University of Barcelona, CIBERNED, 08193 Barcelona, Spain; victor.yuste@uab.cat 5 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; marinagarciamacia@usal.es 6 Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA;   rajat.singh@einsteinmed.org 7 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA Citation: Majuelos-Melguizo, J.; * Correspondence: jmrodriguez@ipb.csic.es (J.M.R.-V.); joliver@ipb.csic.es (F.J.O.) Rodríguez-Vargas, J.M.; † These authors contributed equally to this work. Martínez-López, N.; Delgado-Bellido, ‡ Present address: Department of Radiation Oncology, Albert Einstein College of Medicine, D.; García-Díaz, Á.; Yuste, V.J.; Bronx, NY 10461, USA. García-Macía, M.; López, L.M.; Singh, § Present address: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA. R.; Oliver, F.J. Glioblastoma Cells Counteract PARP Inhibition through Simple Summary: Glioblastoma multiforme (GBM) is the most common and deadly primary brain Pro-Survival Induction of Lipid tumor in adults and one of the most aggressive cancers. The use of Poly ADP-Ribose Polymerase Droplets Synthesis and Utilization. (PARP) inhibitors is being expanded as therapeutic alternative in multiple types of cancer beyond Cancers 2022, 14, 726. https:// BRCA1/2 mutant breast and ovarian cancer. Here we have analyzed glioma cells’ traits that limit doi.org/10.3390/cancers14030726 the efficacy of PARPi as anti-glioma agents and we found that PARPi triggered the synthesis of Academic Editor: David Wong lipid droplets (LDs) that fueled glioma cells by inducing pro-survival lipid consumption. Notably, Received: 12 November 2021 blocking Fatty Acids utilization by inhibition of β-oxidation with etomoxir, increased PARPi-induced Accepted: 27 January 2022 glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of PARPi. We Published: 30 January 2022 uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response Publisher’s Note: MDPI stays neutral to PARP inhibition. with regard to jurisdictional claims in published maps and institutional affil- Abstract: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor iations. in adults. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new class of anti-neoplastic drugs. In the current study, we have characterized the mechanism by which glioblastoma cells evade the effect of PARPi as anti-tumor agents. We have found that suppression of PARP activity Copyright: © 2022 by the authors. exerts an anti-stemness effect and has a dual impact on autophagy, inducing its activation in the Licensee MDPI, Basel, Switzerland. first 24 h (together with down-regulation of the pro-survival mTOR pathway) and preventing This article is an open access article autophagosomes fusion to lysosomes at later time-points, in primary glioma cells. In parallel, PARPi distributed under the terms and triggered the synthesis of lipid droplets (LDs) through ACC-dependent activation of de novo fatty conditions of the Creative Commons acids (FA) synthesis. Notably, inhibiting β-oxidation and blocking FA utilization, increased PARPi- Attribution (CC BY) license (https:// induced glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of creativecommons.org/licenses/by/ PARPi. Moreover, LDs fuel glioma cells by inducing pro-survival lipid consumption as confirmed by 4.0/). Cancers 2022, 14, 726. https://doi.org/10.3390/cancers14030726 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 726 2 of 19 quantitation of oxygen consumption rates using Seahorse respirometry in presence or absence of OA. In summary, we uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response to PARP inhibition. Keywords: glioblastoma stem cells; PARP inhibitors; lipophagy; lipid droplets; Acyl-coA-carboxylase; metabolic adaptation 1. Introduction Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. In spite of some evident progress in strategies against GBM, prognosis remains extremely poor with the average survival reported to be less than 15 months after diagnosis. The nuclear protein PARP-1 functions as a DNA