Enhancing Rectal Cancer Radiosensitivity and Gut Protection through Methionine Restriction

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Miousse, Oscar Zuniga, Sarita Garg, Lokesh Akana, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6497576/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Approximately one-third of colorectal cancer cases involve the rectum, where radiation therapy is an integral part of the treatment of this disease. However, KRAS mutations are associated with poor clinical outcomes and lower therapeutic responses. Previous studies suggest that KRAS mutations may alter the balance of metabolites in the methionine cycle. This study investigates the interplay between the methionine cycle, KRAS mutations, and radiation therapy. Experimental Design We examined the impact of the KRAS mutation and radiation on methionine cycle metabolites. In vitro , we reduced methionine levels in the culture media and assessed the radiosensitivity of KRAS-mutant colorectal cancer cells. In vivo , we used an orthotopic mouse model with KRAS-mutant rectal tumors to evaluate the effects of a methionine-restricted (MR) diet on tumor response to radiation. Additionally, we assessed the impact of MR on normal human intestinal epithelial cells and tissues. Results In vitro , MR increased the radiosensitivity of KRAS-mutant cells, with reduced proliferation and increased DNA damage markers following radiation. In vivo , KRAS mutant tumors in mice fed an MR diet showed an increased response rate to radiation compared to KRASwt tumors. Normal cells and tissues showed reduced DNA damage markers under MR conditions, with MR diet improving villus height and crypt depth following abdominal irradiation in mice. Conclusion KRAS-mutant rectal cancer cells rely on methionine for growth, and MR enhances tumor radiosensitivity while protecting normal tissues from radiation-induced damage. These findings suggest that MR may serve as a potential therapeutic strategy to improve treatment outcomes for rectal cancer, particularly in KRAS-mutant tumors. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) oncogene are common in non-small-cell lung cancer, pancreatic ductal adenocarcinoma, and colorectal cancer( 1 ). Mutations in KRAS lead to the constitutive activation of the molecule and sustained proliferation. In patients, KRAS mutations are associated with a poorer prognosis ( 2 , 3 ). In vitro , KRAS mutant cells show a higher resistance to chemotherapeutic agents ( 4 , 5 ) and radiation ( 6 , 7 ). Previous research has shown that DNA repair is upregulated in KRAS mutant cells ( 4 , 7 ). Studies have also shown a link between KRAS mutations and the metabolism of the essential amino acid methionine. The methionine transporter and the methionine uptake rates are decreased in KRAS mutant cells compared to wild-type cells ( 8 ). Metabolites related to the methionine cycle are also altered in KRAS mutant cells compared to wild-type cells ( 9 , 10 ). Methionine is well known to affect cancer and non-cancer cells differentially ( 11 – 13 ). Most cancer cells are heavily reliant on the availability of methionine for survival. Decreasing dietary methionine (methionine restriction; MR) has been shown to reduce tumor growth in animal models ( 14 – 19 ). In healthy animals, on the other hand, MR is associated with an increase in lifespan and an improvement in glucose and lipid regulation ( 20 – 27 ). This study investigated the relationship between KRAS mutations, methionine metabolism, and the response to radiation in colorectal cancer. First, we confirmed that KRAS mutations alter the methionine cycle. We then examined how MR influences cancer cell proliferation, DNA damage, and repair based on KRAS genotype in vitro and its impact on tumor growth in vivo . Lastly, we explored the effects of MR on normal intestinal fibroblasts in vitro and in vivo . Materials and Methods Cell culture HCT116 (CCL-247) parental and Hs738.St/Int (CRL-7869) cells were obtained from ATCC (Manassas, VA). Genetically engineered HCT116 and SW48 cells with KRAS mutations were obtained from Horizon Discovery (Waterbeach, UK). The parental HCT116 bears the phenotype KRAS G13D/WT , while the parental SW48 is KRAS WT/WT . The genotype of the engineered cells were KRAS G13D/− or KRAS WT/− . HCT116 and SW48 KRAS G13D/− and KRAS WT/− cells were further transfected with a luciferase transgene for luminescence. The cells were maintained in DMEM media. For experiments in low methionine, cells were cultured in high glucose, no glutamine, no methionine, no cystine DMEM (Thermo Fisher Scientific, Waltham MA) supplemented with 5% dialyzed serum (BioTechne, Minneapolis, MN), 100 IU penicillin and streptomycin (Thermo Fisher Scientific), 4 mM L-glutamine (Thermo Fisher Scientific) and 1 mM sodium pyruvate (Thermo Fisher Scientific). L-cystine (Millipore-Sigma, Burlington, MA) was resuspended in PBS with NaOH added until complete solubilization, and added to the cell media at a final concentration of 150 µM. L-methionine (Millipore-Sigma) was resuspended in PBS and added to the cell media at a final concentration of 200 µM in controls, and 5 µM in MR. Amino acid analysis Cell lysates were precipitated with 125 µL of 10% sulfosalicylic acid (SSA), centrifuged, and the supernatant was used to determine essential amino acid concentrations using the internal standard technique and liquid chromatography with tandem mass spectrometry (LCMS: QTrap 5500 MS;AB Sciex, Foster City, CA, USA). Phenylalanine and tyrosine enrichments were measured using the tert-butyldimethylsilyl derivative and gas chromatography-mass spectrometry (models 7890A/5975; Agilent Technologies, Santa Clara, CA, USA). Ions of mass-to-charge ratio of 234, 235, and 239 for phenylalanine and of 466, 467, 468, and 470 for tyrosine were monitored with electron impact ionization and selective ion monitoring. Methylation ratio For the measurement of S-adenosylmethionine and S-adenosylhomocysteine, 50 µL cell lysate samples were extracted in 150 µL cold acetonitrile, vortexed, centrifuged, and the 150 µL supernatant was diluted with the 125 (600 µL) or 200 ng/mL (150 µL) internal standard solution. These different concentrations and volumes of internal standard are used to normalize based on protein concentration but still result in 100 ng/mL internal standards. LC-MS/MS analysis was run on the Agilent Ultivo triple quad coupled to the Agilent Infinity II 1290 with electrospray ionization in positive mode. The injection volume was 10 µL, an Acquity XSelect HSS T3 column (2.5 µm, 2.1 x 100 mm) was used at 25°C with a flow rate of 0.4 mL/min. Mobile phase A was 5 mM PFHA in water, mobile phase B was acetonitrile, with a gradient from 95% A down to 5% A and back up to 95% A over the course of 15 minutes. The analysis was run by Multiple Reaction Monitoring (MRM) with a dwell time of 100 ms, source gas temperature, flow rate, nebulizer pressure, and capillary voltage of 350°C, 13 L/min, 22 psi, and 4000 V, respectively. Radiation Clonogenic Assays Cells were trypsinized to generate single cell suspensions and seeded onto 60 mm tissue culture plates in triplicate. Cells were then irradiated with various doses (0–6 Gy). Ten to 14 days after seeding, colonies were fixed with Methanol/Acetic Acid, stained with 0.5% crystal violet and the numbers of colonies or colony forming units (CFU) containing at least 50 cells were counted using a dissecting microscope (Leica Microsystems, Inc.. Buffalo Grove, IL) and surviving fractions calculated. Experiments were repeated multiple, independent times. Phosphoproteomics Cell pellets were lysed in 0.1 mL of RIPA buffer (Pierce 89900) with protease and phosphatase inhibitor cocktails. Phosphoproteomics sample preparation followed Storey et al. (PMC7423749), where proteins were reduced, alkylated, and digested with trypsin/LysC (Promega VA5071) using Filter-Aided Sample Preparation. Peptides were labeled using TMT 10-plex reagents (Thermo 90113). Most peptides (90%) were enriched using TiO 2 and Fe-NTA phosphopeptide kits (Thermo A32993, A32992), and 10% were used for total proteome analysis. Enriched and un-enriched peptides were separated into 46 fractions on an Acquity BEH C18 column (Waters) using a 50-min gradient, then consolidated into 18 super-fractions. Each super-fraction was further separated on an XSelect CSH C18 column (Waters) using a 60-min gradient. Eluted peptides were ionized by electrospray (2.2 kV) and analyzed on an Orbitrap Fusion Lumos (Thermo) using MS3 with multi-notch parameters. MS data were acquired with the FTMS analyzer in top-speed mode at 120,000 resolution (375–1500 m/z), followed by MS/MS with CID (31.0 normalized collision energy) and HCD (55.0 normalized collision energy) activation. MS3 reporter ion data were acquired at 50,000 resolution (100–500 m/z). MS Data Analysis Proteins were identified, and TMT MS3 reporter ions quantified by searching the UniprotKB Homo sapiens database (June 2018) using MaxQuant (version 1.6.0.16) with a parent ion tolerance of 3 ppm, fragment ion tolerance of 0.5 Da, reporter ion tolerance of 0.001 Da, trypsin/P enzyme with two missed cleavages, and variable modifications including oxidation on M, acetyl on protein N-terminus, phosphorylation on STY, and fixed carbamidomethyl on C. Protein and peptide identifications were accepted with less than 1.0% false discovery. TMT MS3-corrected reporter ion intensity values were analyzed for total protein changes using unenriched lysate samples, while phospho(STY) modifications were analyzed using enriched phosphorylated peptides. The enriched and unenriched samples were multiplexed with two separate TMT10-plex batches. Following data acquisition, the results were normalized, and sample quality verified using ProteiNorm. Protein and phosphopeptide data were normalized using cyclic loss. Linear models were fitted to the expression data using limma 3.46.0, and differential abundance was evaluated using robust empirical Bayes (eBayes). Proteins and phosphopeptides with a fold-change > 2 and an FDR adjusted p-value 75%, peptides with zero values were excluded, and data were log2-transformed before differential abundance analysis. Gene set enrichment analysis was performed using Ensemble of Gene Set Enrichment Analyses, and modified phosphosite-flanking peptides were evaluated using PHOXTRACK to identify kinases and their substrates, assessing kinase activity/enrichment with 50,000 permutations. Gene expression From frozen sections of the distal colon of about 20 mg, we extracted RNA with the QIAzol lysis reagent using a Dounce homogenizer according to the manufacturer’s directions. We washed the RNA pellets with two 75% ethanol washes. We resuspended the RNA pellets in water and quantified the solutions with spectrophotometry (Nanodrop One, Thermo Fisher Scientific). We reverse transcribed 1 µg of each RNA sample into cDNA using the iScript RT Supermix (Bio-Rad, Hercules, CA). We diluted the cDNA at a final concentration of 5 ng/µL and used 20 ng for each real-time quantitative PCR reaction. Gene expression was determined in technical duplicates using the iTaq Universal SYBR® Green Supermix according to the manufacturer’s instructions. We performed amplification on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad). We increased the number of cycles of amplification to 50 to detect low expression in treated samples for the gene Tnf. We analyzed the raw data using the ΔΔCT method relative to the internal control RPLP0. KRAS knockdown In order to ascertain the role of KRAS in the regulation of downstream targets, we downregulated mRNA levels with siRNAs. siRNAs targeting KRAS and a scrambled control (Silencer Select #4390771 s7940, and 4390843, respectively, Thermo Fisher Scientific) were used at a final concentration of 30 nM for a reverse transfection in 6-well