Abstract
Cancer cells depend on nicotinamide adenine dinucleotide phosphate (NADPH) to combat oxidative stress and support reductive biosynthesis. One major NADPH production route is the oxidative pentose phosphate pathway (committed step: glucose-6-phosphate dehydrogenase, G6PD). Alternatives exist and can compensate in some tumors. Here, using genetically-engineered lung cancer mouse models, we show that G6PD ablation significantly suppresses KrasG12D/+;Lkb1-/- (KL) but not KrasG12D/+;P53-/- (KP) lung tumorigenesis. In vivo isotope tracing and metabolomics reveal that G6PD ablation significantly impairs NADPH generation, redox balance, and de novo lipogenesis in KL but not KP lung tumors. Mechanistically, in KL tumors, G6PD ablation activates p53, suppressing tumor growth. As tumors progress, G6PD-deficient KL tumors increase an alternative NADPH source from serine-driven one carbon metabolism, rendering associated tumor-derived cell lines sensitive to serine/glycine depletion. Thus, oncogenic driver mutations determine lung cancer dependence on G6PD, whose targeting is a potential therapeutic strategy for tumors harboring KRAS and LKB1 co-mutations.
Introduction
Tumor cells use nicotinamide adenine dinucleotide phosphate (NADPH) for redox homeostasis and reductive synthesis reactions to sustain their survival and growth1,2. Consumption and production of NADPH are compartmentalized in the mitochondria and cytosol3,4. Cytosolic NADPH is recycled through reduction of NADP+ via the oxidative pentose phosphate pathway (oxPPP) enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), malic enzyme 1 (ME1), isocitrate dehydrogenase 1 (IDH1), and the one-carbon metabolism (folate) enzymes methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1)3,5. The functional importance of different metabolic enzymes involved in cytosolic NADPH homeostasis are not fully understood in cancer in vivo. Better understanding may open therapeutic opportunities.
Pentose phosphate pathway (PPP) flux is the major alternative glucose catabolic pathway to glycolysis. Dysregulation of proteins in this pathway is associated with cancer development, with the master antioxidant transcription factor NRF2 frequently upregulated in human cancers and driving oxPPP gene expression6,7. The oxPPP pathway is essential for mammals, with knockout of the committed enzyme G6PD embryonic lethal. G6PD deficiency is the most common human enzyme defect because it protects against malaria8. G6PD is upregulated in many cancers, and G6PD deficiency is associated with lower cancer risk and mortality for some cancers9,10,11,12, suggesting that cancer cells may depend on G6PD for survival or proliferation. Loss of p53 upregulates G6PD activity and promotes NADPH-driven biosynthetic processes including de novo lipogenesis13. In mouse models, G6PD deficiency significantly reduces melanoma metastasis14. Recently, we employed modern genetic tools to evaluate the role of G6PD in lung, breast, and colon cancer driven by oncogenic KRAS. We found that, in the studied KRAS mutant tumor models, G6PD, at most modestly promotes disease progression and is not strictly essential for solid tumorigenesis or metastatic spread15. In particular, G6PD is not required for KrasG12D/+;P53-/- (KP) lung tumorigenesis15. However, KP tumors further lacking KEAP1, a tumor suppressor whose loss elevates NRF2, show greater dependence on G6PD7. Thus, G6PD is likely to be particularly important in the context of specific tumor types or driver mutations.
Oncogenic KRAS mutation in non-small cell lung cancer (NSCLC) patients confer a poor prognosis and a high risk of cancer recurrence. LKB1 signaling negatively regulates tumor growth through direct phosphorylation and activation of the central metabolic sensor, AMP-activated protein kinase (AMPK), which governs glucose and lipid metabolism in response to alterations in nutrients and intracellular energy levels16,17,18,19. Loss of LKB1 reprograms cancer cell metabolism to efficiently generate energy and biomass components for uncontrolled proliferation and dissemination. Meanwhile, such alterations in turn cause tumor cells to have less plasticity in response to metabolic stress, creating a metabolic vulnerability20,21. p53 and LKB1 co-mutations represent two different subgroups of KRAS-driven NSCLC, with distinct biological properties, metabolic vulnerabilities, and responses to standard therapies22,23,24,25. We have revealed that G6PD is not essential for KP lung tumorigenesis15. Given the distinct features of KL and KP NSCLC, we here further investigated the dispensability of G6PD in KrasG12D/+;Lkb1-/- (KL) lung tumorigenesis. In contrast to KP lung cancers, G6PD showed greater functional importance in KL lung cancers. G6PD deficiency impaired KL lung tumorigenesis, showing increased oxidative stress, and p53 activation-mediated apoptosis and cell cycle arrest. G6PD loss also impaired de novo lipogenesis, whereas fat supplementation rescued the growth of G6PD-deficient KL lung tumors. Moreover, G6PD loss in KL tumors reprogrammed the NADPH generating metabolic pathway by increasing serine uptake to sustain one carbon metabolism-mediated cytosolic NADPH generation. G6PD-deficient KL lung tumor-derived cell lines (TDCLs) were sensitive to serine/glycine depletion, which was associated with increased reactive oxygen species (ROS). Thus, the dependence of G6PD-mediated oxPPP on KRAS-driven lung tumorigenesis is determined by specific oncogenic driver mutations. This also underscores the need for personalized therapies tailored to different subgroups of KRAS-driven lung cancers, especially when considering the application of G6PD inhibitors in cancer treatment.