damage sensor and is also implicated in a broad variety of cellular functions, including metabolic adaptations [1,2]. PARP inhibitors exhibit anti-tumor activity in part due to their ability to induce synthetic lethality in cells deficient for homologous recombination repair [3–6]. Up to date, the studies on GBM and PARPi have focused on the use of these small molecules as radio or chemo-potentiators [7,8]. PARPi enhance the efficacy of Temozolomide (TMZ) in multiple preclinical models and are a promising strategy for GBM [9–11]. In a previous study we have reported that PTEN- deficient glioblastoma cells were particularly sensitive to PARPi because they aggravated homologous recombination repair deficiency and displayed genomic instability leading to mitotic catastrophe [12]. In the current study, we have analyzed glioma cells’ traits that limit the efficacy of PARPi as anti-tumor agents. Emerging evidences demonstrate that lipid metabolism undergoes reprogramming in cancer cells in general and in glioma in particular [13,14]. Here we show that PARPi induces the formation of LDs that serve as fuel for tumor cells, which occurs in parallel with the induction of autophagy. Mecha- nistically, PARPi-induced LDs formation occurs through synthesis de novo, dependent on the activation of Acyl-CoA-Carboxylase after AMPK suppression as a consequence of PARPi-induced ATP recovery. This mechanism occurs concomitantly with the increase in the transcriptional activation of fatty acid synthase (FASN). Moreover, preventing lipid synthesis and utilization by glioma cells increased PARPi-induced glioma cell death sug- gesting that avoidance of metabolic reprogramming might result in potentiation of PARPi antitumor efficacy. 2. Materials and Methods 2.1. Cell Culture and Reagents U87MG (PTEN mutant) cell line was purchased from the European Collection of Authenticated Cell Cultures (Cat. 89081402) through the University of Granada Cell Lines Service. LN929 (PTEN wild type) cell line was a donation from Dr. Joan Seoane (Vall d’Hebron Institute of Oncology, Barcelona, Spain). SW1783 (PTEN mutant) grade III as- trocytoma cell line and MSO4 (mutations not characterized) primary glioblastoma cell line, were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supple- mented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 mg/L Penicillin and 500U Streptomycin (Sigma-Aldrich, St Louis, MO, USA, P4333). Patient-derived Glioma Stem Cells TG1A and TG16 (PTEN wild type) were kindly provided by Hervé Chneiweiss and Marie-Pierre Junier’s lab in Quai Saint-Bernard, Paris, and they were obtained as described previously [15] and cultured in DMEM/F12 supplemented with N2, G5 and B27 (Invitrogen, Waltham, MA, USA). All cells were maintained at 37 ◦ C and 5% CO2 . PJ34 ([N- (6-Oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamine) acetamide hydrochloride]) (Alexis Biochemicals, Barcelona, Spain, ALX-270-289), AZD2281/Olaparib (Selleckchem, Houston, TX, USA, S1060) and BMN 673 (Selleckchem, S7048) were used as PARP inhibitors. Olaparib was dissolved in DMSO, and PJ34 and BMN 673 were dissolved in H2O. Cells Cancers 2022, 14, 726 3 of 19 were treated at the indicated doses and times. mTORC1 inhibitor Rapamycin (Calbiochem, St. Louis, MO, USA, #553210) was dissolved in ethanol and used at a final concentration of 100 nM. Chloroquine (Sigma-Aldrich, C6628), NH4 Cl (American Bioanalytical, New York, NY, USA, AB00161) and Leupeptin (Fisher, Pittsburgh, PA, USA, BP2662100) were used to inhibit lysosomal degradation activity. They all were dissolved in H2 O. Chloroquine was used at a final concentration of 10 µM. NH4 Cl was used at a final concentration of 20 mM and Leupeptin at 100 µM. AMP analog AICAR (Sigma-Aldrich, A9978) was dissolved in H2 O and used at a final concentration of 2 mM. The ROS inhibitor N-acetyl cysteine (NAC) (Sigma-Aldrich A7250) was dissolved in H2 O and used at a final concentration of 3 mM. β-oxidation inhibitor Etomoxir (Sigma-Aldrich, E1905) was dissolved in H2 O and used at a final concentration of 100 µM. Oleic acid (Sigma-Aldrich, O3008) was used at a final concentration of 0.25 mM and was added 12 h before PJ34 treatment. In all cases, cells were plated 24 h before the treatments. 