format in triplicates. siRNA were mixed with lipofectamine RNAiMAX Transfection Reagent and Opti-MEM I reduced serum medium (ThermoFisher Scientific) and added to the wells, then 140,000 cells per well was added to the mixture in media devoid of antibiotics. After 24 hr, the media was changed for test media. After 24 hr, RNA was extracted and gene expression analysis was performed as described above. Western Blot Cell lysates were prepared using RIPA buffer (ThermoFisher, Waltham MA) supplemented with 1x protease (Complete, Roche, Indianapolis, IN) and phosphatase inhibitors (PhosSTOP, Roche, Indianapolis, IN, Roche) followed by protein quantification by the Dc protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded and resolved by SDS/PAGE and transferred to nitrocellulose membranes. Primary antibodies including γH2A.X, RAD51, CTIP, and GAPDH (Cell Signaling, Danvers, MA) were allowed to bind overnight at 4°C, and used at a dilution of 1:100-1,000. After washing in TBS-Tween, membranes were incubated with StarBright Goat Anti-Mouse/Rabbit IgG secondary antibodies (Bio-Rad) diluted 1:2,500-1:5,000 for 1 hour. Membranes were washed with TBS-Tween and then imaged on the ChemiDoc MP Imaging System (Bio-Rad Hercules, CA). DNA damage foci For γH2AX, cells were grown on cover slips within a 10 mm petri dish and then irradiated as described above and then fixed with 2% paraformaldehyde at varying time points, permeabilized with 1% Triton X-100 and blocked with 3% bovine serum albumin (BSA) in PBS. Cells were stained with anti-phospho-H2AX (Cell Signaling, Danvers, MA), washed and incubated with a fluorophore-conjugated secondary antibody (Fisher Scientific, Waltham, MA). Cells were imaged on a confocal microscope (Leica Microsystems, Wetzlar, Germany). For each experiment the total number of foci per cell was determined in at least 100 cells. Orthotopic tumor model All mice experiments were performed under the approved institutional IUCUC protocol #IPROTO202200000033. Athymic mice (NU/J, 5–6 weeks old, The Jackson Laboratory, Bar Harbor, MA) were injected intrarectally with 1×10⁵ cells from one of four cohorts: HCT116 KRAS G13D/− , HCT116 KRAS WT/− , SW48 KRAS G13D/− , or SW48 KRAS WT/− . Each cohort included 20 animals (5 males and 5 females per diet group: radiation plus control diet or radiation plus methionine-restricted (MR) diet, 0.12% vs. 0.65% methionine). Diets began at injection, and one week later, all mice received five daily fractions of 5 Gy X-ray radiation (SARRP) with CBCT-guided treatment planning. Mice were anesthetized with inhaled isoflurane during imaging and radiation delivery. One-week post-irradiation, they returned to a maintenance diet. Tumors were imaged by bioluminescence at baseline and four weeks later. Complete response (CR) was defined as no tumor growth or detectable luminescence. Abdominal Irradiation This study was approved by the UAMS IACUC (protocol #4119). C57BL/6 mice (3–4 months old) were bred on-site and housed under standardized conditions. They were fed a standard diet before being switched to either a methionine-restricted (MR) diet (0.12% methionine) or a control diet (0.65% methionine). After one week, mice underwent local abdominal irradiation (12.5 Gy, SARRP) with CBCT-guided targeting. After seven days, intestinal tissues (segment of proximal jejunum) were collected and fixed in Methanol Carnoy’s reagent, processed, and stained with hematoxylin and eosin (H&E) for histological and morphometric analysis. Slides were scanned using an Aperio Scanner CS2 at 20× magnification (Leica Biosystems). Villous height and crypt depth measurements Assessments of mucosal villus height and crypt depth were obtained by using Image J software (NIH, US). Measurements of villus height and crypt depth were carried out on images captured at 20× magnification in five to six areas of intestinal segments. As described previously, mucosal villus height was measured from the tip to the base of each villus, while crypt depth was measured from the crypt base to the tip opening ( 28 ). In each sample, the average of the measurements was used as a single value for statistical analyses. Statistical analysis For in vitro experiments, data are presented as the mean ± SEM. For in vitro studies, unless specified otherwise, each experiment was conducted in triplicate with a minimum of two independent biological replicates. Statistical comparisons were made between the control and experimental conditions using the two-sided, two-group t-tests with significance assessed at P < 0.05. Results KRAS mutations change metabolites levels in the methionine cycle To explore metabolic differences associated with KRAS mutations in rectal cancer, we performed an untargeted metabolomics analysis of KRASmut versus KRASwt CRC cells. For these studies, we utilized commercially available CRISPR-engineered HCT116 (heterozygous KRAS G13D cell line) human CRC cells with a knockout of either the wild-type KRAS allele (G13D/-) or a monoallelic knockout of the KRAS G13D allele (WT/-). This analysis identified 14 significantly altered metabolites, highlighting key disruptions in metabolic pathways relevant to tumor progression and therapeutic targeting (Table 1 ). Notably, three metabolites within the “sphingosine” pathway, a lipid signaling network implicated in cellular stress responses and tumorigenic adaptation to radiation ( 29 , 30 ), were elevated in KRAS mutant cells. Additionally, we observed significant alterations in amino acid metabolism, with six metabolites belonging to the amino acid super pathway, including three decreased metabolites within the “methionine, cysteine, SAM, and taurine metabolism” pathway—S-methylmethionine, and S-adenosylhomocysteine, and cysteine. These findings suggest a potential metabolic reprogramming in KRASmut rectal cancer cells that may influence tumor biology and response to therapy. Table 1 Differentially Altered Metabolites in KRASmut and KRASwt CRC Cells. 64 Amino Acid Glutamate Metabolism S-1-pyrroline-5-carboxylate 1.85 73 Amino Acid Histidine Metabolism 1-methylhistidine 0.49 444 Amino Acid Methionine, Cysteine, SAM and Taurine Metabolism S-methylmethionine 0.64 452 Amino Acid Methionine, Cysteine, SAM and Taurine Metabolism S-adenosylhomocysteine (SAH) 0.65 461 Amino Acid Methionine, Cysteine, SAM and Taurine Metabolism cysteine 0.72 526 Amino Acid Urea cycle; Arginine and Proline Metabolism N,N,N-trimethyl-alanylproline betaine (TMAP) 2.42 3022 Lipid Sphingolipid Synthesis sphingadienine 1.56 3180 Lipid Sphingosines sphingosine 1.40 3188 Lipid Sphingosines hexadecasphingosine (d16:1)* 1.42 3189 Lipid Sphingosines heptadecasphingosine (d17:1) 1.55 4241 Nucleotide Pyrimidine Metabolism, Uracil containing uracil 0.56 4354 Cofactors and Vitamins Riboflavin Metabolism flavin adenine dinucleotide (FAD) 1.54 4851 Xenobiotics Food Component/Plant beta-guanidinopropanoate 1.96 6271 Xenobiotics Chemical thioproline 0.70 Given the established link between methionine metabolism and tumor response to therapy ( 15 , 31 ) we investigated whether KRAS mutations influence methionine-related metabolites. To test this, we employed two complementary approaches. First, we quantified amino acid concentrations in cell lysates using liquid chromatography-mass spectrometry, analyzing both sham and irradiated cells. Consistent with our untargeted metabolomics findings, methionine levels were highest in WT cells and decreased in KRAS G13D cells (Fig. 1 A). Additionally, irradiation (5 Gy) reduced methionine levels in WT cells compared to sham RT, whereas no significant difference was observed between irradiated and sham-treated KRAS G13D cells. Interestingly, a similar pattern was observed for several essential amino acids, including histidine, isoleucine, leucine, phenylalanine, tryptophan, and valine. Notably, cysteine was excluded from this analysis due to its high propensity for oxidation, which can affect measurement accuracy. Next, we examined methionine metabolism by quantifying S-adenosylmethionine (SAM), a key methyl donor, and S-adenosylhomocysteine (SAH) to determine the methylation ratio (SAM/SAH). SAM levels were significantly elevated in KRAS G13D cells compared to WT cells (Fig. 1 B, p = 0.0025 for genotype), while SAH levels remained unchanged in the targeted analysis (Fig. 1 C). Consequently, the methylation ratio was higher in KRAS G13D cells (Fig. 1 D), with no observed effect of irradiation. These results suggest that KRAS-driven metabolic alterations facilitate the conversion of methionine to S-adenosylmethionine (SAM), potentially influencing cellular methylation dynamics and tumor biology. To validate these findings, we employed RNA interference (siRNA) to reduce KRAS expression in HCT116 KRAS G13D mutant cells, resulting in approximately a 70% decrease in KRAS gene expression (Fig. 2 A). Consistent with previous reports ( 31 ), KRAS knockdown reduced the DNA repair protein RAD51, reinforcing the role of KRAS in promoting DNA repair (Fig. 2 A, B). MR also reduced RAD51 expression compared to control conditions (200 µM methionine) (Fig. 2 B), suggesting a link between methionine availability and DNA repair regulation. Additionally, KRAS knockdown decreased the expression of methionine adenosyltransferase (MAT2A), further supporting the notion that KRAS activity enhances SAM synthesis from methionine (Fig. 2 C). Unexpectedly, we also observed a reduction in SLC7A5 expression, a key transporter responsible for importing large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, histidine, and tyrosine (Fig. 2 D). This suggests that oncogenic KRAS drives methionine metabolism and enhances amino acid import, potentially influencing metabolic reprogramming in tumor cells. Methionine restriction affects cell death DNA damage in KRAS mutant cells Given the observed alterations in methionine metabolism in KRAS mutant cells, we investigated how methionine availability impacts cellular signaling using phosphoproteomics. Comparing HCT116 KRAS G13D mutant cells grown in control versus MR media, total protein analysis identified 54 differentially expressed proteins (≥ 2-fold, adjusted p ≤ 0.05), with pathway analysis indicating cell cycle changes. Notably, MAT2A expression increased, confirming the efficacy of MR treatment ( 32 ). Expanding to phosphoproteomic analysis, nearly 1,000 proteins exhibited altered phosphorylation (≥ 2-fold), which we refined to 192 proteins using a ≥ 5-fold threshold. Enriched pathways included apoptosis, cell cycle checkpoints, ATM signaling, Rho GTPase cycling, pre-mRNA processing, and non-homologous end joining (NHEJ) (Fig. 3 A). DNA repair proteins H2AX, NSD2, PRKDC, TP53BP1, XRCC5, and XRCC6 showed significant changes in phosphorylation, including a 6-fold increase in S139 phosphorylation of H2AX, a key DNA damage marker. ATM, the primary kinase for this modification, was also implicated. Additionally, XRCC6 (Ku70) phosphorylation at S2 increased 40-fold, and XRCC5 (Ku80) phosphorylation at T472 rose 5-fold, while CHEK1 phosphorylation at S284 decreased. These findings suggest that MR disrupts DNA repair and promotes cell death in KRAS mutant cells. KRAS mutant colorectal cancer cells are sensitized to radiation when methionine levels are low To determine whether dysregulation of the methionine pathway is a vulnerability in KRASmut cells, we irradiated HCT116 KRAS G13D mutant cells ± MR with ionizing radiation. In addition to HCT116 KRAS G13D and KRAS +/- WT, we also tested human colorectal SW48 cells. SW48 parental cells are wildtype for KRAS, and we also used a genetically engineered SW48 derivative cell line expressing KRAS G13D. In KRAS WT HCT116 and SW48 cells, there was little to no difference in the fractional survival to doses of radiation from 0 and 6 Gy between cells grown in control media containing 200 µM methionine (CTRL) or