Cells and culture conditions
The Ovcar8, JHOS4, Uwb1.289, and PEO1 cell lines were provided by Benjamin Bitler at the University of Colorado. Ovcar8 cells were grown in RPMI 1640 medium (Gibco, 11875119) with 5% fetal bovine serum (FBS; BioWest, S1620) and 1% penicillin-streptomycin (Fischer Scientific, 15-140-122). JHOS4 cells were maintained in a 1:1 mix of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 11965092) and Ham’s F-12 (DMEM/HamF12), supplemented with 10% FBS, 0.1 mM non-essential amino acids (Gibco, 11140050), and 1% penicillin-streptomycin. Uwb1.289 cells were kept in a 1:1 ratio of RPMI 1640 (Gibco, 11875119) and MEGM (Sigma-Aldrich, C39110), enriched with 3% FBS and 1% penicillin-streptomycin. PEO1 cells were also cultured in RPMI 1640 (Gibco, 11875119) with 10% FBS, alongside 2 mM glutamine (Gibco, 25030081), 2 mM pyruvate (Gibco, 11360070), and 1% penicillin-streptomycin. The Ovcar8-empty vector (EV) and Ovcar8-CCNE1 cell lines were created through transduction with a cyclin E1-overexpression plasmid from Twist Bioscience (pTwist Lenti SFFV Puro) and cultured similarly to Ovcar8 cells but with 1 µg/ml puromycin (Gibco, A1113802). FT282-EV and FT282-CCNE1 cells were courtesy of Ronny Drapkin from the University of Pennsylvania, and these lines were generated by transduction with pCDH-EF1-FHC or pCDH-Flag-c-Myc, respectively. FT282 cells were grown in DMEM:F12 with 2% FBS and 1% penicillin-streptomycin in a low-oxygen environment (2% oxygen), while HepG2 cells, acquired from ATCC, were cultured in DMEM (Fischer Scientific, 12-430-054) with 10% FBS and 1% penicillin-streptomycin. The U2OS-mCherry-LacI-Fok1 line, given by Roger Greenburg from the University of Pennsylvania, was maintained in DMEM (Invitrogen, 11885084) with 10% FBS and 1% penicillin-streptomycin. To induce double-strand breaks (DSBs), cells were treated with Z-(4)-Hydroxytamoxifen (Millipore Sigma, H7904; 1 μM) and Shield-1 (TaKaRa, 632189; 1 μM) for 4 hours in medium containing charcoal-stripped FBS. HEK293FT cells, used for lentiviral packaging, were grown in DMEM (Corning, 10-013-CV) with 10% FBS, in accordance with ATCC guidelines. Kuramochi cells, provided by Ronald Buckanovich from the University of Pittsburgh, were cultured in RPMI 1640 (Gibco, 11875119) with 5% FBS and 1% penicillin-streptomycin. KPCA.B cells came from Robert Weinberg at the Whitehead Institute of Biomedical Research and were maintained in DMEM (Fischer Scientific, 12-430-054) with insulin-transferrin-selenium (Thermo Fisher Scientific; ITS-G, 41400045), EGF (10 µg/ml), 4% heat-inactivated FBS (Thermo Fisher Scientific; IFS, F4135), and 1% penicillin-streptomycin. All cells were incubated at 37 °C in a humidified environment with 5% CO2 and ambient oxygen, except for FT282 cells, which grew in 2% oxygen. The authenticity of cell lines hasn’t been tested, but all were checked for mycoplasma contamination monthly.
Plasmids, antibodies, inhibitors, treatments, and metabolites
All shRNAs were sourced from Sigma-Aldrich with the RNAi consortium numbers (TCRN) as follows: shIDH1 1, TRCN0000027253; shIDH1 2, TRCN0000027249; shTMLHE 1, TRCN0000064804; shTMLHE 2, TRCN0000064807; shBRCA1 1, TRCN0000010305; shBRCA1 2, TRCN0000039833; shCROT 1, TRCN0000036009; shCROT 2, TRCN0000036013; shCRAT 1, TRCN0000035495; shCRAT 2, TRCN0000035496; shACLY, TRCN0000078286. The pLKO.1 shGFP control was obtained from Addgene (30323). A wild-type IDH1 overexpression plasmid was created by Twist Bioscience (pTwist Lenti SFFV Puro).
Antibodies used in this research included: rabbit anti-IDH1, rabbit anti-TMLHE variants, mouse anti-cyclin E1, rabbit anti-MYC, mouse anti-vinculin, and multiple histone modifications, all sourced from respective suppliers with specific dilutions mentioned. For various detection needs, additional reagents were also employed, such as biotin and various HRP-conjugated secondary antibodies.
This study utilized several inhibitors, including GSK864, several PARP inhibitors, cisplatin, mildronate, and Shield-1, along with various metabolites like αKG, diethyl succinate, and others relevant to metabolic function.
Metabolite measurement
Metabolite assessments were done via liquid chromatography–high-resolution mass spectrometry, adjusting prior methods for polar metabolites and acyl-CoAs. For polar metabolomics targeting carnitines and TCA cycle metabolites, samples were quickly quenched in cold methanol:water solutions while spiking with isotope-labeled standards. After thorough processing—dumping the supernatant into a deep-well plate and drying—the samples were reconstituted before being assessed through chromatography linked to mass spectrometry. Numerous meticulous processes ensured accurate readings and quantifications of various metabolites involved in cellular function were obtained, while isotopologue enrichment from isotope tracing was calculated using specialized analysis tools.