Results
G6PD expression level correlates with the survival of lung cancer patients carrying KRAS and LKB1 co-mutations
Tumors exhibit an enormous demand for NADPH due to uncontrolled proliferation2. Mice with same genotype or bearing same TDCLs were randomly assigned to different treatment groups. Sample sizes were chosen based on the power calculation. The investigators were blinded to the group allocation during experiments and when assessing outcomes.
For genetically engineered mouse models generation, G6pdflox/flox;KrasLSL-G12D;Lkb1flox/flox mice were generated by cross-breeding G6pdflox/flox mice with G6pd+/+;KrasLSL-G12D;Lkb1flox/flox mice, G6pdflox/flox;KrasLSL-G12D;P53flox/flox mice were generated by cross-breeding G6pdflox/flox mice with G6pd+/+;KrasLSL-G12D;P53flox/flox mice, G6pd+/+;KrasLSL-G12D;P53flox/flox;Lkb1flox/flox mice were generated by cross-breeding G6pd+/+;KrasLSL-G12D;Lkb1flox/flox mice with G6pd+/+;KrasLSL-G12D;P53flox/flox mice, and G6pdflox/flox;KrasLSL-G12D;P53flox/flox;Lkb1flox/flox mice were generated by cross-breeding G6pdflox/flox mice with G6pd+/+;KrasLSL-G12D;P53flox/flox;Lkb1flox/flox mice. At 6-8 weeks of age, G6pd+/+;KrasG12D;Lkb1-/- (G6pdWT;KL) lung cancer in G6pd+/+;KrasLSL-G12D;Lkb1flox/flox mice, G6pd-/-;KrasG12D;Lkb1-/- (G6pdKO;KL) lung tumor in G6pdflox/flox;KrasLSL-G12D;Lkb1flox/flox mice, G6pd+/+;KrasG12D;P53-/- (G6pdWT;KP) lung tumor in G6pd+/+;KrasLSL-G12D;P53flox/flox mice, G6pd-/-;KrasG12D;P53-/- (G6pdKO;KP) lung tumor in G6pdflox/flox;KrasLSL-G12D;P53flox/flox mice, G6pd+/+;KrasG12D;P53-/-;Lkb1-/- (G6pdWT;KPL) lung tumor in G6pd+/+;KrasLSL-G12D;P53flox/flox;Lkb1flox/flox mice, and G6pd-/-;KrasG12D;P53-/-;Lkb1-/- (G6pdKO;KPL) lung tumor in G6pdflox/flox;KrasLSL-G12D;P53flox/flox;Lkb1flox/flox mice were induced by intranasally infection with Lenti-Cre (University of lowa Viral Vector Core, lowa-28) at 5×106 plaque-forming units (pfu) per mouse, following the methodology employed in our previous investigation58.
Mice were fed with a regular chow diet (LabDiet, Cata#5058). For high-fat diet treatment, on the same day that G6pdWT;KL and G6pdKO;KL lung tumors were induced by intranasal infection with Lenti-Cre, half of mice were fed with the high-fat diet (Bio-Serv Mouse Diet, Cata#F3282) and the other half were fed with the control diet (normal diet) (Bio-Serv Mouse Diet, Cata#S4207). Following a 7-week and 11-week treatment period, the mice were euthanized, and lung tissues were collected for H&E staining, tumor number/burden quantification and IHC. In addition, after 7 weeks treatment of HFD, mice were euthanized, serum and lung tumors were collected for lipidomics analysis.
For TDCLs generation, G6pdWT;KL or G6pdKO;KL TDCLs were made from G6pdWT;KL or G6pdKO;KL lung tumors at 12 weeks post-tumor induction, respectively. TDCLs were cultured in complete RPMI medium (RPMI medium (Gibco, Cata#11875-093) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 0.075% sodium bicarbonate) at 37 °C with 5% CO2. Regular testing using the Universal mycoplasma detection kit (ATCC, Cata#30-1012k) confirmed the absence of mycoplasma contamination in the cell lines.
For allograft tumor induction and high-dose Vit C treatment, G6pdWT;KL or G6pdKO;KL TDCLs were subcutaneously injected into the right and left flank of male C57BL/6 mice with 1 × 106 cells/injection at 6–8 weeks of age. Then the mice bearing allograft tumors were administered Vit C at a dosage of 4 g/kg intraperitoneally (i.p.) daily for 2 weeks. Tumor size was measured using a caliper every other day during the 2-week treatment period. The tumor sizes were not exceeded the maximal tumor size (2000 mm3) permitted by the Institutional Animal Care and Use Committee of Rutgers University. After 2 weeks of treatment, the mice were euthanized, and tumors were collected and weighed for further analysis.