in media containing 5 µM MR. However, in KRAS G13D cells for both cell lines, colony survival was decreased in irradiated cells grown in MR media compared to CTRL media (Fig. 4 ). This indicated that MR treatment sensitized KRAS mutant cells to IR. To investigate why survival was decreased by MR in irradiated KRAS mutant cells, we measured ɣH2A.X, a marker of DNA double strand breaks. We used two different detection methods: western blotting and the detection of foci by microscopy. We used the parental and KRAS mutant engineered HCT116 and SW48 cells. We pretreated the cells with CTRL or MR media for 24h. With this pretreatment, we observed a modest increase in ɣH2A.X in KRAS WT cells in MR versus CTRL. However, ɣH2A.X expression was strongly increased in MR compared to CTRL in KRAS G13D cells, before (0h) or after 5 Gy of X-ray radiation ( Fig. 5A-B ) . A dose of 5 Gy was chosen to mimic the cellular repair response following each fraction in a 5 × 5 Gy fractionated radiation therapy regimen, replicating a standard RT regimen in humans (33,34). We also measured RAD51 and CTIP protein expression in these samples by western blot. Both RAD51 and CTIP expression followed an inverse pattern to ɣH2A.X, where it was decreased in MR samples (Supplementary Figure 1). The MR inhibition of HR repair proteins was more pronounced in the KRAS mutant cells compared to WT. This suggests that MR impairs DNA repair mechanisms selectively in KRAS mutant cells. Methionine restriction improves the tumor response to radiation in vivo We next tested whether the decrease in survival and increase in DNA damage would impact the development of tumors in vivo . For this, we used an orthotopic rectal cancer model. Nude mice were injected intrarectally with HCT116 and SW48, KRAS WT and G13D cells. All mice underwent image-guided localized RT treatment, which involved the delivery of 5 Gy once daily for five consecutive days (25 Gy total). One week before irradiation, mice were placed on a MR diet (0.12% methionine) or a control diet containing a standard amount of 0.65% methionine. Diets were maintained during the week of irradiation. The animals were imaged with bioluminescence four weeks after radiation treatment to estimate tumor response to radiation (Supplementary Fig. 2). In KRAS WT tumors, there was no difference in the rate of complete responders in the MR diet compared to the CTRL diet (Fig. 6 A, C). In the KRAS G13D tumors, however, we observed an increase in the complete response rate in the mice treated with an MR diet (Fig. 6 B, D). These results indicate that the response rate to irradiation in KRAS mutant tumors is improved with MR. Methionine restriction protects non-cancer cells in vitro and in vivo Based on the observed benefit of an MR diet on tumor response, we examined its effect on healthy, non-cancer cells. We focused on the normal fibroblasts in the tissue surrounding the tumor. To assess potential toxicity, we exposed Hs738.St/Int human intestinal fibroblasts to either sham or 5 Gy in CTRL or MR media. Unlike cancer cells, we observed no increase in γH2A.X staining in fibroblasts when methionine was omitted from the media (Fig. 7 A). γH2A.X levels were slightly reduced in the MR group. This suggests that lowering methionine in the media may protect normal gut cells from radiation-induced DNA damage. (A) Methionine restriction in the media decreased the abundance of γH2A.X in human Hs 738.St/Int fibroblasts. (B) One month after receiving 5x5 Gy X-ray treatment, Tnf expression was reduced in the colon of mice on an MR diet compared to a control diet. (C) Representative H&E staining of the proximal jejunum from C57BL/6 mice 7 days after an acute 12.5 Gy X-ray dose, comparing MR and control diets. (D) Measurement of villous height and crypt depth from the images in panel C. Radiation therapy can induce fibrosis in the surrounding tissues over several months. In our irradiated orthotopic tumor experiment, tissues were collected one month post-irradiation, a time-point where we did not expect detectable collagen accumulation. However, we investigated potential fibrosis-related changes by measuring Tnf gene expression, which encodes tumor necrosis factor, in the distal colon of our irradiated tumor-bearing mice. Tnf expression was lower in the MR diet group than the control diet group (Fig. 7 B), indicating reduced inflammation in the colon of MR mice. To further investigate the impact of MR on healthy tissue in vivo , we used an immunocompetent C57BL/6 mouse model. Mice were fed either a CTRL or MR diet for one week, followed by a single 12.5 Gy dose of abdominal irradiation. We collected tissue from the proximal jejunum, an organ particularly sensitive to radiation, on day 7 post-radiation. Histological analysis of CTRL diet tissue showed expected damage, including reduced villous and crypt integrity. In contrast, tissue from MR diet animals maintained better morphology (Fig. 7 C). Measurement of villous height revealed a two-fold increase in MR animals compared to CTRL animals (Fig. 7 D). Crypt depth was also significantly deeper in the MR group (Fig. 7 D). These results indicate that the MR diet may preserve the intestinal epithelium's integrity following irradiation. Discussion Our study provides strong evidence that KRAS mutations create a distinct dependence on elevated methionine levels compared to KRASwt cells. We validated prior findings linking KRAS mutations to altered methionine metabolism, as demonstrated by Gao et al. ( 15 ), and explored the therapeutic implications of this relationship. Notably, our analysis of cell viability under varying methionine conditions revealed a heightened sensitivity of KRASmut cells to MR. Compared to KRASwt cells, KRASmut cells exhibited a more pronounced radiosensitivity under MR conditions. This effect correlated with increased DNA damage marker γH2AX levels and decreased expression of repair proteins such as RAD51. Phosphoproteomic analysis further confirmed impaired double-strand break repair in KRASmut cells following MR. The in vivo experiments using an orthotopic rectal cancer model provided translational relevance to our findings. Mice with KRAS mutant tumors exhibited an increased rate of complete responders to radiation therapy when maintained on an MR diet compared to the control diet. Notably, our study considered the impact of MR on non-cancer cells. Contrary to the observed sensitization of KRAS mutant cells, normal fibroblasts exposed to radiation in MR media showed no increase in ɣH2A.X staining and displayed a slight decrease. This indicates a protective effect of MR on healthy cells, supporting previous observations by Wanders et al. ( 35 ), highlighting the potential for a selective therapeutic approach. In an immunocompetent model, we further demonstrated the protective effect of MR on the intestinal epithelium following irradiation. The maintenance of tissue morphology, increased villous height, and reduced crypt depth in the MR group underscore the potential benefits of this dietary intervention in minimizing radiation-induced damage to normal tissues. We also offer some preliminary indications that MR may mitigate inflammation in irradiated gut tissues, as seen in the reduction in Tnf expression three weeks after irradiation. This adds to growing evidence of MR protective effects on normal tissue during radiation therapy ( 36 ). The implementation of MR in human patients in combination with radiation remains to be explored. A phase 1 trial evaluated the safety and feasibility of an MR diet in combination with radiation therapy in patients with non-skin cancers was attempted. While no grade 3 or higher toxicities were observed, poor patient adherence led to early trial closure, highlighting the challenge of implementing MR in clinical settings despite its potential to enhance RT efficacy ( 37 ). Despite these promising findings, our study has several limitations. First, while our in vitro and in vivo models provide strong preclinical evidence, the translational potential of MR in combination with radiation therapy remains uncertain due to the lack of clinical validation. Second, the metabolic adaptations of KRAS mutant tumors to prolonged MR were not fully explored, which may influence long-term treatment efficacy and resistance mechanisms. Additionally, our immunocompetent model did not fully characterize the effects of MR on the tumor microenvironment, including potential interactions with immune cells. Finally, the feasibility challenges observed in the pilot clinical trial ( 37 ) highlight the need for alternative strategies, such as pharmacologic methionine depletion, to improve patient adherence and clinical applicability. Future studies should address these limitations to refine the therapeutic potential of MR in KRAS-driven cancers. Our comprehensive study sheds light on the intricate interplay between KRAS mutations, methionine metabolism, and radiation response. The findings propose a novel therapeutic strategy for KRAS mutant tumors and emphasize the protective effects of MR on healthy tissues, opening avenues for further exploration of dietary interventions in cancer treatment. Declarations Availability of data and materials All models are available to academic centers under an institutional MTA Financial support and sponsorship This work was supported by the Winthrop P. Rockefeller Cancer Institute (WPRCI), the Arkansas Tobacco Settlement Commission, the National Center for Advancing Translational Sciences grant number KL2TR003108 (ARW), the American Cancer Society and American Society for Radiation Oncology ASTRO-CSDG-23-1037280-01-CDP (ARW) and NIGMS grant P20 GM139768-01 (IRM). The National Resource for Quantitative Proteomics is supported by the NIGMS grant R24GM137786. The UAMS Bioinformatics core is supported by NIGMS grant P20GM121293 and WPRCI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflicts of interest: All authors declared that there are no conflicts of interest. Ethical approval and consent to participate: Experiments were conducted using protocols and conditions approved by the Institutional Biosafety Committee at UAMS. Translational Relevance: The results of this study highlight the potential of methionine restriction (MR) as a therapeutic strategy to sensitize rectal cancer to radiation therapy. Our findings suggest that MR-induced metabolic alterations in cancer cells can enhance the efficacy of radiation treatment, overcoming resistance mechanisms that limit current therapeutic options. By identifying key metabolic pathways affected by MR, we provide insights into novel strategies for potentiating radiation response in rectal cancer, particularly in KRAS-mutant tumors. These results have significant clinical implications, offering the potential to develop combination therapies that integrate MR with conventional treatment regimens. Such therapies could improve treatment outcomes, reduce toxicity to normal tissues, and provide personalized, targeted approaches for patients with rectal cancer. Ultimately, this research paves the way for advancing precision medicine, particularly in the context of radiation oncology, where metabolic reprogramming could be exploited to optimize therapeutic efficacy and overcome treatment resistance in colorectal cancers. References Huang L, Guo Z, Wang F, Fu L. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther. Nature Publishing Group; 2021;6:1–20. Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study. J Natl Cancer Inst. 1998;90:675–84. Chatila WK, Kim JK, Walch H, Marco MR, Chen C-T, Wu F, et al. Genomic and transcriptomic determinants of response to neoadjuvant therapy in rectal cancer. Nat Med. 2022;28:1646–55. Caiola E, Salles D, Frapolli R, Lupi M, Rotella G, Ronchi A, et al. Base excision repair-mediated resistance to cisplatin in KRAS(G12C) mutant NSCLC cells. Oncotarget. 