2.2. Western Blot Analysis Cells were plated in 6-well plates at a density of 1.5 × 105 cells per well. After the treatments, cells were washed twice with 1× Phosphate Buffered Saline (PBS) and resus- pended in lysis buffer RIPA (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8), supplemented with protease and phosphatase inhibitors. Lysates were sonicated, centrifuged at maximum velocity for 30 min at 4 ◦ C and the supernatant was collected. Protein quantification was determined using the Lowry assay (Bio-Rad laborato- ries, Hercules, CA, USA, #500-0112). Proteins were resolved on SDS-polyacrylamide gels and transferred onto PVDF Membrane (Bio-Rad laboratories). Membranes were blocked with 5% nonfat dry milk powder in TBST (1× TBS containing 0.1% Tween-20) for 1 h and incubated overnight with 1% milk TBST with the following antibodies: Anti-PARP-1 (C2-10 mouse, ALEXIS, LA, ALX-804-210), anti-PAR (10H) (Millipore, Burlington, MA, USA, MABC547), anti-P-PTEN (S380) (Cell Signaling Technology, Danvers, MA, USA, #9551), anti-PTEN (A2B1) (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7974), anti-P- AKT (Ser473) (Cell Signaling Technology, #9271), anti-AKT (Cell Signaling Technology, #4691), anti-P-S6 (Ser235/6) (Cell Signaling Technology, #2211), anti-P-GSK3β (Ser9) (Cell Signaling Technology, #9336), anti-β-CATENIN (Sigma-Aldrich, C2206), anti-LC3B (Cell sig- naling Technology, #2775), anti-SOX2 (Millipore, AB5603), anti-OCT4 (Abcam, Cambridge, UK, ab18976), anti-NANOG (Millipore, PA1-097) anti-P-P70S6k (Thr389) (Cell Signaling Technology, #9205), anti-P70S6k (Cell Signaling Technology, #9202), anti-ILK (Millipore, MABT66), anti-P-ACC (Ser79) (Millipore, 07-303), anti-P-AMPK (Thr172) (Cell Signaling Technology, #2535S), anti-AMPK (D5A2) (Cell Signaling Technology, #5831), anti-ATG1 (Cell Signaling Technology, #4776). Anti-α-Tubulin (Santa Cruz Biotechnology, sc-8035), anti-GAPDH (Sigma-Aldrich, St G9545) and anti-β-Actin (Sigma-Aldrich, A5316) were used as loading control. Bands were visualized with AmershamTM ECLTM Western Blot- ting Detection Reagent (GE Healthcare, Chicago, IL, USA, RPN2106) and ImmobilonTM Western (Millipore, WBKLS0500). Pictures were taken with the imaging system ChemiDoc XRS System (Bio-Rad) or medical X-ray films (AGFA, Mortsel, Belgium). To quantify west- ern blot signals we used ImageJ and all the films were exposed for different times to make sure that the bands are in the linear range of detection. In all the cases we compare the relative abundance of a specific protein (Bands) based in two methods for normalizing WBs: (i) housekeeping protein normalization α-tubulin or β-actin; (ii) total protein normalization, total Akt, AMPK, P70S6k . In all the cases, 3 independent experiments were migrated to highlight the results. Most representative’s gels of each experiment have been shown including molecular weight according with antibodies data sheet. 2.3. Transfection of Small Interfering (si) RNA (siRNA) Cells were plated in 6-well plates at a density of 1 × 105 cells per well; 24 h later, cells were transfected with the indicated siRNAs at 50 nM using Lipofectamine® 2000 (Invitrogen, 11668) or Jet PRIME (Polyplus Transfection® , Alsace, France, 114-75), ac- Cancers 2022, 14, 726 4 of 19 cording to the manufacturer’s guide. Double-stranded RNA duplexes corresponding to a non-targeted control (scramble) (50 -CCUACAUCCCGAUCGAUGAUGUU-30 ) and ATG1 (50 -CAGCAUCACUGCCGAGAGGUU-30 , 50 -CCACGCAGGUGCAGAACUAUU-30 , 50 -GCACAGAGACCGUGGGCAAUU-30 , 50 -UCACUGACCUGCUCCUUAAUU-30 ) were ordered to Sigma-Aldrich. Double-stranded RNA duplexes corresponding to human PARP1 were from Ambion Applied Biosystems (Burlington, MA, USA), and double-stranded RNA duplexes corresponding to human ACC were from Thermo Scientific (Burlington, MA, USA) (siRNA against ACACA #s883; siRNA against ACACB #s887). 