Histology and IHC
Mice were euthanized via cervical dislocation at the designated time points following Lenti-Cre infection. Lung tissues were collected and placed in formaldehyde (Fisher Scientific, Cata#SF93-4) for a period of 12–24 hours. Afterward, the tissues were transferred to 70% ethanol solution and stored at 4 °C. Paraffin-embedded tissue sections were prepared using the methodology described in a previous study for histology and IHC60. For histology, the tissue sections were first deparaffinized using xylene and then rehydrated through a graded series of ethanol and water. Subsequently, the sections were stained with hematoxylin (Sigma, Cata#GHS216) and eosin (Sigma, Cata#1170811000), commonly referred to as H&E staining. Following the staining procedure, the sections were dehydrated and mounted onto slides using Cytoseal 60 mounting medium (Thermo Scientific, Cata#23-244256) for further microscopic examination. For IHC, the tissue sections were deparaffinized and rehydrated following the protocol for H&E staining. The sections were then heated at 95 °C in citrate buffer (Diagnostic Biosystems, Cata#K035) for 20 minutes. Subsequently, the sections were incubated with 3% hydrogen peroxide (Walgreens, Cata#715333) for 10 minutes to block endogenous peroxidase activity, followed by blocking in 10% goat serum (Fisher Scientific, Cata#16210064) for 1 hour at room temperature. The sections were then incubated overnight at 4 °C with the anti-G6PD (Abcam, Cata#AB993, Clone#Polyclonal, Lot#GR274589-46, 1:2000 dilution, https://www.abcam.com/products/primary-antibodies/glucose-6-phosphate-dehydrogenase-antibody-ab993.html), anti-Ki67 (Abcam, Cata#ab15580, Clone#Polyclonal, Lot#GR3375556-1, 1:2000 dilution, https://www.abcam.com/products/primary-antibodies/ki67-antibody-ab15580.html), anti-pS6 (Cell Signaling, Cata#4858 S, Clone#D57.2.2E, Lot#21, 1:500 dilution, https://www.cellsignal.com/products/primary-antibodies/phospho-s6-ribosomal-protein-ser235-236-d57-2-2e-xp-rabbit-mab/4858), anti-P-p42/44 MAPK (pERK) (Cell Signaling, Cata#9101 S, Clone#NA, Lot#26, 1:500 dilution, https://www.cellsignal.com/products/primary-antibodies/phospho-p44-42-mapk-erk1-2-thr202-tyr204-antibody/9101), anti-cleaved caspase3 (Cell Signaling, Cata#9661 S, Clone#NA, Lot#47, 1:150 dilution, https://www.cellsignal.com/products/primary-antibodies/cleaved-caspase-3-asp175-antibody/9661), anti-p53 (Leica, Cata#NCL-L-p53-CM5p, Clone#POLYCLONAL, Lot#6065476, 1:2000 dilution, https://shop.leicabiosystems.com/us/ihc-ish/ihc-primary-antibodies/pid-p53-protein-cm5), anti-p21 (Santa Cruz Biotech, Cata#sc-6246, Clone#F-5, Lot#I1020, 1:1000 dilution, https://www.scbt.com/p/p21-antibody-f-5), anti-8-oxo-dG (R&D systems, Cata#4354-MC-050, Clone#15A3, Lot#P323432, 1:1000 dilution, https://www.rndsystems.com/products/8-oxo-dg-antibody-15a3_4354-mc-050), anti-γ-H2AX (Cell Signaling, Cata#9718, Clone#20E3, Lot#21, 1:1000 dilution, https://www.cellsignal.com/products/primary-antibodies/phospho-histone-h2a-x-ser139-20e3-rabbit-mab/9718), anti-NQO1 (Invitrogen, Cata#PA5-21290, Clone#AB_11153144, Lot#YL4152869, 1:1000 dilution, https://www.thermofisher.com/antibody/product/NQO1-Antibody-Polyclonal/PA5-21290), anti-NRF2 (Invitrogen, Cata#PA5-27882, Clone#AB_2545358, Lot#YF3956921A, 1:1000 dilution, https://www.thermofisher.com/antibody/product/Nrf2-Antibody-Polyclonal/PA5-27882), anti-pACC (S79) (Cell Signaling, Cata#3661, Clone#NA, Lot#10, 1:1000 dilution, https://www.cellsignal.com/products/primary-antibodies/phospho-acetyl-coa-carboxylase-ser79-antibody/3661), or anti-pAMPK (Cell Signaling, Cata#50081, Clone#D4D6D, Lot#6, 1:1000 dilution, https://www.cellsignal.com/products/primary-antibodies/phospho-ampka-thr172-d4d6d-rabbit-mab/50081) antibodies. The following day, the sections were incubated with biotin-conjugated secondary antibody for 30 minutes (Vector, Cata#BA-1000), horseradish peroxidase streptavidin for 10 minutes (Vector Laboratories, Cata#SA-5704-100) and developed by DAB (Agilent/Dako, Cata#K346811-2,) followed by hematoxylin staining. Sections were then dehydrated, mounted in Cytoseal 60 mounting medium for further analysis.
For the quantification of IHC for Ki67, pS6, pERK, cleaved caspase3, p53, p21, 8-oxo-dG, γ-H2AX, NQO1, NRF2, pACC, and pAMPK, more than 10 representative images from each group were obtained using a Nikon Eclipse 80i microscope and scored using the ImageJ (Version 1.52a) software.
Tumor number/burden quantification
H&E-stained lung specimens were imaged using an Olympus VS120 whole-slide scanner (Olympus Corporation of the Americas) at 20 × magnification at the Rutgers Cancer Institute Biomedical Informatics shared resource. Image analysis was conducted using a custom-developed protocol on the Visiopharm image analysis platform (Visiopharm A/S). The protocol facilitated the identification of tissue area and the computation of tumor burden based on semiautomatically detected tumors. Low-resolution image maps, extracted from the whole-slide images, were utilized to generate tumor masks and whole-tissue masks. These masks were generated for each slide, enabling the segmentation of tumor burden ratios.