2015;6:30072–87. Tao S, Wang S, Moghaddam SJ, Ooi A, Chapman E, Wong PK, et al. Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2. Cancer Res. 2014;74:7430–41. Wang M, Han J, Marcar L, Black J, Liu Q, Li X, et al. Radiation resistance in KRAS-mutated lung cancer is enabled by stem-like properties mediated by an osteopontin-EGFR pathway. Cancer Res. 2017;77:2018–28. Yang L, Shen C, Estrada-Bernal A, Robb R, Chatterjee M, Sebastian N, et al. Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair. Nucleic Acids Res. 2021;49:11067–82. De Sanctis G, Spinelli M, Vanoni M, Sacco E. K-Ras Activation Induces Differential Sensitivity to Sulfur Amino Acid Limitation and Deprivation and to Oxidative and Anti-Oxidative Stress in Mouse Fibroblasts. PloS One. 2016;11:e0163790. Varshavi D, Varshavi D, McCarthy N, Veselkov K, Keun HC, Everett JR. Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines. Metabolomics. 2021;17:104. Varshavi D, Varshavi D, McCarthy N, Veselkov K, Keun HC, Everett JR. Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines. Metabolomics. 2020;16:51. Wallis KF, Morehead LC, Bird JT, Byrum S, Miousse IR. Differences in cell death in methionine versus cysteine depletion. Environ Mol Mutagen. 2021;62:216–26. Hoffman RM, Erbe RW. High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine. Proc Natl Acad Sci. 1976;73:1523–7. Aoki Y, Han Q, Tome Y, Yamamoto J, Kubota Y, Masaki N, et al. Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation. Front Oncol [Internet]. Frontiers; 2022 [cited 2024 Dec 9];12. Available from: https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2022.1009548/full Morehead LC, Garg S, Wallis KF, Siegel ER, Tackett AJ, Miousse IR. Increased response to immune checkpoint inhibitors with dietary methionine restriction. Cancers. 2023;15:4467. Gao X, Sanderson SM, Dai Z, Reid MA, Cooper DE, Lu M, et al. Dietary methionine influences therapy in mouse cancer models and alters human metabolism. Nature. 2019;572:397–401. Hens JR, Sinha I, Perodin F, Cooper T, Sinha R, Plummer J, et al. Methionine-restricted diet inhibits growth of MCF10AT1-derived mammary tumors by increasing cell cycle inhibitors in athymic nude mice. BMC Cancer [Internet]. 2016 [cited 2016 Jun 6];16. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891836/ Ji M, Xu X, Xu Q, Hsiao Y-C, Martin C, Ukraintseva S, et al. Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota. Nat Metab. Nature Publishing Group; 2023;1–18. Komninou D, Leutzinger Y, Reddy BS, Richie JP. Methionine restriction inhibits colon carcinogenesis. Nutr Cancer. 2006;54:202–8. Li T, Tan Y-T, Chen Y-X, Zheng X-J, Wang W, Liao K, et al. Methionine deficiency facilitates antitumour immunity by altering m6A methylation of immune checkpoint transcripts. Gut. BMJ Publishing Group; 2022;72:501–11. Richie JP, Leutzinger Y, Parthasarathy S, Malloy V, Orentreich N, Zimmerman JA. Methionine restriction increases blood glutathione and longevity in F344 rats. FASEB J. 1994;8:1302–7. Lee BC, Kaya A, Ma S, Kim G, Gerashchenko MV, Yim SH, et al. Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino-acid status. Nat Commun [Internet]. 2014 [cited 2017 Jul 26];5. Available from: http://www.nature.com/doifinder/10.1038/ncomms4592 Bárcena C, Quirós PM, Durand S, Mayoral P, Rodríguez F, Caravia XM, et al. Methionine Restriction Extends Lifespan in Progeroid Mice and Alters Lipid and Bile Acid Metabolism. Cell Rep. 2018;24:2392–403. Feng C, Jiang Y, Wu G, Shi Y, Ge Y, Li B, et al. Dietary Methionine Restriction Improves Gastrocnemius Muscle Glucose Metabolism through Improved Insulin Secretion and H19/IRS-1/Akt Pathway in Middle-Aged Mice. J Agric Food Chem [Internet]. American Chemical Society; 2023 [cited 2023 Apr 3]; Available from: https://doi.org/10.1021/acs.jafc.2c08373 Luo T, Yang Y, Xu Y, Gao Q, Wu G, Jiang Y, et al. Dietary methionine restriction improves glucose metabolism in the skeletal muscle of obese mice. Food Funct. The Royal Society of Chemistry; 2019;10:2676–90. Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell. 2005;4:119–25. Wanders D, Forney LA, Stone KP, Burk DH, Pierse A, Gettys TW. FGF21 Mediates the Thermogenic and Insulin-Sensitizing Effects of Dietary Methionine Restriction but Not Its Effects on Hepatic Lipid Metabolism. Diabetes. 2017;66:858–67. Malloy VL, Krajcik RA, Bailey SJ, Hristopoulos G, Plummer JD, Orentreich N. Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction. Aging Cell. 2006;5:305–14. Garg S, Garg TK, Miousse IR, Wise SY, Fatanmi OO, Savenka AV, et al. Effects of Gamma-Tocotrienol on Partial-Body Irradiation-Induced Intestinal Injury in a Nonhuman Primate Model. Antioxid Basel Switz. 2022;11:1895. Gault CR, Eblen ST, Neumann CA, Hannun YA, Obeid LM. Oncogenic K-Ras regulates bioactive sphingolipids in a sphingosine kinase 1-dependent manner. J Biol Chem. 2012;287:31794–803. Nava VE, Cuvillier O, Edsall LC, Kimura K, Milstien S, Gelmann EP, et al. Sphingosine enhances apoptosis of radiation-resistant prostate cancer cells. Cancer Res. 2000;60:4468–74. Miousse IR, Tobacyk J, Quick CM, Jamshidi-Parsian A, Skinner CM, Kore R, et al. Modulation of Dietary Methionine Intake Elicits Potent, yet Distinct, Anticancer Effects on Primary Versus Metastatic Tumors. Carcinogenesis. 2018;39:1117–26. Garg S, Morehead LC, Bird JT, Graw S, Gies A, Storey AJ, et al. Characterization of methionine dependence in melanoma cells. Mol Omics [Internet]. The Royal Society of Chemistry; 2023; Available from: http://dx.doi.org/10.1039/D3MO00087G Bahadoer RR, Dijkstra EA, van Etten B, Marijnen CAM, Putter H, Kranenbarg EM-K, et al. Short-course radiotherapy followed by chemotherapy before total mesorectal excision (TME) versus preoperative chemoradiotherapy, TME, and optional adjuvant chemotherapy in locally advanced rectal cancer (RAPIDO): a randomised, open-label, phase 3 trial. Lancet Oncol. 2021;22:29–42. Dahlberg M, Glimelius B, Påhlman L. Improved survival and reduction in local failure rates after preoperative radiotherapy: evidence for the generalizability of the results of Swedish Rectal Cancer Trial. Ann Surg. 1999;229:493–7. Wanders D, Hobson K, Ji X. Methionine Restriction and Cancer Biology. Nutrients. 2020;12:684. Miousse IR, Ewing LE, Skinner CM, Pathak R, Garg S, Kutanzi KR, et al. Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome. Am J Physiol - Gastrointest Liver Physiol. 2020;318:G439–50. Mattes MD, Koturbash I, Leung CN, Wen S, Jacobson GM. A Phase I Trial of a Methionine Restricted Diet with Concurrent Radiation Therapy. Nutr Cancer. Routledge; 2024;0:1–6. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresWolfe.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6497576","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448802753,"identity":"1c7091b8-2231-422f-bca9-6cc7e27cef4f","order_by":0,"name":"Isabelle R. Miousse","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"R.","lastName":"Miousse","suffix":""},{"id":448802754,"identity":"c8a206da-ba05-44de-ab14-b8adf9f2ac06","order_by":1,"name":"Oscar Zuniga","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Zuniga","suffix":""},{"id":448802755,"identity":"1feab86d-5ab6-4b6f-8882-378672f95850","order_by":2,"name":"Sarita Garg","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sarita","middleName":"","lastName":"Garg","suffix":""},{"id":448802756,"identity":"5acf7304-e28e-4871-af99-4212dfd8e7e7","order_by":3,"name":"Lokesh Akana","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lokesh","middleName":"","lastName":"Akana","suffix":""},{"id":448802757,"identity":"fc8dc961-e5d1-47b3-a4f7-3ff460cddc2c","order_by":4,"name":"Henrique Rodrigues","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Henrique","middleName":"","lastName":"Rodrigues","suffix":""},{"id":448802758,"identity":"929f145d-6a68-406d-9d26-da9db53c2953","order_by":5,"name":"Saaimatul Huq","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Saaimatul","middleName":"","lastName":"Huq","suffix":""},{"id":448802759,"identity":"0dc6865c-f277-4488-8bf7-2b661c48597b","order_by":6,"name":"Kimberley J. Krager","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kimberley","middleName":"J.","lastName":"Krager","suffix":""},{"id":448802760,"identity":"c22783b9-0685-4532-a7cf-6555fe49d5ae","order_by":7,"name":"David Church","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Church","suffix":""},{"id":448802761,"identity":"b5855945-dc73-47c6-9d5a-ed6e68a06050","order_by":8,"name":"Tatiana Wolfe","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tatiana","middleName":"","lastName":"Wolfe","suffix":""},{"id":448802762,"identity":"bbac940b-02e5-4bb6-84ff-d0b8a4b3844e","order_by":9,"name":"Nükhet Aykin-Burns","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nükhet","middleName":"","lastName":"Aykin-Burns","suffix":""},{"id":448802763,"identity":"36ad336f-b9d5-4d4f-969e-e7b4ce38319c","order_by":10,"name":"Alan Tackett","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"","lastName":"Tackett","suffix":""},{"id":448802764,"identity":"08168348-ef40-4ef1-83e5-615f705fb056","order_by":11,"name":"Adam R. Wolfe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYHACxgMVDDZQNhuReg6cYUgjXcthErTwz0g+cOBAxfnE/mnHHzB8KDtMUAeDxI20hAMHztxOnHE7x4BxxjkitDAAVR7+2HY7seF2DgMzbxsRWuSBWg4c/Hcucf7t9AfMf4nRYgDW0nAgccPtBANmRmK0GN5/BvTLsWTjjUC9B3vOpRPWInfm8MEHB2rsZOfdTn/44EeZNWEtKOAAiepHwSgYBaNgFOACAKmvSH4rpeiDAAAAAElFTkSuQmCC","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Adam","middleName":"R.","lastName":"Wolfe","suffix":""}],"badges":[],"createdAt":"2025-04-21 16:10:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6497576/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6497576/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82142505,"identity":"64214eae-fa80-4326-bc9a-bba544dbce6b","added_by":"auto","created_at":"2025-05-07 06:38:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":433529,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of methionine metabolism and methylation dynamics in WT and KRAS G13D cells.\u003c/p\u003e\n\u003cp\u003e(A) Liquid chromatography-mass spectrometry was used to measure amino acid concentrations in cell lysates from sham and irradiated (5 Gy) WT and KRAS G13D colorectal cancer cells. (B) SAM levels were quantified to assess methionine metabolism in WT and KRAS G13D cells. (C) SAH levels were measured to evaluate downstream methionine cycle alterations. (D) The SAM/SAH ratio was calculated as an indicator of methylation potential.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/cb2c71d370802103c797043e.png"},{"id":82140360,"identity":"2b19c717-8c73-4d41-871c-fd722cb85703","added_by":"auto","created_at":"2025-05-07 06:30:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279936,"visible":true,"origin":"","legend":"\u003cp\u003eEffects and KRAS and methionine in human HCT116 cells. (A-D) Gene expression in scrambled versus siRNA against KRAS in HCT116 KRAS G13D cells in control (200uM methionine) and MR (5 uM methionine) media.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/4c8b8d427bc8010d4bcc9cf3.png"},{"id":82138083,"identity":"04a9f190-a4f1-4689-8191-df59e8fcda38","added_by":"auto","created_at":"2025-05-07 06:22:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKRAS G13D cells have alterations in DNA damage response.\u003c/strong\u003e (A) Quantitative phosphoproteomics of HCT116 KRAS mutant cells in control nutrient-rich versus MR media.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/091db63c4799a6f8daeb5b8f.png"},{"id":82138086,"identity":"0d3eb09a-c470-4c00-934f-0a675ff67bce","added_by":"auto","created_at":"2025-05-07 06:22:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":162743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of methionine restriction on colony survival in SW48 and HCT116 cells following radiation.