Reagents
TMZ (#T2577-25MG) was purchased from Sigma-Aldrich and resuspended in DMSO (ThermoFisher Scientific, #D/4125/PB08). PANK4 (#12055, 1:1000), MCL1 (#5453, 1:1000), caspase-3 (#9665, 1:1000), cleaved caspase-3 (#9664, 1:1000), GAPDH (#5174, 1:1000) as well as anti-rabbit (#7074P2, 1:4000) and anti-mouse (#7076P2, 1:4000) HRP-linked antibodies were purchased from Cell Signaling Technology. Alpha-tubulin (#A01410-100, 1:1000) was purchased from GenScript. The anti-Ki-67 antibody (#ab15580, 1:150) was purchased from Abcam and the anti-PANK4 antibody (#HPA027961, 1:300)was purchased from Sigma-Aldrich. The pCMV6-PANK4 overexpressing plasmid (#RC208116) and the pCMV6 empty vector (#PS100001) were purchased from Origene. The pLKO.1-puro PANK4 shRNA (#SH0111; targeting sequence: GGACTCTTCTGCTTGTCACTT) and the pLKO.1-puro non-targeting scrambled shRNA (#SHC016) were purchased from Sigma-Aldrich. The pMD2.G (#12259) and psPAX2 (#12260) packaging plasmids were obtained from Addgene. All other reagents, if not otherwise specified, were purchased from ThermoFisher Scientific.
Cell lines and culture conditions
The TMZ-resistant GBM cell lines (U87MGRes, T98GRes and LN229Res) and their parental counterparts (U87MGPar, T98GPar and LN229Par) used in this study were derived from the “Resistant Cancer Cell Line (RCCL) collection” (https://research.kent.ac.uk/industrial-biotechnology-centre/the-resistant-cancer-cell-line-rccl-collection/; University of Kent, UK) (21) and established by continuous exposure to increasing drug concentrations as described before (61). These cell lines as well as the control and stably PANK4-depleted T98GRes cells were cultured in Iscove's Modified Dulbecco's Medium(IMDM; ThermoFisher Scientific, #21980-032)supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, #F7524-500ML), 1% Penicillin-Streptomycin solution (Sigma-Aldrich, #P0781-100ML) and 4mM L-glutamine (Sigma-Aldrich, #G7513). The U251Res and U251Par cell lines were kindly provided by Dr Corinne Griguer (University of Iowa, Iowa City, USA) and were generated as previously described (22). These cells were grown in DMEM/F-12 medium (ThermoFisher Scientific, #11320-033) supplemented with 7% heat-inactivated FBS (Sigma-Aldrich, #F7524-500ML) and 1% Penicillin-Streptomycin solution (Sigma-Aldrich, #P0781-100ML). All drug-resistant cells mentioned above were maintained in culture in the presence of TMZ as previously described (https://research.kent.ac.uk/industrial-biotechnology-centre/the-resistant-cancer-cell-line-rccl-collection/) (21, 22). The U3027MG and U3031MG patient-derived GBM cell lines were obtained from the “Human Glioblastoma Cell Culture (HGCC) biobank” (https://www.hgcc.se; Uppsala University, Uppsala, Sweden) (23). These cell lines were cultured in Neurobasal (ThermoFisher Scientific, # 21103-049) and DMEM/F-12 GlutaMAX (ThermoFisher Scientific, #31331-028) medium (1:1), supplemented with B-27 (ThermoFisher Scientific, #12587010), N2 (ThermoFisher Scientific, #17502048), EGF (PeproTech, #AF-100-15-100UG), FGF (PeproTech, #100-18B-100UG), 1% Penicillin-Streptomycin solution (Sigma-Aldrich, #P0781-100ML) and grown on laminin-coated Corning Primaria Cell Culture plates (Corning, #353846 & 353872), as previously described (23). HEK-293T cells were purchased from ATCC and maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, #D6046-500ML) supplemented with 10% FBS (Sigma-Aldrich, #F7524-500ML) and 1% Penicillin-Streptomycin solution (Sigma-Aldrich, #P0781-100ML). All cell lines were incubated at 37°C with 5% CO2 and regularly subjected to mycoplasma testing.