D2O, [U-13C6]-glucose and [2,2,3-2H]-serine infusion
Before the infusion experiments, venous catheters were surgically implanted into the jugular veins of tumor-bearing mice, with a 3 to 4 days interval. The infusions were conducted on conscious, freely moving mice. For the infusion of D2O (Cambridge Isotope, Cata#DLM-4-50) and [2,2,3-2H]-serine (Cambridge Isotope, Cata#DLM-582-0.5), mice were fed continuously throughout the infusion period (8:00 PM - 8:00 AM). For the infusion of [U-13C6]-glucose (Cambridge Isotope, Cata#CLM-1396-1), food was removed from the mice at approximately 9:00 AM, and infusion was commenced between 2:30 PM-5:00 PM. Mice were infused with D2O saline (0.9% NaCl) at a rate of 0.1 mL/g/minute, or [2,2,3-2H]-serine (200 mmol/L) at a rate of 0.2 mL/g/minute for 12 hours overnight, or [U-13C6]-glucose (200 mmol/L) at a rate of 0.1 mL/g/minute for 2.5 hours before being euthanized for rapid lung tumors collection. Blood samples for serum analysis were collected from the mice’s cheeks into 1.5 mL Eppendorf Tubes (Flex-Tubes, Cata#20901-551). Lung tumors were swiftly dissected and frozen using a liquid-nitrogen cold clamp to halt metabolic activity and then stored at −80 °C until further metabolites extraction.
cBioPortal data processing
The overall survival analysis comparing the low and high expression levels of G6PD, IDH1, ME1, MTHFD1, and NFE2L2 (NRF2) in lung cancer patients was conducted using the cBioPortal datasets61 (https://www.cbioportal.org/, accessed on December 09, 2023). Data from 28 studies (as listed in Supplementary Table 1) available in the cBioPortal datasets were utilized for the present analysis.
For the overall survival analysis, the “gene specific” option was chosen, adding gene names including G6PD, IDH1, ME1, MTHFD1, and NRF2. The mRNA data type selected was “mRNA expression z-scores relative to all samples (log RNA Seq V2 RSEM)”. A chart was then generated to compare the two groups based on the median expression of the indicated gene’s mRNA. Subsequently, the overall survival was compared between the mRNA low expression group and the mRNA high expression group of the indicated gene.
For the analysis of mRNA expression levels of G6PD, IDH1, ME1, and MTHFD1, data were obtained from a 586 samples study on lung cancer (Lung Adenocarcinoma, TCGA, Firehose Legacy) available in the cBioPortal datasets (https://www.cbioportal.org/, accessed on December 09, 2023). Sample information for those with KRAS/TP53 co-mutations and KRAS/LKB1 co-mutations was extracted from the study, and the mRNA expression levels of the indicated genes were compared between these two groups. The mRNA expression levels were represented as mRNA expression z-scores relative to all samples (log RNA Seq V2 RSEM).
mRNA-seq and GSEA analysis
G6pdWT;KL and G6pdKO;KL lung tumors were induced by intranasal infection with Lenti-Cre. At 12 weeks post-tumor induction, mice were euthanized by cervical dislocation. The lung tumors were rapidly dissected and snap-frozen in liquid nitrogen. Efforts have been made to collect the predominant portion of tumor tissues from each mouse lung. Subsequently, the frozen samples were pulverized to a powder using a Cryomill (Retsch). High-quality total RNA was extracted from the above samples, and mRNA enrichment were performed using RNeasy Min Kit (QIAGEN, Cata#74104). cDNA library was prepared and sequenced at Novogene.
For GSEA analysis, the gene set for “Oxidative stress” was downloaded from GeneCards (https://www.genecards.org/, accessed on April 09, 2023), and the gene sets for “GOBP positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator”, “GOBP lipid biosynthetic process” and “GOBP fatty acids biosynthetic process” were downloaded from mSigDB website (https://www.gsea-msigdb.org/, accessed on April 09, 2023). A dataset containing mRNA expression profiles of all genes for G6pdWT;KL and G6pdKO;KL lung tumors was prepared. The GSEA software (Version 4.3.2), using the classic setting recommended for mRNA-seq data in the GSEA manual, was employed to perform the GSEA analysis.
Western blot
Western blot was performed as previously described62. Briefly, TDCLs protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween 20) for 1 hour at room temperature, followed by incubation with primary antibody anti-G6PD (Abcam, Cata#AB993, Clone#Polyclonal, Lot#GR274589-46, 1:1000 dilution, https://www.abcam.com/products/primary-antibodies/glucose-6-phosphate-dehydrogenase-antibody-ab993.html) overnight at 4 °C. After washing, membranes were incubated with appropriate HRP-conjugated secondary antibody anti-β-actin (Sigma, Cata#A1978, Clone#AC-15, Lot#109M4849V, 1:100,000 dilution) for 1 hour at room temperature. Detection was carried out using ChemiDox Touch Imaging System (BIO-RAD). Uncropped scan of the Western blot for G6PD and β-actin is provided in the Source Data file.
Cell proliferation assay
For IncuCyte measurement, G6pdWT;KL or G6pdKO;KL TDCLs were seeded at 4 × 104 cells per well in 12-well plates in complete RPMI medium. The IncuCyte live-cell imaging system automatically quantified cell surface area coverage to determine the percentage of confluence in one well of 12-well plate every 2 hours over 4 days, and the slope of the time-course changes in the percentage of confluence was utilized to reflect the proliferation rate.