\u003cbr\u003e\n \u003c/strong\u003eColony formation assays were performed in SW48 (A, B) and HCT116 (C, D) cells to compare the effects of methionine control (Meth Ctrl) and methionine restriction (MR) on radiation sensitivity.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/8b70aa6bacd17b087b70523f.png"},{"id":82142501,"identity":"a8b0de04-486d-400b-a2b1-1947a82743ea","added_by":"auto","created_at":"2025-05-07 06:38:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":456739,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/e46407621a9336bf5fc7f0b4.png"},{"id":82142495,"identity":"850a2923-575b-42f3-84c7-a1de3ad22f10","added_by":"auto","created_at":"2025-05-07 06:38:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":268257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKRAS G13D tumors are sensitized to radiation with a MR diet. \u003c/strong\u003e\u0026nbsp;Rates of complete response 4 weeks post radiation in A) SW48 WT B) SW48 KRAS G13D C)HCT116 KRAS +/- and D) HCT116 G13D. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/4f6547ca2614e885b9365cf6.png"},{"id":82138097,"identity":"b1d85ee9-aadd-4922-9752-52073f804202","added_by":"auto","created_at":"2025-05-07 06:22:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":705612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormal cell and tissue protection.\u003c/strong\u003e\u003cbr\u003e\n(A) Methionine restriction in the media decreased the abundance of γH2A.X in human Hs 738.St/Int fibroblasts.\u003cbr\u003e\n(B) One month after receiving 5x5 Gy X-ray treatment, Tnf expression was reduced in the colon of mice on an MR diet compared to a control diet.\u003cbr\u003e\n(C) Representative H\u0026amp;E staining of the proximal jejunum from C57BL/6 mice 7 days after an acute 12.5 Gy X-ray dose, comparing MR and control diets.\u003cbr\u003e\n(D) Measurement of villous height and crypt depth from the images in panel C.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/b3700c94af3f013da3d2eea9.png"},{"id":103050381,"identity":"e6947d19-56cf-47c8-bac0-e106c7ad571b","added_by":"auto","created_at":"2026-02-20 07:49:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3353419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/06771207-34af-4e13-9a33-4e72d9690d6c.pdf"},{"id":82142499,"identity":"ae01d94d-d887-4ac3-ac93-d997d521e807","added_by":"auto","created_at":"2025-05-07 06:38:18","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1330781,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresWolfe.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6497576/v1/7c51c3bfb80ca6d5affabb55.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Rectal Cancer Radiosensitivity and Gut Protection through Methionine Restriction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eActivating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) oncogene are common in non-small-cell lung cancer, pancreatic ductal adenocarcinoma, and colorectal cancer(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Mutations in KRAS lead to the constitutive activation of the molecule and sustained proliferation. In patients, KRAS mutations are associated with a poorer prognosis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e, KRAS mutant cells show a higher resistance to chemotherapeutic agents (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and radiation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Previous research has shown that DNA repair is upregulated in KRAS mutant cells (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Studies have also shown a link between KRAS mutations and the metabolism of the essential amino acid methionine. The methionine transporter and the methionine uptake rates are decreased in KRAS mutant cells compared to wild-type cells (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Metabolites related to the methionine cycle are also altered in KRAS mutant cells compared to wild-type cells (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Methionine is well known to affect cancer and non-cancer cells differentially (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Most cancer cells are heavily reliant on the availability of methionine for survival. Decreasing dietary methionine (methionine restriction; MR) has been shown to reduce tumor growth in animal models (\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In healthy animals, on the other hand, MR is associated with an increase in lifespan and an improvement in glucose and lipid regulation (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study investigated the relationship between KRAS mutations, methionine metabolism, and the response to radiation in colorectal cancer. First, we confirmed that KRAS mutations alter the methionine cycle. We then examined how MR influences cancer cell proliferation, DNA damage, and repair based on KRAS genotype \u003cem\u003ein vitro\u003c/em\u003e and its impact on tumor growth \u003cem\u003ein vivo\u003c/em\u003e. Lastly, we explored the effects of MR on normal intestinal fibroblasts \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHCT116 (CCL-247) parental and Hs738.St/Int (CRL-7869) cells were obtained from ATCC (Manassas, VA). Genetically engineered HCT116 and SW48 cells with KRAS mutations were obtained from Horizon Discovery (Waterbeach, UK). The parental HCT116 bears the phenotype KRAS\u003csup\u003eG13D/WT\u003c/sup\u003e, while the parental SW48 is KRAS\u003csup\u003eWT/WT\u003c/sup\u003e. The genotype of the engineered cells were KRAS\u003csup\u003eG13D/\u0026minus;\u003c/sup\u003e or KRAS\u003csup\u003eWT/\u0026minus;\u003c/sup\u003e. HCT116 and SW48 KRAS\u003csup\u003eG13D/\u0026minus;\u003c/sup\u003e and KRAS\u003csup\u003eWT/\u0026minus;\u003c/sup\u003e cells were further transfected with a luciferase transgene for luminescence. The cells were maintained in DMEM media. For experiments in low methionine, cells were cultured in high glucose, no glutamine, no methionine, no cystine DMEM (Thermo Fisher Scientific, Waltham MA) supplemented with 5% dialyzed serum (BioTechne, Minneapolis, MN), 100 IU penicillin and streptomycin (Thermo Fisher Scientific), 4 mM L-glutamine (Thermo Fisher Scientific) and 1 mM sodium pyruvate (Thermo Fisher Scientific). L-cystine (Millipore-Sigma, Burlington, MA) was resuspended in PBS with NaOH added until complete solubilization, and added to the cell media at a final concentration of 150 \u0026micro;M. L-methionine (Millipore-Sigma) was resuspended in PBS and added to the cell media at a final concentration of 200 \u0026micro;M in controls, and 5 \u0026micro;M in MR.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAmino acid analysis\u003c/h3\u003e\n\u003cp\u003eCell lysates were precipitated with 125 \u0026micro;L of 10% sulfosalicylic acid (SSA), centrifuged, and the supernatant was used to determine essential amino acid concentrations using the internal standard technique and liquid chromatography with tandem mass spectrometry (LCMS: QTrap 5500 MS;AB Sciex, Foster City, CA, USA). Phenylalanine and tyrosine enrichments were measured using the tert-butyldimethylsilyl derivative and gas chromatography-mass spectrometry (models 7890A/5975; Agilent Technologies, Santa Clara, CA, USA). Ions of mass-to-charge ratio of 234, 235, and 239 for phenylalanine and of 466, 467, 468, and 470 for tyrosine were monitored with electron impact ionization and selective ion monitoring.\u003c/p\u003e\n\u003ch3\u003eMethylation ratio\u003c/h3\u003e\n\u003cp\u003eFor the measurement of S-adenosylmethionine and S-adenosylhomocysteine, 50 \u0026micro;L cell lysate samples were extracted in 150 \u0026micro;L cold acetonitrile, vortexed, centrifuged, and the 150 \u0026micro;L supernatant was diluted with the 125 (600 \u0026micro;L) or 200 ng/mL (150 \u0026micro;L) internal standard solution. These different concentrations and volumes of internal standard are used to normalize based on protein concentration but still result in 100 ng/mL internal standards.\u003c/p\u003e \u003cp\u003eLC-MS/MS analysis was run on the Agilent Ultivo triple quad coupled to the Agilent Infinity II 1290 with electrospray ionization in positive mode. The injection volume was 10 \u0026micro;L, an Acquity XSelect HSS T3 column (2.5 \u0026micro;m, 2.1 x 100 mm) was used at 25\u0026deg;C with a flow rate of 0.4 mL/min. Mobile phase A was 5 mM PFHA in water, mobile phase B was acetonitrile, with a gradient from 95% A down to 5% A and back up to 95% A over the course of 15 minutes. The analysis was run by Multiple Reaction Monitoring (MRM) with a dwell time of 100 ms, source gas temperature, flow rate, nebulizer pressure, and capillary voltage of 350\u0026deg;C, 13 L/min, 22 psi, and 4000 V, respectively.\u003c/p\u003e\n\u003ch3\u003eRadiation Clonogenic Assays\u003c/h3\u003e\n\u003cp\u003eCells were trypsinized to generate single cell suspensions and seeded onto 60 mm tissue culture plates in triplicate. Cells were then irradiated with various doses (0\u0026ndash;6 Gy). Ten to 14 days after seeding, colonies were fixed with Methanol/Acetic Acid, stained with 0.5% crystal violet and the numbers of colonies or colony forming units (CFU) containing at least 50 cells were counted using a dissecting microscope (Leica Microsystems, Inc.. Buffalo Grove, IL) and surviving fractions calculated. Experiments were repeated multiple, independent times.\u003c/p\u003e\n\u003ch3\u003ePhosphoproteomics\u003c/h3\u003e\n\u003cp\u003eCell pellets were lysed in 0.1 mL of RIPA buffer (Pierce 89900) with protease and phosphatase inhibitor cocktails. Phosphoproteomics sample preparation followed Storey et al. (PMC7423749), where proteins were reduced, alkylated, and digested with trypsin/LysC (Promega VA5071) using Filter-Aided Sample Preparation. Peptides were labeled using TMT 10-plex reagents (Thermo 90113). Most peptides (90%) were enriched using TiO\u003csub\u003e2\u003c/sub\u003e and Fe-NTA phosphopeptide kits (Thermo A32993, A32992), and 10% were used for total proteome analysis. Enriched and un-enriched peptides were separated into 46 fractions on an Acquity BEH C18 column (Waters) using a 50-min gradient, then consolidated into 18 super-fractions. Each super-fraction was further separated on an XSelect CSH C18 column (Waters) using a 60-min gradient. Eluted peptides were ionized by electrospray (2.2 kV) and analyzed on an Orbitrap Fusion Lumos (Thermo) using MS3 with multi-notch parameters. MS data were acquired with the FTMS analyzer in top-speed mode at 120,000 resolution (375\u0026ndash;1500 m/z), followed by MS/MS with CID (31.0 normalized collision energy) and HCD (55.0 normalized collision energy) activation. MS3 reporter ion data were acquired at 50,000 resolution (100\u0026ndash;500 m/z).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMS Data Analysis\u003c/h2\u003e \u003cp\u003eProteins were identified, and TMT MS3 reporter ions quantified by searching the UniprotKB Homo sapiens database (June 2018) using MaxQuant (version 1.6.0.16) with a parent ion tolerance of 3 ppm, fragment ion tolerance of 0.5 Da, reporter ion tolerance of 0.001 Da, trypsin/P enzyme with two missed cleavages, and variable modifications including oxidation on M, acetyl on protein N-terminus, phosphorylation on STY, and fixed carbamidomethyl on C. Protein and peptide identifications were accepted with less than 1.0% false discovery. TMT MS3-corrected reporter ion intensity values were analyzed for total protein changes using unenriched lysate samples, while phospho(STY) modifications were analyzed using enriched phosphorylated peptides. The enriched and unenriched samples were multiplexed with two separate TMT10-plex batches.