Crystal violet assays
In all experimental setups, an equal number of cells was seeded into plates. Specific inhibitor studies were carried out with various doses over a span of five days, while glutamine-starvation and other treatments were administered, monitoring survival relative to controls through crystal violet staining and subsequent spectrophotometric analysis.
Western blotting
Cell lysates prepared in sample buffer underwent boiling and sonication before determining protein concentration and resolving on SDS-PAGE gels. Following transfer to membranes, primary antibodies targeted specific proteins overnight, and visualization was done after several washing steps with the appropriate secondary antibodies. Chemiluminescence aided in detecting signals on film after substrate exposure.
In vivo mouse experiments
Female mice, aged 8–12 weeks, were obtained from Jackson Laboratories and kept in regulated conditions. Ethical guidelines were closely followed, ensuring animal well-being. The number of animals used was decided based on previous studies, with randomization into treatment groups. For xenograft experiments, specific cells were injected intraperitoneally and various treatments were administered, with careful monitoring. After euthanization, tumour samples were collected for further analysis.
αKG-dependent dioxygenases CRISPR library construction
A pooled sgRNA library targeting 64 αKG-dependent dioxygenase genes was constructed. Essential design tools were employed for optimal targeting, and sequencing ensured robust representation. This library aimed to facilitate subsequent research and experimentation.
CRISPR drop-out screen
The designed knockout library was screened in various isogenic cell lines. Infections with pooled libraries were sequenced, and bioinformatics processed data to highlight negatively enriched genes for analysis, revealing insights into metabolic functions under study.
IHC
Immunohistochemistry was done to visualize specific proteins within tissues following established protocols. Tissues underwent fixation and blocking before primary antibody incubation, visualized using HRP/DAB and other reagents for detailed analysis. Scoring was performed using appropriate software tools.
Stable isotope labeling of essential nutrients
Stable isotope labeling was applied to analyze subcellular components, especially focusing on HepG2 cells and ensuring fractionation purity through meticulous procedures.
Mass spectrometry analysis of histone modifications
Histones were isolated and prepared for mass spectrometry analysis, ensuring a detailed understanding of protein modifications associated with chromatin dynamics, informing on cellular processes and regulatory mechanisms.
Immunofluorescence and BrdU labeling
Cells were prepared for immunofluorescence to visualize specific proteins and DNA replication activity, with careful protocol adherence to ensure accurate quantification of results. Variables were closely monitored, providing insight into treatment-induced responses.
DSB assay in Xenopus egg extracts
Double-strand break reactions in Xenopus egg extracts were performed, mixing various components to understand the repair processes under diverse conditions and inhibitions, ultimately addressing crucial aspects of DNA repair mechanisms.
Plasmid pull-down from Xenopus egg extracts
This step involved isolating DNA-bound proteins from egg extracts through magnetic bead affinity methods, allowing for subsequent analysis of protein interactions relative to DNA content, contributing to our understanding of repair mechanisms.
Metabolite analysis of Xenopus egg extracts
Samples were carefully processed and analyzed via mass spectrometry post-extraction, ensuring insights into the metabolic changes occurring within these extracts under varying conditions and treatments, enriching our understanding of cellular metabolism.
Oxygen consumption analysis of Xenopus egg extracts
Respiration assessments in egg extracts were conducted using dedicated metabolic analyzers, focusing on mitochondrial functions and responses to various treatments, while gathering data through intricately controlled experiments.
MitoTracker analysis of Xenopus egg extracts
MitoTracker staining contributed to the assessment of mitochondrial dynamics within extracts, followed by imaging to complement findings about mitochondrial presence and functionality, further enhancing the breadth of analysis.
RNA isolation, quantitative PCR, sequencing, and analysis
Total RNA isolation and quantification were completed, facilitating subsequent analyses like cDNA synthesis and quantitative PCR, ensuring reproducibility in gene expression assessments through rigorous controls and methods established for accurate quantification.
DNA fibre analysis
Specific protocols were employed to evaluate single-stranded DNA gaps, using dual labeling approaches to precisely monitor DNA replication activities across treatments, allowing for a deeper understanding of the mechanisms underpinning replication stress.
ChIP
Chromatin immunoprecipitation (ChIP) was conducted following detailed protocols to precipitate chromatin from treated cells, enabling the evaluation of specific DNA-protein interactions and epigenetic modifications in response to treatment stimuli and conditions.
TMAs
Two tissue microarrays were utilized, collected under appropriate ethical guidelines to explore various serous tumors, contributing valuable clinical insights related to patient outcomes and treatment responses, as documented in extensive previous studies.
Patient serum samples and outcomes data
Blood was collected from patients under approved protocols for metabolomics analysis, ensuring careful collection and processing to assess metabolite levels accurately while aligning with comprehensive clinical data for rigorous outcome evaluation.
Quantification and statistical analysis
Statistical analyses were performed using dedicated software packages, with provisions to exclude outliers ensuring data integrity. Comparisons of groups were performed using appropriate statistical methods, facilitating robust conclusions drawn from the presented research findings.
Reporting summary
Additional details regarding the study’s design are available through linked material summaries, offering further context and insights into the methodologies employed and findings reported.