Kinome-wide RNAi screen
The “Silencer Select Human Kinase siRNA Library V4” (ThermoFisher Scientific, #4397918), targeting 709 human kinase and kinase-related genes was used. U87MGRes cells (3000/well) were reverse transfected in 96-well plates with either a pool of 3 siRNAs targeting each gene of the library (25nM/siRNA) or non-targeting negative control siRNAs (ThermoFisher Scientific, #4390844). The Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, #L3000015) was used according to the manufacturer’s instructions. 24 hours after transfection, cells were treated with either DMSO or a sublethal dose of TMZ (IC20), and incubated for 96 hours. Cell proliferation was determined using the CyQUANT Direct assay (ThermoFisher Scientific, #C35011), following the manufacturer’s instructions. Two independent primary screens (biological repeats) were performed. Data were background corrected and normalised to their respective control (siCTRL DMSO). Normalised values were used to calculate z-scores as previously described (62). Gene candidates displaying an effect of >20% on cell proliferation alone were excluded from further analysis.
PANK4 silencing and overexpression
Cells were reverse transfected with a pool of 3 siRNAs (25nM each) using the Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, #L3000015), according to the manufacturer’s instructions. Non-targeting negative control siRNA (ThermoFisher Scientific, #4390843) and PANK4 siRNAs (ThermoFisher Scientific, #4392420; IDs: s224353, s30501, s30502) were used. Briefly, a mix of siRNAs, Opti-MEM medium (ThermoFisher Scientific, #31985062) and Lipofectamine 3000 was prepared following the manufacturer’s instructions. After formation of the transfection complexes, the transfection mix was spotted into the wells and cells were subsequently seeded. For PANK4 overexpression, cells were seeded into wells and transfected with the pCMV6-PANK4 overexpressing plasmid (Origene, #RC208116) or the pCMV6 empty vector (Origene, #PS100001), using the Fugene HD transfection reagent (Promega, #E2311), according to the manufacturer’s instructions. To achieve long-term PANK4 knockdown, lentiviral-mediated shRNA transfection was performed. Briefly, HEK-293T cells were transiently co-transfected with pLKO.1-puro PANK4 shRNA (targeting sequence: GGACTCTTCTGCTTGTCACTT) (Sigma-Aldrich, #SH0111) or pLKO.1-puro non-targeting scrambled shRNA (Sigma-Aldrich, #SHC016), and pMD2.G, psPAX2 packaging plasmids (Addgene, #12259 and #12260, respectively). Transient transfection was performed using Lipofectamine 3000 (ThermoFisher Scientific, #L3000015), as described above. Non-replicating viral particles were harvested and concentrated using PEG-it (5x) (System Biosciences, #LV810A-1) overnight at 4°C. The concentrated virus was dispensed to T98GRes cells, and TransDux (200x) (System Biosciences, #LV850A-1) was added to increase transduction efficiency. 72-hours post transduction, cells were incubated in the presence of 0.8 μg/mL puromycin (Gibco, #A1113803) to ensure effective positive selection. For all the experiments performed, PANK4 silencing and overexpression were confirmed by western blotting, as indicated in the respective figure legends.
Cell proliferation assays
Briefly, cells were reverse transfected with siCTRL or siPANK4, as described above. After 24 hours, cells were treated with DMSO or TMZ, as specified in the figures and their respective legends. Cell proliferation was evaluated by the CyQUANT Direct assay (ThermoFisher Scientific, #C35011), following the manufacturer’s instructions. Fluorescence intensity was measured on a SpectraMax i3x microplate reader (Molecular Devices). Alternatively, the crystal violet assay was used. Following fixation of cells with 4% paraformaldehyde solution (Santa Cruz Biotechnology, #sc-281692) in 1x PBS, and staining with 0.5% crystal violet (ThermoFisher Scientific, #B21932.14), absorbance was measured using the GloMax-Multi detection system (Promega).