For manual cell counting, cells were treated with H2O2 (Sigma-Aldrich, Cata#88597-100ML-F) at concentrations of 0, 20, 40, and 80 μmol/L for 24 hours. Subsequently, the cells were trypsinized off the culture plates and counted using a Vi-cell XR cell viability analyzer (Beckman coulter). The relative proliferation rate for cells treated with different concentrations of H2O2 was calculated by normalizing the cell number to the corresponding cells without H2O2 treatment.
MTS assay
G6PDWT;KL TDCLs were seeded at 2 x 104 cells per well in 96-well plates and G6PDKO;KL TDCLs were seeded at 5 x 104 cells per well in 96-well plates. And 2 mg/mL MTS reagent (VWR, Cata#PAG1112) and 0.92 mg/mL PMS reagent were added to RPMI medium (0.2 mL of MTS reagent for per mL of RPMI and 0.01 mL of PMS reagent per mL of RPMI) to make MTS/PMS solution freshly before each assay. At the day of assay, each well was aspirated and 200 µL of MTS/PMS solution was added with minimal light exposure. An incubation period of 1 hour was performed before the first measurement. OD measurements were obtained at an excitement wavelength of 490 nm and were performed daily up to three days. The number of replicates in each group was specified in the figure legends.
For the experiments that utilized G6PDi-1 (Cayman, Cata#31484), the day following KL TDCLs seeding on 96-well plates, TDCLs were treated with vehicle control or G6PDi-1 at concentrations of 20 and 40 µmol/L, and MTS assay was performed at the indicated time points. The number of replicates in each group was specified in the figure legends. The relative proliferation rate for cells treated with different concentrations of G6PDi-1 was calculated by normalizing the cell number to the corresponding cells without G6PDi-1 treatment.
Apoptosis/necrosis assay
G6pdWT;KL and G6pdKO;KL TDCLs were seeded in 96-well plates at 3 x 104 cells per well. Blank control wells contained culture mediums without cells. Complete RPMI medium kept at 37 °C was used to dilute detecting reagents from the Promega RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega, Cata#JA1011) 1000-fold and added to each seeded well (100 µL) during measurement. Measurements were obtained at 22 and 46 hours after seeding. Luminescence measurements were obtained simultaneously to fluorescence which was optically measured at an excitement wavelength of 485 nm and collected at an emission wavelength of 530 nm for apoptosis. Fluorescence emissions were measured multiple times for each well and the mean values were used for data analysis for necrosis.
De novo fatty acid synthesis analysis in vitro
G6pdWT;KL and G6pdKO;KL TDCLs were cultured in 6-cm dishes in RPMI medium without glucose (Gibco, Cata#11879-020) supplemented with 10% fetal FBS, 1% Penicillin-Streptomycin, 0.075% sodium bicarbonate, and 2 g/L [U-13C6]-glucose for 24 hours and assessed in triplicate. Afterward, saponified fatty acids were extracted and subjected to LC-MS analysis for further analysis and calculation of 13C labeling fraction for fatty acids.
Serine consumption assay
G6pdWT;KL or G6pdKO;KL TDCLs were seeded at 0.5 × 105 or 1 × 105 cells per well in 24-well plates in complete RPMI medium, respectively. The following day, fresh complete RPMI medium was replaced, and medium was collected at 0, 24, 36, 48, and 60 hours. Each timepoint set up duplicate wells for both G6pdWT;KL and G6pdKO;KL TDCLs. The pool size levels of serine in medium were measured using LC-MS. A Vi-cell XR cell viability analyzer was used to measure cell number at each time point. Based on the following formula: the reduction in serine amount in the well (serine amount at the 0-hour timepoint minus the serine amount at the indicated timepoint) divided by the increase in cell number in the same well (cell number at the indicated timepoint minus the cell number at the 0-hour timepoint), the serine consumption (μg) per one million cells increase at the indicated timepoint can be calculated.
Serine and glycine depletion assay
G6pdWT;KL and G6pdKO;KL TDCLs were cultured in customized complete RPMI medium (RPMI medium without glucose, serine, and glycine (Teknova, Cata#R9660), supplemented with 2 g/L glucose (Sigma, Cata#G8270-1KG), 10 mg/L glycine (Sigma, Cata#50046-50 G) and 30 mg/L serine (Sigma, Cata#S4311-25G)) with 10% fetal FBS, 1% Penicillin-Streptomycin, 0.075% sodium bicarbonate, at 37°C with 5% CO2. After 2 days, G6pdWT;KL TDCLs were trypsinized and seeded at 0.5 × 105 cells per well, while G6pdKO;KL TDCLs were trypsinized and seeded at 1 × 105 cells per well in 24-well plates. For Serine and glycine depletion assay, the TDCLs were cultured in the serine/glycine free RPMI medium (RPMI medium without glucose, serine and glycine, supplemented with 2 g/L glucose) with 10% fetal FBS, 1% Penicillin-Streptomycin, 0.075% sodium bicarbonate, and the complete RPMI medium as control. After 2 days, TDLCs were trypsinized off plates and then counted using a Vi-cell XR cell viability analyzer.