\u003c/p\u003e \u003cp\u003eFollowing data acquisition, the results were normalized, and sample quality verified using ProteiNorm. Protein and phosphopeptide data were normalized using cyclic loss. Linear models were fitted to the expression data using limma 3.46.0, and differential abundance was evaluated using robust empirical Bayes (eBayes). Proteins and phosphopeptides with a fold-change\u0026thinsp;\u0026gt;\u0026thinsp;2 and an FDR adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Phosphosites were filtered for a localization probability\u0026thinsp;\u0026gt;\u0026thinsp;75%, peptides with zero values were excluded, and data were log2-transformed before differential abundance analysis. Gene set enrichment analysis was performed using Ensemble of Gene Set Enrichment Analyses, and modified phosphosite-flanking peptides were evaluated using PHOXTRACK to identify kinases and their substrates, assessing kinase activity/enrichment with 50,000 permutations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene expression\u003c/h3\u003e\n\u003cp\u003eFrom frozen sections of the distal colon of about 20 mg, we extracted RNA with the QIAzol lysis reagent using a Dounce homogenizer according to the manufacturer\u0026rsquo;s directions. We washed the RNA pellets with two 75% ethanol washes. We resuspended the RNA pellets in water and quantified the solutions with spectrophotometry (Nanodrop One, Thermo Fisher Scientific). We reverse transcribed 1 \u0026micro;g of each RNA sample into cDNA using the iScript RT Supermix (Bio-Rad, Hercules, CA). We diluted the cDNA at a final concentration of 5 ng/\u0026micro;L and used 20 ng for each real-time quantitative PCR reaction. Gene expression was determined in technical duplicates using the iTaq Universal SYBR\u0026reg; Green Supermix according to the manufacturer\u0026rsquo;s instructions. We performed amplification on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad). We increased the number of cycles of amplification to 50 to detect low expression in treated samples for the gene Tnf. We analyzed the raw data using the ΔΔCT method relative to the internal control RPLP0.\u003c/p\u003e\n\u003ch3\u003eKRAS knockdown\u003c/h3\u003e\n\u003cp\u003eIn order to ascertain the role of KRAS in the regulation of downstream targets, we downregulated mRNA levels with siRNAs. siRNAs targeting KRAS and a scrambled control (Silencer Select #4390771 s7940, and 4390843, respectively, Thermo Fisher Scientific) were used at a final concentration of 30 nM for a reverse transfection in 6-well format in triplicates. siRNA were mixed with lipofectamine RNAiMAX Transfection Reagent and Opti-MEM I reduced serum medium (ThermoFisher Scientific) and added to the wells, then 140,000 cells per well was added to the mixture in media devoid of antibiotics. After 24 hr, the media was changed for test media. After 24 hr, RNA was extracted and gene expression analysis was performed as described above.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eCell lysates were prepared using RIPA buffer (ThermoFisher, Waltham MA) supplemented with 1x protease (Complete, Roche, Indianapolis, IN) and phosphatase inhibitors (PhosSTOP, Roche, Indianapolis, IN, Roche) followed by protein quantification by the Dc protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded and resolved by SDS/PAGE and transferred to nitrocellulose membranes. Primary antibodies including γH2A.X, RAD51, CTIP, and GAPDH (Cell Signaling, Danvers, MA) were allowed to bind overnight at 4\u0026deg;C, and used at a dilution of 1:100-1,000. After washing in TBS-Tween, membranes were incubated with StarBright Goat Anti-Mouse/Rabbit IgG secondary antibodies (Bio-Rad) diluted 1:2,500-1:5,000 for 1 hour. Membranes were washed with TBS-Tween and then imaged on the ChemiDoc MP Imaging System (Bio-Rad Hercules, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDNA damage foci\u003c/h2\u003e \u003cp\u003eFor γH2AX, cells were grown on cover slips within a 10 mm petri dish and then irradiated as described above and then fixed with 2% paraformaldehyde at varying time points, permeabilized with 1% Triton X-100 and blocked with 3% bovine serum albumin (BSA) in PBS. Cells were stained with anti-phospho-H2AX (Cell Signaling, Danvers, MA), washed and incubated with a fluorophore-conjugated secondary antibody (Fisher Scientific, Waltham, MA). Cells were imaged on a confocal microscope (Leica Microsystems, Wetzlar, Germany). For each experiment the total number of foci per cell was determined in at least 100 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOrthotopic tumor model\u003c/h2\u003e \u003cp\u003e All mice experiments were performed under the approved institutional IUCUC protocol #IPROTO202200000033. Athymic mice (NU/J, 5\u0026ndash;6 weeks old, The Jackson Laboratory, Bar Harbor, MA) were injected intrarectally with 1\u0026times;10⁵ cells from one of four cohorts: HCT116 KRAS\u003csup\u003eG13D/\u0026minus;\u003c/sup\u003e, HCT116 KRAS\u003csup\u003eWT/\u0026minus;\u003c/sup\u003e, SW48 KRAS\u003csup\u003eG13D/\u0026minus;\u003c/sup\u003e, or SW48 KRAS\u003csup\u003eWT/\u0026minus;\u003c/sup\u003e. Each cohort included 20 animals (5 males and 5 females per diet group: radiation plus control diet or radiation plus methionine-restricted (MR) diet, 0.12% vs. 0.65% methionine). Diets began at injection, and one week later, all mice received five daily fractions of 5 Gy X-ray radiation (SARRP) with CBCT-guided treatment planning. Mice were anesthetized with inhaled isoflurane during imaging and radiation delivery. One-week post-irradiation, they returned to a maintenance diet. Tumors were imaged by bioluminescence at baseline and four weeks later. Complete response (CR) was defined as no tumor growth or detectable luminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAbdominal Irradiation\u003c/h2\u003e \u003cp\u003eThis study was approved by the UAMS IACUC (protocol #4119). C57BL/6 mice (3\u0026ndash;4 months old) were bred on-site and housed under standardized conditions. They were fed a standard diet before being switched to either a methionine-restricted (MR) diet (0.12% methionine) or a control diet (0.65% methionine). After one week, mice underwent local abdominal irradiation (12.5 Gy, SARRP) with CBCT-guided targeting. After seven days, intestinal tissues (segment of proximal jejunum) were collected and fixed in Methanol Carnoy\u0026rsquo;s reagent, processed, and stained with hematoxylin and eosin (H\u0026amp;E) for histological and morphometric analysis. Slides were scanned using an Aperio Scanner CS2 at 20\u0026times; magnification (Leica Biosystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVillous height and crypt depth measurements\u003c/h2\u003e \u003cp\u003eAssessments of mucosal villus height and crypt depth were obtained by using Image J software (NIH, US). Measurements of villus height and crypt depth were carried out on images captured at 20\u0026times; magnification in five to six areas of intestinal segments. As described previously, mucosal villus height was measured from the tip to the base of each villus, while crypt depth was measured from the crypt base to the tip opening (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In each sample, the average of the measurements was used as a single value for statistical analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e experiments, data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For \u003cem\u003ein vitro\u003c/em\u003e studies, unless specified otherwise, each experiment was conducted in triplicate with a minimum of two independent biological replicates. Statistical comparisons were made between the control and experimental conditions using the two-sided, two-group t-tests with significance assessed at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKRAS mutations change metabolites levels in the methionine cycle\u003c/h2\u003e \u003cp\u003eTo explore metabolic differences associated with KRAS mutations in rectal cancer, we performed an untargeted metabolomics analysis of KRASmut versus KRASwt CRC cells. For these studies, we utilized commercially available CRISPR-engineered HCT116 (heterozygous KRAS G13D cell line) human CRC cells with a knockout of either the wild-type KRAS allele (G13D/-) or a monoallelic knockout of the KRAS G13D allele (WT/-). This analysis identified 14 significantly altered metabolites, highlighting key disruptions in metabolic pathways relevant to tumor progression and therapeutic targeting (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, three metabolites within the \u0026ldquo;sphingosine\u0026rdquo; pathway, a lipid signaling network implicated in cellular stress responses and tumorigenic adaptation to radiation (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), were elevated in KRAS mutant cells. Additionally, we observed significant alterations in amino acid metabolism, with six metabolites belonging to the amino acid super pathway, including three decreased metabolites within the \u0026ldquo;methionine, cysteine, SAM, and taurine metabolism\u0026rdquo; pathway\u0026mdash;S-methylmethionine, and S-adenosylhomocysteine, and cysteine. These findings suggest a potential metabolic reprogramming in KRASmut rectal cancer cells that may influence tumor biology and response to therapy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferentially Altered Metabolites in KRASmut and KRASwt CRC Cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlutamate Metabolism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS-1-pyrroline-5-carboxylate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHistidine Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1-methylhistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethionine, Cysteine, SAM and Taurine Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS-methylmethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethionine, Cysteine, SAM and Taurine Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS-adenosylhomocysteine (SAH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethionine, Cysteine, SAM and Taurine Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUrea cycle; Arginine and Proline Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN,N,N-trimethyl-alanylproline betaine (TMAP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphingolipid Synthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esphingadienine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphingosines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esphingosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphingosines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehexadecasphingosine (d16:1)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLipid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphingosines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eheptadecasphingosine (d17:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNucleotide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePyrimidine Metabolism, Uracil containing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euracil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCofactors and Vitamins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRiboflavin Metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eflavin adenine dinucleotide (FAD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXenobiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFood Component/Plant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebeta-guanidinopropanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXenobiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChemical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ethioproline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGiven the established link between methionine metabolism and tumor response to therapy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) we investigated whether KRAS mutations influence methionine-related metabolites. To test this, we employed two complementary approaches. First, we quantified