48 h after transfection, cells were treated with the correspondent treatment as indicated above. 2.4. Transfection of mCherry-GFP-LC3 Cells were plated in 6-well plates at a density of 1 × 105 cells per well on glass cover-slips. 24 h later, transfection was performed with 0.5 µg mCherry-GFP-LC3 plasmid (kindly provided by Dr S. Hilfiker (Department of Immunology and Cell Biology. Institute of Parasitology and Biomedicine “López-Neyra”, IPBLN, CSIC, Granada, Spain) and ideated by Dr. T. Johansen (The Arthic University of Norway, Tromsø, Norway)) using Jet PRIME (Polyplus Transfection® , 114-75) according to the manufacturer’s protocol. Cells were treated 48 h after the transfection. After the treatments, cells were fixed with paraformaldehyde (PFA) solution (4% PFA (w/v), 2% (w/v) sucrose in 1× PBS) for 10 min at room temperature and permeabilized with 1× PBS containing 0.5% Triton X-100 for 5 min at room temperature. Nuclei were stained with DAPI and immunostaining was visualized with Confocal Leica LCS SP5 (Wetzlar, Hesse, Germany) using LAS AF software. Quantification of the yellow and red puncta was performed using at least five fields of view (at least 150 cells) per condition in three independent experiments. 2.5. Autophagy Flux Assay Cells were plated in 6-well plates at a density of 1.25 × 105 cells per well. Cells were treated with in presence or absence of lysosomal inhibitors (Leupeptin (200 mM) and ammonium chloride (20 mM)) for 4 h. Net autophagy/LC3-II flux was determined by subtracting the densitometric value of Inh-untreated LC3-II from corresponding Inh- treated LC3-II value. Steady state LC3 was calculated as LC3-II/LC3-I ratio. Rate of fusion is the result of dividing the densitometric value of Inh-treated LC3-II by corresponding Inh-untreated LC3-II value. All results were normalized to the correspondent control in 3 independent experiments. 2.6. RT-qPCR Primers were designed using NCBI primers design software as follows: human FASN→Forward→50 -ACCAGAGCAGCCATGGAGGAG-30 ; →Reverse→5’-CGTAGAGCC CCGCCTTCCAG-30 . Cells were plated in 6 wells at a density of 105 cells. RNA extraction was carried out using RNeasy MiniKit (Qiagen Inc., Venlo, The Netherlands) and genomic DNA was eliminated via gDNA Eliminator spin columns. RNA quantification was performed using NanoDROP© ND-1000 Spectrophotometer (Thermo Fisher). Complementary DNA or cDNA obtaining was performed with Invitrogen M-MLV Reverse Transcriptase (Invitrogen #10338842) and qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems). Expression of Fatty acid synthase (FASN) was normalized to TATA-binding protein (TBP) and all reactions were performed in triplicate. 2.7. GICs Immunofluorescence Cells grown as neurospheres were dropped on poly d-lysine-coated slides (Microscope slide Polysine adhesion Thermo Scientific J2800AMNZ) and fixed in PBS-PFA 4%. Next, cells were permeabilized with 1× PBS containing 0.5% Triton X-100. SOX2 and NESTIN (Millipore) were used at a dilution of 1:100, and nuclear counterstaining with DAPI was Cancers 2022, 14, 726 5 of 19 performed after removal of excess secondary antibody. Immunostaining was visualized with Confocal Leica LCS SP5. At least 250 cells in 10 independent fields of view per condition and replicate were analyzed in 3 independent experiments. 2.8. LAMP1-Bodipy Immunofluorescence 2.5 × 104 cells were seeded on poly d-lysine-coated coverslips in 12-well plates (Sigma- Aldrich, P0899). After the treatments, cells were fixed with 4% PFA in 1× PBS for 30 min at room temperature and washed three times in 1× PBS. Cells were permeabilized with 0.5% Triton X-100, blocked for 1 h and incubated with primary antibody against LAMP1 (Abcam, #241170) and secondary antibody (Alexa Fluor 647 conjugated) (Invitrogen). For lipid droplet detection, cells were incubated with BODIPY 493/503 (Molecular Probes, Eugene, OR, USA, D-3922) for 20 min at room temperature. Coverslips were mounted using mount- ing medium contained DAPI (40 ,6-diamidino-2-phenylindole) (Invitrogen). Immunos- taining was visualized with Leica DMi8 wide field fluorescence (Inverted) microscope. Images were deconvolved using the Huygens software (https://svi.nl/Huygens-Software, accessed on 12 November 2021). All images were subjected to identical post-acquisition processing. Percentage colocalization was calculated using the JACoP plugin in single Z-stack sections of deconvolved images using at least 10 fields of view per condition in 2 independent experiments. 