ROS levels measurement
The CM-H2DCFDA assay (Invitrogen, Cata#C6827) was performed to measure cellular ROS levels. G6pdWT;KL or G6pdKO;KL TDCLs were seeded at 0.5 x 105 or 1 x 105 cells per well in 24-well plates in complete RPMI medium, respectively. After 2 days, the cells were washed twice with HBSS (Corning, Cata#21-022-CV). Then, 0.5 mL of CM-H2DCFDA solution with a concentration of 5 μmol/L in HBSS was added to each well, and the cells were incubated at 37 °C for 45 minutes in the dark. Following incubation, the medium was changed to RPMI medium (Gibco, Cata#11875-093) for 30 minutes to allow for recovery. Subsequently, the medium was replaced with HBSS, and the fluorescence intensity was measured using a microplate reader (Tecan). The excitation wavelength was set to 493 nm, and the emission wavelength was set to 520 nm. After measuring the fluorescence intensity, TDLCs were trypsinized off the plates and counted using a Vi-cell XR cell viability analyzer. The ROS levels were calculated using the following formula: the fluorescence intensity (the fluorescence intensity of each well stained with CM-H2DCFDA minus the fluorescence intensity without staining) was divided by the cell number (x 106) in the same well.
For the ROS levels measurement under the H2O2 treatment condition, cells were treated with H2O2 at concentrations of 0, 20 μmol/L for 24 hours. Subsequently, the cells were stained with CM-H2DCFDA following the aforementioned method.
To measure ROS levels under serine and glycine depletion conditions, cells were treated according to the “serine and glycine depletion assay” method. Subsequently, the cells were stained with CM-H2DCFDA following the aforementioned method.
Serine uptake measurement in vitro
G6pdWT;KL and G6pdKO;KL TDCLs were seeded in 6-cm dishes with regular complete RPMI medium for serine uptake measurement. The following day, the medium was replaced with the RPMI medium without glucose, serine, and glycine (Teknova, Cata#R9660), supplemented with 10% FBS, 1% penicillin-streptomycin, 0.075% sodium bicarbonate, 2 g/L glucose, 10 mg/L glycine, and 30 mg/L [2,2,3-2H]-serine. The cells were incubated for 4 hours, and the experiment was performed in triplicate. Subsequently, water-soluble metabolites were extracted and subjected to LC-MS analysis to further analyze and calculate 2H labeling for serine.
Sample preparation of water-soluble metabolites for LC-MS analysis
For the extraction of water-soluble metabolites from lung tumors, following the methodology described in a previous study62. Approximately 20–30 mg of tumor samples were precisely weighed and placed into a pre-cooled tube. The samples were then pulverized using the Cryomill. Pre-cooled extraction buffer consisting of methanol: acetonitrile: H2O (40:40:20, V/V) with 0.5% formic acid (Sigma-Aldrich, Cata#F0507-100ML) was added to the resulting powder (40 μL of solvent per mg of tumors). The samples were then vortexed for 15 seconds and incubated on ice for 10 minutes. Subsequently, 15% NH4HCO3 solution (5% V/V of the extraction buffer) was used to neutralize the samples. Then all samples were vortexed again for 10 seconds and centrifuged at 4 °C, 13,000 × g for 20 minutes. The resulting supernatant was transferred to LC-MS vials for subsequent analysis.
For the extraction of water-soluble metabolites from serum, following the methodology described in a previous study62, pre-cooled methanol was added to the serum samples in a 1.5 mL Eppendorf Tube (Tube A). The mixture was vortexed for 10 seconds and left at −20 °C for 20 minutes. Afterward, the tube was centrifuged at 4 °C for 10 minutes. The top supernatant was carefully transferred to another 1.5 mL Eppendorf Tube (Tube B) as the first extract. Then, 200 μL of an extraction buffer composed of methanol: acetonitrile: H2O (40:40:20, V/V) was added to Tube A, which still contained the pellet at the bottom. The tube was vortexed for 10 seconds and placed on ice for 10 minutes. Subsequently, the tube was centrifuged at 4 °C, 13,000 × g for 10 minutes. The top supernatant from this step was combined with the first extract in Tube B, resulting in a final extract volume of 250 μL. The extract was loaded onto the Phree Phospholipid Removal Tabbed 1 mL tube (Phenomenex, Cata#8B-S133-TAK) and centrifuged at 4°C, 13,000 × g for 10 minutes. The extract was transferred to LC-MS vials for subsequent analysis.
For the extraction of water-soluble metabolites from cultured cell samples, following the methodology described in a previous study58, G6pdWT;KL and G6pdKO;KL TDCLs cultured in triplicate in 6-cm dishes were washed twice with PBS. Subsequently, the cells were incubated with 1 mL of pre-cooled extraction buffer containing methanol: acetonitrile: H2O in a ratio of 40:40:20, along with 0.5% formic acid solution, on ice for 5 minutes. The extraction process was followed by neutralization with 50 µL of 15% ammonium bicarbonate. The cells were then scraped from the plates and transferred to 1.5 mL tubes. Afterward, the tubes were centrifuged at 4°C, 13,000 × g for 10 minutes. The resulting supernatant was transferred to LC-MS vials for subsequent analysis.