Clonogenic survival assays
Clonogenic survival assays were performed as previously described (63, 64). Briefly, cells were transfected with either siCTRL or siPANK4 and subsequently seeded at predetermined densities. After 24 hours, cells were treated with DMSO or TMZ, as indicated. When colony size reached more than 50 cells per colony, cells were fixed with 4% paraformaldehyde solution (Santa Cruz Biotechnology, #sc-281692) in 1x PBS, followed by staining with 0.5% crystal violet (ThermoFisher Scientific, #B21932.14). The surviving fraction was determined using the plating efficiencies of the respective controls as reference.
Cell death and apoptosis
The assay was performed as previously described (65, 66). Cells were transfected with either siCTRL or siPANK4 as described above and subjected to drug treatments as specified in the corresponding figures and figure legends. After 96 hours, cells were stained using the Muse Annexin V Dead Cell Kit according to the manufacturer’s instructions (Luminex, #MCH100105). Cells were then analysed using the Muse Cell Analyzer (Millipore).
ROS detection
The assay was performed using the DCFDA / H2DCFDA - Cellular ROS Assay Kit (Abcam, #ab113851). Briefly, cells were transfected with either siCTRL or siPANK4 as described above and treated with a sublethal concentration of TMZ, as previously determined. After 96 hours cells were stained with DCFDA solution according to the manufacturer’s instructions and fluorescence was measured using the BMG Labtech CLARIOstar Microplate Reader at Ex/Em = 485/535 nm.
RNA extraction and RT-qPCR
Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen, #12183018A) following the manufacturer’s instructions (62, 67). All RNA samples were subjected to DNase treatment. The concentration and purity of RNA was determined using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). Complementary DNA (cDNA) synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s instructions (ThermoFisher Scientific #4368814). Quantitative real-time PCR was carried out using the SYBR green gene expression assay (Applied Biosystems, #4367659). Samples were run on a StepOne thermal cycler (Applied Biosystems) and analyzed with the SDS 1.9 software (Applied Biosystems) (n = 3 biological replicates and n = 3 technical replicates). GAPDH was used as an internal control. Primer sequences are listed in Supplementary Table S4.
Western Blotting
Western Blotting was performed as previously described (62, 65, 68). Briefly, cells were lysed in RIPA buffer (Sigma-Aldrich, #R0278) supplemented with protease and phosphatase inhibitors (Roche, #11697498001 and #4906845001, respectively). Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific, #23227). Proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose transfer membrane (ThermoFisher Scientific, #IB23001) using the iBlot 2 dry blotting system (ThermoFisher Scientific, #IB21001). Following blocking of membranes in TBS containing 0.1% (v/v) Tween 20 and 5% (w/v) non-fat milk for 1 hour, incubation with primary antibodies was performed overnight at 4°C. Anti-mouse (#7076P2, 1:4000) and anti-rabbit (#7074P2, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies were used (Cell Signaling Technology) and binding was detected using the SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific, #34577). Emission was captured using the UVP ChemStudio Imaging Systems (Analityk jena). Densitometric analysis of western blots was performed using the ImageJ software.
Animal experiments
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (stock no: 005557; NSG) (69, 70) mice were purchased from the Jax repository (Bar Harbor, ME, USA) and bred in-house in individually ventilated cages under specific pathogen-free conditions. All animal studies were performed in full compliance with FELASA (Federation of Laboratory Animal Science Associations) recommendations in the Animal House Facility of the Biomedical Research Foundation of the Academy of Athens (BRFAA, Greece). All procedures for the care and treatment of the animals were approved by the Institutional Committee on Ethics of Animal Experiments. The license for the animal handling protocol for this project is: 1385947/27-12-2022. To produce the ectopic tumour xenograft model, 10x106 T98GRes cells in 10% matrigel (Corning) were subcutaneously injected in the right and left flank of mice. After establishing palpable tumours (~30 mm3), mice were randomly assigned to groups. Tumour volume was measured twice a week with caliper and calculated as V=axb2/2, “a” being the largest diameter, “b” the smallest. Tumour specimens up to 50 mm3 from non-treated mice were transplanted subcutaneously in new NSG mice under anaesthesia in order to produce mirror images of the primary tumour.