amino acid concentrations in cell lysates using liquid chromatography-mass spectrometry, analyzing both sham and irradiated cells. Consistent with our untargeted metabolomics findings, methionine levels were highest in WT cells and decreased in KRAS G13D cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, irradiation (5 Gy) reduced methionine levels in WT cells compared to sham RT, whereas no significant difference was observed between irradiated and sham-treated KRAS G13D cells. Interestingly, a similar pattern was observed for several essential amino acids, including histidine, isoleucine, leucine, phenylalanine, tryptophan, and valine. Notably, cysteine was excluded from this analysis due to its high propensity for oxidation, which can affect measurement accuracy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined methionine metabolism by quantifying S-adenosylmethionine (SAM), a key methyl donor, and S-adenosylhomocysteine (SAH) to determine the methylation ratio (SAM/SAH). SAM levels were significantly elevated in KRAS G13D cells compared to WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, p\u0026thinsp;=\u0026thinsp;0.0025 for genotype), while SAH levels remained unchanged in the targeted analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Consequently, the methylation ratio was higher in KRAS G13D cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), with no observed effect of irradiation. These results suggest that KRAS-driven metabolic alterations facilitate the conversion of methionine to S-adenosylmethionine (SAM), potentially influencing cellular methylation dynamics and tumor biology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate these findings, we employed RNA interference (siRNA) to reduce KRAS expression in HCT116 KRAS G13D mutant cells, resulting in approximately a 70% decrease in KRAS gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consistent with previous reports (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), KRAS knockdown reduced the DNA repair protein RAD51, reinforcing the role of KRAS in promoting DNA repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). MR also reduced RAD51 expression compared to control conditions (200 \u0026micro;M methionine) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting a link between methionine availability and DNA repair regulation.\u003c/p\u003e \u003cp\u003eAdditionally, KRAS knockdown decreased the expression of methionine adenosyltransferase (MAT2A), further supporting the notion that KRAS activity enhances SAM synthesis from methionine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Unexpectedly, we also observed a reduction in SLC7A5 expression, a key transporter responsible for importing large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, histidine, and tyrosine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This suggests that oncogenic KRAS drives methionine metabolism and enhances amino acid import, potentially influencing metabolic reprogramming in tumor cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMethionine restriction affects cell death DNA damage in KRAS mutant cells\u003c/h2\u003e \u003cp\u003eGiven the observed alterations in methionine metabolism in KRAS mutant cells, we investigated how methionine availability impacts cellular signaling using phosphoproteomics. Comparing HCT116 KRAS G13D mutant cells grown in control versus MR media, total protein analysis identified 54 differentially expressed proteins (\u0026ge;\u0026thinsp;2-fold, adjusted p\u0026thinsp;\u0026le;\u0026thinsp;0.05), with pathway analysis indicating cell cycle changes. Notably, MAT2A expression increased, confirming the efficacy of MR treatment (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Expanding to phosphoproteomic analysis, nearly 1,000 proteins exhibited altered phosphorylation (\u0026ge;\u0026thinsp;2-fold), which we refined to 192 proteins using a\u0026thinsp;\u0026ge;\u0026thinsp;5-fold threshold. Enriched pathways included apoptosis, cell cycle checkpoints, ATM signaling, Rho GTPase cycling, pre-mRNA processing, and non-homologous end joining (NHEJ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). DNA repair proteins H2AX, NSD2, PRKDC, TP53BP1, XRCC5, and XRCC6 showed significant changes in phosphorylation, including a 6-fold increase in S139 phosphorylation of H2AX, a key DNA damage marker. ATM, the primary kinase for this modification, was also implicated. Additionally, XRCC6 (Ku70) phosphorylation at S2 increased 40-fold, and XRCC5 (Ku80) phosphorylation at T472 rose 5-fold, while CHEK1 phosphorylation at S284 decreased. These findings suggest that MR disrupts DNA repair and promotes cell death in KRAS mutant cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKRAS mutant colorectal cancer cells are sensitized to radiation when methionine levels are low\u003c/h2\u003e \u003cp\u003eTo determine whether dysregulation of the methionine pathway is a vulnerability in KRASmut cells, we irradiated HCT116 KRAS G13D mutant cells\u0026thinsp;\u0026plusmn;\u0026thinsp;MR with ionizing radiation. In addition to HCT116 KRAS G13D and KRAS +/- WT, we also tested human colorectal SW48 cells. SW48 parental cells are wildtype for KRAS, and we also used a genetically engineered SW48 derivative cell line expressing KRAS G13D. In KRAS WT HCT116 and SW48 cells, there was little to no difference in the fractional survival to doses of radiation from 0 and 6 Gy between cells grown in control media containing 200 \u0026micro;M methionine (CTRL) or in media containing 5 \u0026micro;M MR. However, in KRAS G13D cells for both cell lines, colony survival was decreased in irradiated cells grown in MR media compared to CTRL media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This indicated that MR treatment sensitized KRAS mutant cells to IR.\u003c/p\u003e\u003cp\u003eTo investigate why survival was decreased by MR in irradiated KRAS mutant cells, we measured ɣH2A.X, a marker of DNA double strand breaks. We used two different detection methods: western blotting and the detection of foci by microscopy. We used the parental and KRAS mutant engineered HCT116 and SW48 cells. We pretreated the cells with CTRL or MR media for 24h. With this pretreatment, we observed a modest increase in ɣH2A.X in KRAS WT cells in MR versus CTRL. However, ɣH2A.X expression was strongly increased in MR compared to CTRL in KRAS G13D cells, before (0h) or after 5 Gy of X-ray radiation (\u003cstrong\u003eFig. 5A-B\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e A dose of 5 Gy was chosen to mimic the cellular repair response following each fraction in a 5 \u0026times; 5 Gy fractionated radiation therapy regimen, replicating a standard RT regimen in humans (33,34). We also measured RAD51 and CTIP protein expression in these samples by western blot. Both RAD51 and CTIP expression followed an inverse pattern to ɣH2A.X, where it was decreased in MR samples (Supplementary Figure 1). The MR inhibition of HR repair proteins was more pronounced in the KRAS mutant cells compared to WT. This suggests that MR impairs DNA repair mechanisms selectively in KRAS mutant cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e \u003cb\u003eMethionine restriction improves the tumor response to radiation in vivo\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next tested whether the decrease in survival and increase in DNA damage would impact the development of tumors \u003cem\u003ein vivo\u003c/em\u003e. For this, we used an orthotopic rectal cancer model. Nude mice were injected intrarectally with HCT116 and SW48, KRAS WT and G13D cells. All mice underwent image-guided localized RT treatment, which involved the delivery of 5 Gy once daily for five consecutive days (25 Gy total). One week before irradiation, mice were placed on a MR diet (0.12% methionine) or a control diet containing a standard amount of 0.65% methionine. Diets were maintained during the week of irradiation. The animals were imaged with bioluminescence four weeks after radiation treatment to estimate tumor response to radiation (Supplementary Fig.\u0026nbsp;2). In KRAS WT tumors, there was no difference in the rate of complete responders in the MR diet compared to the CTRL diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C). In the KRAS G13D tumors, however, we observed an increase in the complete response rate in the mice treated with an MR diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, D). These results indicate that the response rate to irradiation in KRAS mutant tumors is improved with MR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMethionine restriction protects non-cancer cells in vitro and in vivo\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the observed benefit of an MR diet on tumor response, we examined its effect on healthy, non-cancer cells. We focused on the normal fibroblasts in the tissue surrounding the tumor. To assess potential toxicity, we exposed Hs738.St/Int human intestinal fibroblasts to either sham or 5 Gy in CTRL or MR media. Unlike cancer cells, we observed no increase in γH2A.X staining in fibroblasts when methionine was omitted from the media (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). γH2A.X levels were slightly reduced in the MR group. This suggests that lowering methionine in the media may protect normal gut cells from radiation-induced DNA damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(A) Methionine restriction in the media decreased the abundance of γH2A.X in human Hs 738.St/Int fibroblasts.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(B) One month after receiving 5x5 Gy X-ray treatment, Tnf expression was reduced in the colon of mice on an MR diet compared to a control diet.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(C) Representative H\u0026amp;E staining of the proximal jejunum from C57BL/6 mice 7 days after an acute 12.5 Gy X-ray dose, comparing MR and control diets.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(D) Measurement of villous height and crypt depth from the images in panel C.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eRadiation therapy can induce fibrosis in the surrounding tissues over several months. In our irradiated orthotopic tumor experiment, tissues were collected one month post-irradiation, a time-point where we did not expect detectable collagen accumulation. However, we investigated potential fibrosis-related changes by measuring Tnf gene expression, which encodes tumor necrosis factor, in the distal colon of our irradiated tumor-bearing mice. Tnf expression was lower in the MR diet group than the control diet group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating reduced inflammation in the colon of MR mice.\u003c/p\u003e \u003cp\u003eTo further investigate the impact of MR on healthy tissue \u003cem\u003ein vivo\u003c/em\u003e, we used an immunocompetent C57BL/6 mouse model. Mice were fed either a CTRL or MR diet for one week, followed by a single 12.5 Gy dose of abdominal irradiation. We collected tissue from the proximal jejunum, an organ particularly sensitive to radiation, on day 7 post-radiation. Histological analysis of CTRL diet tissue showed expected damage, including reduced villous and crypt integrity. In contrast, tissue from MR diet animals maintained better morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Measurement of villous height revealed a two-fold increase in MR animals compared to CTRL animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Crypt depth was also significantly deeper in the MR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results indicate that the MR diet may preserve the intestinal epithelium's integrity following irradiation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study provides strong evidence that KRAS mutations create a distinct dependence on elevated methionine levels compared to KRASwt cells. We validated prior findings linking KRAS mutations to altered methionine metabolism, as demonstrated by Gao et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and explored the therapeutic implications of this relationship. Notably, our analysis of cell viability under varying methionine conditions revealed a heightened sensitivity of KRASmut cells to MR. Compared to KRASwt cells, KRASmut cells exhibited a more pronounced radiosensitivity under MR conditions. This effect correlated with increased DNA damage marker γH2AX levels and decreased expression of repair proteins such as RAD51. Phosphoproteomic analysis further confirmed impaired double-strand break repair in KRASmut cells following MR.