2.9. Sudan-Red Staining Cells were grown in 12 well-plates at a density of 2.5 × 104 cells per well on glass coverslips. After the treatments, cells were fixed with PFA solution (4% PFA (w/v), 2% (w/v) sucrose in 1× PBS) for 10 min at room temperature and permeabilized with 1× PBS containing 0.5% Triton X-100 for 5 min at room temperature. Next, lipid droplets were labeled with 0.2% Sudan III for 20 min at room temperature. Nuclear counterstaining with DAPI was performed after removal of excess of the lipidic dye. Immunostaining was visualized with Confocal Leica LCS SP5 Fluorescence Microscope using LAS AF software. Quantification of lipid droplets was performed with ImageJ in at least 150 cells, using at least 5 fields of view per condition in 3 independent experiments. 2.10. ATP Determination Cells were grown in 6 well-plates at a density of 5 × 105 cells. Intracellular ATP was measured using a luciferin/luciferase-based assay (Adenosine 50 -triphosphate (ATP) Biolu- minescent Assay Kit, Sigma-Aldrich, FLAA-1KT) following manufacturer’s instructions. Protein concentration was determined using Lowry protein assay reagent (Bio-Rad). The content of ATP was normalized to protein content and expressed as percentage of control. 2.11. Short-Term Proliferation Assay: MTT For MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide) assay, cells were grown in 96 well-plates at a density of 8 × 103 cells. MTT assay was performed using Cell Proliferation Kit I (MTT, 1-65-007, Roche, Mannheim, Germany) following manufacturer’s instructions. Plate absorbance was read at a wavelength of 570 nm in VERSAmax plate reader (Molecular Devices, St José, CA, USA). 2.12. SubG1 Analysis: Propidium Iodide Cells were grown in DMEM without FBS in 6 well-plates at a density of 1.5 × 105 cells. After the indicated treatments, cells were trypsinized, washed with 1× PBS, permeabilized with 70% ice cold ethanol for at least 10 min, washed again with 1× PBS and incubated with Propidium iodide 40 µg/mL and RNAase A 100 µg/mL (Ribonuclease A from bovine pancreas, Sigma-Aldrich, R6513-10MG) at 37 ◦ C in dark for 20 min. Cells were analyzed on a FACScalibur using CellQuest software (Becton Dickinson, Mountain View, CA, USA) and cell cycle was determined using FlowJo software. Cancers 2022, 14, 726 6 of 19 2.13. Seahorse Respirometry Cells bioenergetics was determined using a Seahorse respirometer (Seahorse Bio- science, North Billerica, MA, USA). Briefly, cells were grown in a XF96 plate at a density of 7.5 × 103 cells. Treatments with PJ34, lysosomal inhibitors and etomoxir were per- formed as indicated. One hour before the end of the experiment, DMEM was replaced by freshly prepared Seahorse Assay Medium XF base medium (Sigma-Aldrich D5030), 5 mM Glucose, 2 mM L-glutamine (GIBCO 35050), 1 mM Sodium pyruvate (GIBCO 11360), Phenol Red (Sigma-Aldrich P5530) and incubated in a CO2 -free incubator for 1 h. 1.25 µM Oligomycin (Sigma-Aldrich 75351), 2 µM FCCP (Sigma-Aldrich C2920), 1.8 µM Antimycin (Sigma-Aldrich A8674) were then added as indicated, and basal oxygen consumption rates (OCR) values were normalized to protein quantification using the Lowry assay (Bio-Rad laboratories, #500-0112). 