In vivo transfections
In Vivo Ready non-targeting negative control siRNA (ThermoFisher Scientific, #4404020) and PANK4 siRNAs (ThermoFisher Scientific, #4404010; HPLC-IVR IDs: s224353, s30501) were used in this study. siRNAs were encapsulated using Invivofectamine 3.0 reagent (ThermoFisher Scientific, #IVF3001), a cationic liposome-based formulation, according to the manufacturer’s instructions. Animals were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (12 mg/kg). The injection site was swabbed with 70% ethanol prior to injection. siRNA:Invivofectamine 3.0 complexes were injected intratumorally at a concentration of 6μg/tumour at day 1 and 5. The siRNA concentrations used were based on previously published studies (71, 72) and knockdown efficiency was assessed by western blot.
In vivo TMZ treatments
TMZ (MedChemExpress, #HY-17364/CS-0943) was administered intraperitoneally (IP) at a concentration of 1.5mg/kg on day 2 and every other day for the following 12 days. The concentration of TMZ used was based on the dose response curve generated after IP administration of TMZ at concentrations of 0 (vehicle), 1, 3 and 10 mg/kg. TMZ was dissolved in 5% DMSO / 5% solutol (Sigma-Aldrich) in PBS. At the end of each treatment, mice were euthanized in accordance with standard protocols. A small part of the freshly dissected tumour was fresh frozen for molecular analysis and the rest was fixed in a 10% formalin solution.
Immunoblotting of xenograft tumour tissues
Tumours were lysed in 8M UREA/50mM TEAB with protease inhibitors (Calbiochem) using mild sonication on ice followed by homogenization with a 26 G syringe. Total protein concentration was determined with the Bio-Rad protein assay. Lysates were subjected to SDS-PAGE followed by immunoblot analysis. The primary antibodies used were PANK4 (Cell Signaling Technology, #12055, 1:1000) and GAPDH (Cell Signaling Technology, #5174, 1:1000). The anti-rabbit HRP-conjugated secondary antibody (Cell Signaling Technology) was used at a 1:4000 dilution. Densitometric analysis of western blots was performed using the ImageJ software.
Immunohistochemistry of xenograft tumour tissues
Immunohistochemistry (IHC) was performed according to standard procedures (65, 73). Rabbit anti-Ki-67 antibody (Abcam, #ab15580, 1:150) was used for overnight incubation at 4°C in humidified chambers. The anti-rabbit secondary antibody (Cell Signaling Technology, 1:400) was HRP-conjugated and was detected with DAB (Vector Laboratories).
Immunohistochemical analysis of clinical specimens
Immunohistochemistry staining for PANK4 and hematoxylin and eosin (H&E) staining were performed as previously described (65, 74, 75). The anti-PANK4 antibody (Sigma-Aldrich, #HPA027961) was used at a 1:300 dilution. The study was approved by the ethics committee of Renmin Hospital of Wuhan University (March 15th, 2022). The approval number is: WDRY2022-K064. Clinical GBM specimens (n= 79 GBM, IDH-wildtype patients) were collected in the cancer centre at Renmin Hospital of Wuhan University, and processed at the hospital research laboratories after de-identification of the samples. PANK4 immunoreactivity was assessed semi-quantitatively on a 0-2 scale, with 0=negative, 1+=mild, 2+=moderate staining (https://www.proteinatlas.org). Percentages of 0, 1+, 2+ cells were recorded. H-scores were calculated as follows: % of (1+) cells + 2x[% of (2+) cells]. All cases were scored without knowledge of the clinicopathological data. Patients’ information is provided in Supplementary Table S5.
Sample preparation for the TMT-based proteomic experiment
Briefly, T98GRes cells were reverse transfected with either siCTRL or siPANK4. After 24 hours, cells were treated with either DMSO or TMZ. Following 96h, cells were washed (×3 in PBS) and pelleted. Cell pellets were lysed separately with freshly prepared lysis buffer containing 50 mM HEPES, pH 8.0, 2% SDS, 1 mM PMSF, supplemented with phosphatase and protease inhibitor cocktail (Sigma-Aldrich). Samples were thawed at room temperature (RT) for 20 minutes (min) before heating to 99°C for 5 min. After cooling to RT, DNA was sheared by sonication. Cell debris was removed by centrifugation at 20,000×g for 15 min at 20 °C. Protein concentration was determined using the BCA protein assay kit (Applichem GmbH, Darmstadt, Germany).