Sample preparation of saponified fatty acids for LC-MS analysis
To extract saponified fatty acids from lung tumors and serum, samples were collected under two states, as indicated in the figure legend: fed state and fasted state. In fed state, the food was available in cages, and mice were euthanized with samples collected at 8:00 AM. In fasted state, food was removed from the mice at approximately 9:00 AM, and mice were euthanized with samples collected at 3:00 PM. The extraction methodology described in a previous study was followed35. Pre-cooled methanol was added to the resulting powder or serum (12 μL of methanol per mg of lung tumors or μL of serum). The samples were vortexed for 10 seconds, followed by adding −20 °C MTBE (40 μL of MTBE per mg of tumors or μL of serum). After another 10 seconds vortexing step, the samples were incubated on a shaker at 4 °C for 6 minutes. Next, H2O (10 μL of H2O per mg of tumors or μL of serum) was added, and the samples were centrifuged at 4 °C, 13,000 × g for 2 minutes. Following centrifugation, 160 μL of the top MTBE layer was transferred to a new 1.5 mL Eppendorf Tube. The liquid was then dried by nitrogen. Subsequently, the sample was resuspended in 1 mL of saponification solvent (0.3 mol/L KOH in 90:10 methanol/H2O), and the entire volume was transferred to 4 mL glass vials. The vials were placed in a water bath at 80 °C for 1 hour. After incubation, the vials were cooled on ice for 3 minutes, and 100 μL of formic acid was added. Then, 300 μL of hexanes was added, and the samples were vortexed for 10 seconds, resulting in two layers. The top layer was transferred to a new 1.5 mL Eppendorf Tube, and this step was repeated to obtain a final volume of 600 μL. The liquid was then dried in the 1.5 mL Eppendorf Tube under air. To resuspend the extracted sample, 150 μL of resuspension solvent (50:50 isopropanol/methanol) was added. The samples were centrifuged at 4 °C, 13,000 × g for 10 minutes, and the resulting supernatant was transferred to LC-MS vials for further analysis.
For the extraction of saponified fatty acids from cultured cell samples, following the methodology described in a previous study58, G6pdWT;KL and G6pdKO;KL TDCLs cultured in triplicate in 6-cm dishes were washed twice with PBS, followed by the addition of 1 mL of −20 °C 90% methanol containing 0.3 mmol/L KOH. The resulting liquid, along with the cell debris, was scraped into 4 mL glass tubes. The samples were then heated at 80 °C for 1 hour to saponify the fatty acids. After saponification, the samples were acidified with 100 µL of formic acid, followed by 1 minute of vortexing. The samples were extracted twice with 1 mL of hexane, and the organic phase was collected. The extracts were dried under air and dissolved in a mixture of methanol, chloroform, and isopropanol in a 1:1:1 ratio. All samples were vortexed for 20 seconds and then centrifuged at 4 °C, 13,000 × g for 10 minutes. The resulting supernatant was transferred to LC-MS vials for subsequent analysis.
LC-MS methods
For the LC-MS analysis of water-soluble metabolites, following the methodology described in a previous study63, the experimental conditions were optimized using an HPLC-ESI-MS system consisting of a Thermo Scientific Vanquish HPLC coupled with a Thermo Q Exactive Plus MS. The HPLC system was equipped with a Waters XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size) and a Waters XBridge BEH XP VanGuard cartridge (2.1 mm x 5 mm, 2.5 μm particle size, 130 Å pore size) guard column. The column temperature was maintained at 25 °C. The mobile phase A consisted of a mixture of H2O: acetonitrile (95:5, V/V) with 20 mmol/L NH3AC and 20 mmol/L NH3OH at pH 9. The mobile phase B consisted of a mixture of acetonitrile: H2O (80:20, V/V) with 20 mmol/L NH3AC and 20 mmol/L NH3OH at pH 9. The composition of mobile phase B varied over time as follows: 0 minutes, 100%; 3 minutes, 100%; 3.2 minutes, 90%; 6.2 minutes, 90%; 6.5 minutes, 80%; 10.5 minutes, 80%; 10.7 minutes, 70%; 13.5 minutes, 70%; 13.7 minutes, 45%; 16 minutes, 45%; 16.5 minutes, 100%. The flow rate was set to 300 μL/minute, and the injection volume was 5 μL. The column temperature was maintained at 25 °C throughout the analysis. MS scans were acquired in negative ionization mode, with a resolution of 70,000 at m/z 200. The automatic gain control (AGC) target was set to 3 x 106, and the mass-to-charge ratio (m/z) scan range was set from 72 to 1000 35.
For the LC-MS analysis of NADPH and NADP+, the gradient consisted of the following steps: 0 minutes, 85% B; 2 minutes, 85% B; 3 minutes, 60% B; 9 minutes, 60% B; 9.5 minutes, 35% B; 13 minutes, 5% B; 15.5 minutes, 5% B; 16 minutes, 85% B. The run was stopped at 20 minutes, and the injection volume was 15 µL. As described previously, full scans were alternated with targeted scans in the m/z range of 640-765, with a resolution of 35,000 at m/z 200, and with AGC target of 5 × 105.
For the LC-MS analysis of fatty acids samples, following the methodology described in a previous study58, a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA) with a Poroshell 120 EC-C18 column (150 mm × 2.1 mm, 2.7 μm particle size, Agilent Infinity Lab, Santa Clara, CA) was employed using a gradient of solvent A (90%:10% H2O: methanol with 34.2 mmol/L acetic acid, 1 mmol/L ammonium acetate, pH 9.4), and solvent B (75%:25% IPA: methanol with 34.2 mmol/L acetic acid, 1 mmol/L ammonium acetate, pH 9.4). The gradient program was as follows: 0 minutes, 25% B; 2 minutes, 25% B; 5.5 minutes, 65% B; 12.5 minutes, 100% B; 19.5 minutes, 100% B; 20 minutes, 25% B; 30 minutes, 25% B. The flow rate was set to 200 μL/minute, and the column temperature was maintained at 55°C. MS/MS data were acquired using a Thermo Q Exactive PLUS mass spectrometer with heated electrospray ionization source. The spray voltage was set to −2.7 KV in negative mode. The sheath gas, auxiliary gas, and sweep gas flow rates of 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300 °C, and the auxiliary gas heater was set to 360 °C. The S-lens RF level was 45. In negative ionization mode, the m/z scan range was set from 200 to 2,000. The AGC target was set to 1 x 106 and the maximum injection time was 200 milliseconds. The resolution was set to 140,000. Data-dependent MS/MS scans were acquired from pooled samples in negative ionization mode with a loop count of 3, an AGC target of 1 x 106, and the maximum IT of 50 milliseconds. The mass resolution was set to 17,500, and the normalized collision energy was stepped at 20, 30, and 40. Dynamic exclusion was set to 10 seconds. The MS/MS data were processed using MS-DIAL64,65, and the fatty acids species annotations were performed by matching the built-in MS/MS database. The annotated fatty acids species were quantified from MS1 runs for better accuracy66 using El-MAVEN67,68.
