P53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression in response to thymidylate synthase (TS) targeting

Nucleotide metabolism in cancer cells can influence malignant behavior and intrinsic resistance to therapy. Here we describe p53-dependent control of the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU treatment of cells. Consequently, decrease in Histone H3K9AC and increase in H3K27me3 marks at the DPYD promoter are observed concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition and DPYD repression by p53 is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. In-vivo, liver specific Tp53 loss increases the conversion of 5-FU to 5-FUH2 in plasma and elicits a diminished 5-FU therapeutic response in a syngeneic colorectal tumor model consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through repression of DPYD expression, following metabolic stress imposed by nucleotide imbalance. These findings have implications for the toxicity and efficacy of the cancer therapeutic 5-FU.

Measurement of free dUTP in cell culture in vitro. The measurement of dUTP has been previously described by Wilson et al. 2011 22 .
For a more detailed description of the methods see supplementary information.

Statistical analysis.
All results are presented as the mean ± SEM of data. Statistical analyses were performed by the Student's-t-test (Two tail) and P values as mentioned in the figures were analyzed by Graph Pad Prism V5, unless otherwise mentioned in figure legends. Western blots are representative images of 3 independent experiments. All other experiments were conducted in triplicate unless otherwise mentioned in figure legends.

Results
Fluorouracil (5-FU) has been a mainstay of cancer chemotherapy for over 50 years. Based on prior work implicating p53 in 5-FU induced apoptosis 19 , we hypothesized that p53 may regulate the cellular toxicity of 5-FU potentially through effects on 5-FU metabolism. In order to gain insight into how p53 could influence metabolic pathways with particular relevance for the cellular response to 5-FU, we employed an in-silico approach [Genomatix (http://www.Genotmatix.de/index.html)] to identify genes with potential p53 binding sites (p53BS) within and ± 20 kb upstream and downstream of the genes involved in 5-FU metabolism. We screened for p53 binding as defined by the presence of two copies of 5′-PuPuPu-CWWG-PyPyPy-3′ with or without an intervening spacer 23 . We found an enrichment of genes encoding rate-limiting enzymes in the pyrimidine catabolic pathway with p53BS. These include enzymes involved in 5-FU metabolism, whose regulation currently is poorly understood. Three genes in particular stood out, having multiple p53BS within either the 5′ promoter region or within gene introns; DPYS, DPYD and UBP1. We focused on DPYD as the key rate-limiting enzyme in the pyrimidine catabolic pathway.
P53 binds to a conserved high-affinity p53BS downstream of the DPYD gene and represses gene expression. Our in silico screen identified several putative half-and full-p53BS in the DPYD gene.
However, based on the highest scoring matrix conforming to the consensus binding motif, we selected six (6) putative p53BS for further characterization (Fig. 1A). ChIP analysis on genomic DNA isolated from the livers of mice subjected to treatment with 5-FU showed enrichment for the downstream p53BS R-0.840 (Chr1: 119451237-119451256; 5′-ACA-CATG-CTC-CAC-CATG-TTC-3′) (Fig. 1B). R-0.840 was found to be conserved as a p53BS downstream (p53hDBS; chr1: 97299933-97299953; 5′-TGG-CTTG-CCT-GGG-CATG-CCT-3′) of the DPYD gene that was previously identified 24 . These two sequences are located at 18,319 bp (R-0.840) (p53DBS) and 15,955 bp (p53hDBS) downstream of the mouse and human DPYD genes respectively. In our ChIP analysis, we found that R-0.840 was enriched more than 2-fold following treatment of the mice with 5-FU (Fig. 1C). Thus it is clear that p53 increasingly occupies a p53BS downstream of the DPYD gene following 5-FU. We have previously established a mouse model for assessing the in vivo response to 5-FU-based toxicities and gene expression changes in the gut 25 . Using this model, we assessed the p53-dependent expression of DPYD in the liver, a key organ in the biotransformation of 5-FU. Following administration of IV 5-FU, we observed that liver DPYD mRNA and DPYD protein expression was reduced by 2.2-and 1.7-fold respectively in p53 +/+ as compared to liver mRNA and protein isolated from p53 −/− mice ( Fig. 2A,B). Thus, our data indicate that p53 can modulate the expression of DPYD by binding to a p53BS downstream of the DPYD gene.
The R72P polymorphism in TP53 modulates DPYD expression following 5-FU exposure. We hypothesized that polymorphisms in the TP53 gene that modulate the capacity of p53 to bind DNA could influence its ability to repress DPYD. Codon-72 polymorphism is a frequent polymorphism observed in the TP53 gene and is characterized by an arginine (R) or proline (P) substitution at codon position 72. The P72 variant of p53 is capable of increased DNA binding and activation of transcription of target genes 26 . To address the impact of the R72 and P72 alleles on inhibiting the expression of DPYD, we used MEF's isolated from humanized knock-in p53 mice (HUPKI) carrying variant alleles of the codon R72P polymorphism in TP53. Indeed, we found that the P72 allele of p53 suppresses DPYD expression when compared to the R72 p53 allele following 5-FU treatment in HUPKI MEF's (Fig. 2C). Furthermore, human fibroblasts taken from two different patients with carrying homozygous polymorphic alleles (P/P and R/R) confirmed the enhanced ability of the P72 TP53 allele to repress DPYD expression following 5-FU treatment (Fig. 2D). Taken together these results suggest that p53 inhibits This was observed at the protein level where a gradual increase in protein was noted over time following 5-FU treatment in the TP53 −/− cells (Fig. 3A, right panel and Fig. S7). This was further supported by immunofluorescence staining for DPYD whose expression decreased following 5-FU exposure ( Fig. S6A right panel). Since we observed increased p53 binding in-vivo in mouse liver to the conserved DNA-binding site, we evaluated the importance of the binding site to overall DPYD repression using HCT-116 WT cells. We knocked-out half of the binding site using CRISPR/Cas9 technology and analyzed for DPYD protein expression (Fig. S1). We found that repression of DPYD was not rescued suggesting this site alone is not sufficient to account for the observed repression. A possible reason is, since the DPYD gene is 850 kb in length, potentially there are other unknown p53 non-canonical binding regions that may cooperatively bring about transcriptional repression or there could be different mechanism involved inherent to tumor and normal cells. This would not at all be unexpected for a p53-regulated gene that typically harbors multiple p53 response elements scattered throughout the genomic sequence. This may include sites within promoters, several hundred base pairs or several Kb upstream of the promoters, within introns or several kb downstream of the gene. However, in order to confirm the generality of the p53-dependent DPYD repression after cellular exposure to 5-FU, we used the A549, H460, U87MG and HT-1080 cancer cell lines that are wild-type for TP53 and trigger the expression of canonical p53 target genes. Indeed, the mRNA and proteins levels of DPYD decreased at 24 hr following treatment of the lung cancer cell lines A549 and H460 cells with 5-FU as compared to cells subjected to siRNA targeting of TP53 ( Fig. 3B and Fig. S6A,B). In a similar manner, the U87MG (glioblastoma) and HT-1080 (fibrosarcoma) cell lines also repressed DPYD expression in a TP53-dependent manner (Fig. 3D,E,F). The correlative decrease of DPYD protein to its decrease in mRNA were not so apparent in the liver as it was in cancer cells, something which has previously observed [28][29][30][31] . To further investigate the transcriptional mechanism of DPYD repression, we evaluated H3K9 acetylation, H3K4me3 and H3K27me3 at the DPYD promoter region following 5-FU administration in HCT-116 and in H460 cells. H3K9 acetylation progressively decreased and was lowest at 24 hr following 5-FU treatment consistent with lower DPYD promoter activity (Fig. 3C). H3K27me3 was also increased at 24 hr but no changes were observed in H3K4m3 (Fig. 3G and Fig. S6C). Taken together our data indicate, regardless of the genetic or epigenetic background, that p53 negatively regulates the expression of the DPYD gene in human cells in vitro and that loss of TP53 abrogates the repression of DPYD expression.