Protein digestion and offline fractionation
FASP digestion was performed according to the procedure described by Wisniewski et al. (76). TMT labelling was performed with TMTpro 16-plex reagents (Lot#WA314599) according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). Offline Fractionation of peptides into 12 fractions was performed via RP-HPLC at high pH as described by Gilar et al. (77). After solvent removal in a vacuum concentrator, samples were reconstituted in 0.1% TFA for LC-MS/MS analysis.
Liquid chromatography-mass spectrometry analysis
Mass spectrometry was performed on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) coupled to a Dionex Ultimate 3000RSLC nano system (ThermoFisher Scientific, San Jose, CA, USA) via a nanoflex source. Tryptic peptides were separated on a 50 cm, 75 µm i.d. analytical column (self-packed with ReproSil-Pur 120 C18-AQ, 3 μm, Dr. Maisch, Ammerbuch Entringen, Germany) and a 90 min acetonitrile gradient (5-90%) at a flow rate of 230 nL/min. Analysis was performed in a data-dependent acquisition mode using a TopN dependent scan method with a cycle time of up to 20 scans for precursor ion selection. MS1 data were acquired in the orbitrap at a resolution of 120,000 (at 200 m/z). Automatic gain control (AGC) was set to a target of 2.5E4 and a maximum injection time of 86 ms. MS2 spectra were acquired in the orbitrap (FT) using a quadrupole isolation window of 0.5 Da and higher-energy collision induced dissociation (HCD) at a normalised collision energy (NCE) of 34%. The resolution was 50,000 (at 200 m/z) with a fixed first mass of 110 m/z, an AGC target of 5E4, and a maximum injection time of 110 ms. Dynamic exclusion for selected ions was 90 s. A single lock mass at m/z 445.120024 was employed (78).
Proteomic data analysis
Protein identification and comparative quantification of TMTpro 16-plex labelled proteins from MS and MS/MS raw data were performed using the MaxQuant software suit (version 1.6.12.0) (Max Planck Institute of Biochemistry, Planegg, Germany) with the implemented peptide search engine Andromeda (79) against a reference proteome database of Homo sapiens (Human/Uniprot proteome ID: UP000005640, Version 7 March 2021). Statistical analysis was performed using the Perseus software (version 1.6.14.0). Unpaired t-test was employed to determine the significance of the observed differences. Differences were considered statistically significant at p < 0.05 (95% confidence interval, * p < 0.05; ** p < 0.01; ***p < 0.001; ****p < 0.0001).
Gene Set Enrichment Analysis
Functional annotation and Gene Set Enrichment Analysis (GSEA) were performed using clusterProfiler (v 4.4.4) (80). Enrichment representations were plotted with the dotplot, cnetplot and gseaplot2 functions. Gene Ontology (GO) terms were considered significantly enriched in over-representation analysis when Benjamini-Hochberg adjusted p values were below < 0.05(*). For all analyses and plots, R (v 4.2.0) and Bioconductor (v 3.15) were used.
Statistical analysis
Graphics and statistical analysis were performed using the GraphPad Prism 9 software. Each experiment was conducted at least three times and results are expressed as mean ± SEM, unless otherwise specified. Statistical significance was evaluated using unpaired Student’s t-test when two groups were compared. More than two groups were compared using two-way ANOVA, unless otherwise specified. For assessment of biological reproducibility of the two primary screens, correlation was determined using the Pearson correlation coefficient. The Cooperativity Index (CI) was calculated as previously described (81). CI values < 1 indicate a synergistic effect, when CI values = 1 the effect is additive, and CI values > 1 indicate an antagonistic effect. The statistical significance of the Kaplan-Meier survival plots was evaluated by log-rank analysis. The statistical details and p-values of each experiment are indicated in the corresponding figures and figure legends [p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and “ns” indicates not significant (p>0.05)].