NADPH active-H labeling calculation
NADPH and NADP+ features were extracted in EI-MAVEN software. The 2H isotope natural abundance and impurity of labeled substrate were corrected using AccuCor written in R69. NADPH active-H labeling p from [2,3,3-2H]-serine was determined based on labeling of NADPH-NADP+ pair and calculated using the previously described Eq. (1)35, the matrix on the left side of Eq. (1) contains the experimentally measured mass 2H isotope distribution for NADP+, while the right side contains the experimentally measured mass 2H isotope distribution for NADPH.
Water-soluble metabolites and fatty acids labeling fraction calculation
Water-soluble metabolites and fatty acids data were obtained using the EI-MAVEN software package68 with each labeled isotope fraction. The isotope natural abundance and tracer isotopic impurity were corrected using AccuCore written in R69. Additionally, the labeling fraction and enrichment was calculated automatically by the AccuCor69. Fractional 13C-enrichment of indicated water-soluble metabolite in the tumor was calculated by dividing the labeling fraction of the indicated water-soluble metabolite from tumor by [U-13C6]-glucose enrichment in the serum from same mouse.
Statistics and reproducibility
GraphPad Prism 9.1.0 (GraphPad Software Inc., La Jolla, CA) was employed for data analysis. The normal distribution of variables was estimated using Shapiro-Wilk (W) test, while Friedman’s test was utilized to assess the homogeneity of variance. The data underwent analysis using appropriate statistical tests, including the two-tailed unpaired t-test, two-tailed unpaired t-test with Welch’s correction, Mann-Whitney test, one-way and two-way ANOVA followed by t-test, one-way ANOVA followed by t-test with Welch’s correction, and one-way ANOVA followed by Bonferroni’s multiple comparisons test, as specified in the figure legends. The log-rank test determined the significance of Kaplan-Meier analyses for survival. Data were presented as the mean ± SEM, and P < 0.05 was considered statistically significant. GSEA statistical test was performed by GSEA software (Version 4.3.2), gene set with |NES | > 1, NOM p-value < 0.05, FDR q-value < 0.25 was considered as significant. All experiments were repeated independently three times with similar results.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The raw data of mRNA-seq have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE253613. Additionally, full list of RNA-seq result is provided in Supplementary Data 1. The metabolomics and lipidomics raw data have been deposited in Metabolomics Workbench under project ID PR001843. Source data are provided with this paper.
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Acknowledgements
We are grateful to Eric Chiles and Yujue Wang in the ** Wang, Samuel Wang, Vrushank Bhatt, Eduardo Cararo Lopes, Zhixian Hu, Michael Sun, Xuefei Luo, **aoyang Su, Joshua D. Rabinowitz, Eileen White & Jessie Yanxiang Guo
Contributions
J.Y.G. was the lead principal investigator who conceived and supervised the project. J.Y.G. and T.L. designed the experiments, performed the data analysis, and interpreted the data. J.M.G. and J.D.R. provided G6pdflox/flox mouse strain, T.L., S.A., J.L., H.K., W.W., S.W., E.C.L., M.S., and V.B. performed most experiments. S.W. and M.S. performed IHC quantification. S.A., S.W., X.L., and J.L. maintained mouse colonies and mouse genoty**. Z.H. performed mouse surgery. X.S. provided technical support for metabolomics and lipidomics measurements and analyses. J.D.R, and E.W. provided intellectual input in project development. J.Y.G. and T.L. wrote the manuscript that was reviewed and edited by all authors.
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E.W. is a stockholder in a founder of Vescor Therapeutics. J.D.R. is an advisor and stockholder in Colorado Research Partners, L.E.A.F. Pharmaceuticals, Bantam Pharmaceuticals, Rafael Pharmaceuticals; a paid consultant of Third Rock Ventures; a founder, director and stockholder of Farber Partners, Serien Therapeutics and Sofro Pharmaceuticals; a founder and stockholder in Empress Therapeutics; and a director of the Princeton University–PKU Shenzhen collaboration. The Rabinowitz lab at Princeton University and the Princeton University-PKU Shenzhen collaboration have discovered and generated intellectual property regarding G6PD inhibitors. Other authors have no conflict of interest to declare.
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Lan, T., Arastu, S., Lam, J. et al. Glucose-6-phosphate dehydrogenase maintains redox homeostasis and biosynthesis in LKB1-deficient KRAS-driven lung cancer. Nat Commun 15, 5857 (2024). https://doi.org/10.1038/s41467-024-50157-8
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DOI: https://doi.org/10.1038/s41467-024-50157-8
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