2.14. Transmission Electron Microscopy Cells were grown in 10 cm plates at a density of 1 × 106 cells. After the treatments, cells were trypsinized, washed with 1× PBS and fixed in fixation solution (2% Glutaraldehyde, 1% Formaldehyde in 0.05 M Cacodilate buffer pH 7.4) for 5 h. Next, cells were incubated three times in washing solution (Cacodilate 0.1 M pH 7.4) for 15 min. Finally, samples were stained with uranil acetate (Uranyl Acetate 2% in miliQ H2 O). The ultrathin sections were performed with a diamond knife in an ultra-microtome (Reichert Ultracut S, Leica Microsystems, Wetzlar, Hesse, Germany). The samples were analyzed in a TEM Zeiss 902 (Jena, Turingia, Germany) with 80 Kv of voltage acceleration (CIC-UGR, Granada, Spain). 2.15. Statistical Analysis Independent experiments were pooled when the coefficient of variance could be assumed identical. Statistical significance was evaluated using Student’s t test (n = number of independent experiments). P values below 0.05 were considered significant. (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01). 3. Results 3.1. PARP Inhibition Down-Regulates Pro-Survival Pathways in Glioblastoma The presence and involvement of Glioma Stem-like Cells (GSCs), also named Glioma Initiating Cells (GICs), in the initiation and propagation of GBM is broadly accepted [16], and the comprehension of their basic biology is critical to understand tumor relapse and failure of treatments. Currently, any anti-tumor approaches against GBM must necessarily target, one way or another, GSCs population. Consequently, to fully recognize the effect of PARPi against GBM we aimed to evaluate their action on GSCs. For this purpose, we used to patient-derive primary glioma stem-like cells TG1A and TG16 [15]. Due to the intrinsic resistance of GSCs to different treatments, we exposed cells to PARPi for one week and re-added PARPi during the first three days of the experiment to counteract the effect of surface ABC transporters [17]. We first analyzed the activity of the AKT/mTOR axis after PARP inhibition. Treatment with either PJ34 or Olaparib resulted in AKT/mTOR pathway down-regulation (Figure 1A). We observed decreased phosphorylation of PTEN (Ser380), which reflects PTEN activation and consequently constitutes a brake for AKT/mTOR signaling. In addition, we detected decreased phospho-AKT (Ser473) levels accompanied by diminished phospho-GSK3β (Ser9) and β-catenin levels, both downstream effectors of AKT. mTOR inactivation was further confirmed by decreased phosphorylation of the mTOR signaling target ribosomal protein P70S6k (Thr389) and increased LC3 II/I levels (Figure 1A). We observed decreased phosphorylation of PTEN (Ser380), which reflects PTEN activa- tion and consequently constitutes a brake for AKT/mTOR signaling. In addition, we de- tected decreased phospho-AKT (Ser473) levels accompanied by diminished phospho- Cancers 2022, 14, 726 GSK3β (Ser9) and β-catenin levels, both downstream effectors of AKT. mTOR inactivation 7 of 19 was further confirmed by decreased phosphorylation of the mTOR signaling target ribo- somal protein P70S6k (Thr389) and increased LC3 II/I levels (Figure 1A). Figure 1. PARP inhibition compromises glioma stem-like phenotype. (A) Immunoblots (IB) and Figure 1. PARP inhibition compromises glioma stem-like phenotype. (A) Immunoblots (IB) and graphical representation for the indicated proteins in TG1A and TG16 Glioma Initiating Cells graphical treated representation with PARP inhibitors for PJ34the indicatedfor or Olaparib proteins one week.in TG1A and (B) IB of TG16 SOX2, Gliomaand NANOG Initiating OCT4 inCells treated withand TG1A PARPTG16 inhibitors PJ34with cells treated or Olaparib for week. PJ34 for one one week. (B) IB of Full Western SOX2, Blot NANOG images and OCT4 can be found in in TG1A Figure S8. (C)cells and TG16 Immunofluorescence treated with PJ34 (IF)for of one SOX2 (red) Full week. and Western NESTIN (green) in TG1A Blot images cancells treatedin Figure S8. be found with PJ34 for one week; at least 10 fields of view per condition and replicate were analyzed and (C) Immunofluorescence (IF) of SOX2 (red) and NESTIN (green) in TG1A cells treated with PJ34 for quantified. Data are representative of 3 independent experiments. Values are mean ± SEM. * p < 0.05, ** one week; p < 0.01, *** pat