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e experiments using an orthotopic rectal cancer model provided translational relevance to our findings. Mice with KRAS mutant tumors exhibited an increased rate of complete responders to radiation therapy when maintained on an MR diet compared to the control diet.\u003c/p\u003e \u003cp\u003eNotably, our study considered the impact of MR on non-cancer cells. Contrary to the observed sensitization of KRAS mutant cells, normal fibroblasts exposed to radiation in MR media showed no increase in ɣH2A.X staining and displayed a slight decrease. This indicates a protective effect of MR on healthy cells, supporting previous observations by Wanders et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), highlighting the potential for a selective therapeutic approach.\u003c/p\u003e \u003cp\u003eIn an immunocompetent model, we further demonstrated the protective effect of MR on the intestinal epithelium following irradiation. The maintenance of tissue morphology, increased villous height, and reduced crypt depth in the MR group underscore the potential benefits of this dietary intervention in minimizing radiation-induced damage to normal tissues. We also offer some preliminary indications that MR may mitigate inflammation in irradiated gut tissues, as seen in the reduction in Tnf expression three weeks after irradiation. This adds to growing evidence of MR protective effects on normal tissue during radiation therapy (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe implementation of MR in human patients in combination with radiation remains to be explored. A phase 1 trial evaluated the safety and feasibility of an MR diet in combination with radiation therapy in patients with non-skin cancers was attempted. While no grade 3 or higher toxicities were observed, poor patient adherence led to early trial closure, highlighting the challenge of implementing MR in clinical settings despite its potential to enhance RT efficacy (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these promising findings, our study has several limitations. First, while our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models provide strong preclinical evidence, the translational potential of MR in combination with radiation therapy remains uncertain due to the lack of clinical validation. Second, the metabolic adaptations of KRAS mutant tumors to prolonged MR were not fully explored, which may influence long-term treatment efficacy and resistance mechanisms. Additionally, our immunocompetent model did not fully characterize the effects of MR on the tumor microenvironment, including potential interactions with immune cells. Finally, the feasibility challenges observed in the pilot clinical trial (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) highlight the need for alternative strategies, such as pharmacologic methionine depletion, to improve patient adherence and clinical applicability. Future studies should address these limitations to refine the therapeutic potential of MR in KRAS-driven cancers.\u003c/p\u003e \u003cp\u003eOur comprehensive study sheds light on the intricate interplay between KRAS mutations, methionine metabolism, and radiation response. The findings propose a novel therapeutic strategy for KRAS mutant tumors and emphasize the protective effects of MR on healthy tissues, opening avenues for further exploration of dietary interventions in cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll models are available to academic centers under an institutional MTA\u003c/p\u003e\n\u003cp\u003eFinancial support and sponsorship\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Winthrop P. Rockefeller Cancer Institute (WPRCI), the Arkansas Tobacco Settlement Commission, the National Center for Advancing Translational Sciences grant number KL2TR003108 (ARW), the American Cancer Society and American Society for Radiation Oncology ASTRO-CSDG-23-1037280-01-CDP (ARW) and NIGMS grant P20 GM139768-01 (IRM). The National Resource for Quantitative Proteomics is supported by the NIGMS grant R24GM137786. The UAMS Bioinformatics core is supported by NIGMS grant P20GM121293 and WPRCI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eAll authors declared that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate:\u003c/strong\u003e Experiments were conducted using protocols and conditions approved by the Institutional Biosafety Committee at UAMS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranslational Relevance:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this study highlight the potential of methionine restriction (MR) as a therapeutic strategy to sensitize rectal cancer to radiation therapy. Our findings suggest that MR-induced metabolic alterations in cancer cells can enhance the efficacy of radiation treatment, overcoming resistance mechanisms that limit current therapeutic options. By identifying key metabolic pathways affected by MR, we provide insights into novel strategies for potentiating radiation response in rectal cancer, particularly in KRAS-mutant tumors. These results have significant clinical implications, offering the potential to develop combination therapies that integrate MR with conventional treatment regimens. Such therapies could improve treatment outcomes, reduce toxicity to normal tissues, and provide personalized, targeted approaches for patients with rectal cancer. Ultimately, this research paves the way for advancing precision medicine, particularly in the context of radiation oncology, where metabolic reprogramming could be exploited to optimize therapeutic efficacy and overcome treatment resistance in colorectal cancers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang L, Guo Z, Wang F, Fu L. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther. Nature Publishing Group; 2021;6:1\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eAndreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer: the multicenter \u0026ldquo;RASCAL\u0026rdquo; study. J Natl Cancer Inst. 1998;90:675\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eChatila WK, Kim JK, Walch H, Marco MR, Chen C-T, Wu F, et al. Genomic and transcriptomic determinants of response to neoadjuvant therapy in rectal cancer. 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Methionine Restriction Extends Lifespan in Progeroid Mice and Alters Lipid and Bile Acid Metabolism. Cell Rep. 2018;24:2392\u0026ndash;403. \u003c/li\u003e\n\u003cli\u003eFeng C, Jiang Y, Wu G, Shi Y, Ge Y, Li B, et al. Dietary Methionine Restriction Improves Gastrocnemius Muscle Glucose Metabolism through Improved Insulin Secretion and H19/IRS-1/Akt Pathway in Middle-Aged Mice. J Agric Food Chem [Internet]. American Chemical Society; 2023 [cited 2023 Apr 3]; Available from: https://doi.org/10.1021/acs.jafc.2c08373\u003c/li\u003e\n\u003cli\u003eLuo T, Yang Y, Xu Y, Gao Q, Wu G, Jiang Y, et al. Dietary methionine restriction improves glucose metabolism in the skeletal muscle of obese mice. Food Funct. The Royal Society of Chemistry; 2019;10:2676\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003eMiller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell. 2005;4:119\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eWanders D, Forney LA, Stone KP, Burk DH, Pierse A, Gettys TW. FGF21 Mediates the Thermogenic and Insulin-Sensitizing Effects of Dietary Methionine Restriction but Not Its Effects on Hepatic Lipid Metabolism. Diabetes. 2017;66:858\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eMalloy VL, Krajcik RA, Bailey SJ, Hristopoulos G, Plummer JD, Orentreich N. Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction. Aging Cell. 2006;5:305\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eGarg S, Garg TK, Miousse IR, Wise SY, Fatanmi OO, Savenka AV, et al. 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Nutrients. 2020;12:684. \u003c/li\u003e\n\u003cli\u003eMiousse IR, Ewing LE, Skinner CM, Pathak R, Garg S, Kutanzi KR, et al. Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome. Am J Physiol - Gastrointest Liver Physiol. 2020;318:G439\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eMattes MD, Koturbash I, Leung CN, Wen S, Jacobson GM. A Phase I Trial of a Methionine Restricted Diet with Concurrent Radiation Therapy. Nutr Cancer. Routledge; 2024;0:1\u0026ndash;6. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6497576/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6497576/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately one-third of colorectal cancer cases involve the rectum, where radiation therapy is an integral part of the treatment of this disease. However, KRAS mutations are associated with poor clinical outcomes and lower therapeutic responses. Previous studies suggest that KRAS mutations may alter the balance of metabolites in the methionine cycle. This study investigates the interplay between the methionine cycle, KRAS mutations, and radiation therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined the impact of the KRAS mutation and radiation on methionine cycle metabolites. \u003cem\u003eIn vitro\u003c/em\u003e, we reduced methionine levels in the culture media and assessed the radiosensitivity of KRAS-mutant colorectal cancer cells. \u003cem\u003eIn vivo\u003c/em\u003e, we used an orthotopic mouse model with KRAS-mutant rectal tumors to evaluate the effects of a methionine-restricted (MR) diet on tumor response to radiation. Additionally, we assessed the impact of MR on normal human intestinal epithelial cells and tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e, MR increased the radiosensitivity of KRAS-mutant cells, with reduced proliferation and increased DNA damage markers following radiation. \u003cem\u003eIn vivo\u003c/em\u003e, KRAS mutant tumors in mice fed an MR diet showed an increased response rate to radiation compared to KRASwt tumors. Normal cells and tissues showed reduced DNA damage markers under MR conditions, with MR diet improving villus height and crypt depth following abdominal irradiation in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKRAS-mutant rectal cancer cells rely on methionine for growth, and MR enhances tumor radiosensitivity while protecting normal tissues from radiation-induced damage. These findings suggest that MR may serve as a potential therapeutic strategy to improve treatment outcomes for rectal cancer, particularly in KRAS-mutant tumors.\u003c/p\u003e","manuscriptTitle":"Enhancing Rectal Cancer Radiosensitivity and Gut Protection through Methionine Restriction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:22:13","doi":"10.21203/rs.3.rs-6497576/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0063b73-36b4-4996-82c4-31a619cce6d3","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-20T00:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 06:22:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6497576","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6497576","identity":"rs-6497576","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}