Liver specific deletion of TP53 increases systemic catabolism of 5-FU and accelerates syngeneic tumor growth.
Approximately eighty (80) percent of administered 5-FU is eliminated through catabolism by hepatic DPYD 32 . In order to establish the significance of liver DPYD in limiting the bioavailability of intravenously (IV) administered 5-FU treatment we subjected mice to treatment with IV 5-FU in the presence or absence of the specific DPYD inhibitor gimeracil and monitored parameters of the acute toxic response to the drug (Fig. S2). Indeed, administration of gimeracil along with IV 5-FU triggered increased loss of body weight of mice over the course of ten (10) days as compared to IV 5-FU alone (Fig. S2A). The group of mice subjected to the 5-FU/gimeracil combination treatment were also more moribund (Fig. S2B). In addition, we monitored the levels of leukopenia and thrombocytopenia in mice subjected to the combination of 5-FU/gimeracil (Fig. S3). These data indicate that DPYD has a profound effect on the toxicity of accumulated high doses of 5-FU administered IV to mice in vivo.
We further explored if the repression of DPYD expression by p53 in vivo could modulate the pharmacokinetics of 5-FU. We hypothesized that targeting of DPYD-expression through deletion of TP53 in hepatocytes in vivo may impact the systemic bioavailability of 5-FU. To this end we generated mice with liver-specific deletion of the TP53 gene i.e., through the use of mice expressing Cre recombinase under the control of the Albumin promoter 33 . Indeed, Albcre;mT/mg;p53 Δ/Δ mice showed expression of Cre recombinase in hepatocytes and no histological changes were detected in other organs following deletion of TP53 in the liver (Fig. 4A). The plasma half-life of 5-FU is approximately ~20 min 34,35 . Therefore, in order to measure the impact of DPYD repression on 5-FU bioavailability, we followed a treatment schedule in which mice were either given Vehicle or 5-FU for the first 6 hrs, i.e. a time point at which DPYD expression is repressed (Fig. 2B), followed by second dose of 5-FU for 30 min. As expected, a lower ratio of 5-FUH 2 /5-FU was observed in Albcre;mT/mg;p53 Δ/+ mice as compared to the Albcre;mT/mG;p53 Δ/Δ mice (Fig. 4B,C). Western blot analysis revealed expression levels of DPYD in these mice consistent with our earlier observation of repression of DPYD by p53 (Fig. 4F). We further investigated whether the apparent decrease in 5-FU catabolism indicated by a lower plasma ratio of 5-FUH 2 /5-FU in the presence of wild-type TP53 could have a functional consequence on the in vivo therapeutic response to 5-FU. We used syngeneic (C57BL6/J) malignantly transformed mouse colonocytes to model colorectal cancer with  36 . p53dmc-Ras-Myc cells were injected subcutaneously in the flanks of mice and the mice were subjected to treatment with 5-FU (100 mg/kg/week for 6 weeks) with a follow-up time of up to 40 days. Indeed, Albcre;mT/mg;p53 Δ/+ mice showed a significant increase in the tumor doubling-time (vehicle versus 5-FU-treated tumor doubling-times were 6.22 versus 11.06 days, respectively) following treatment with 5-FU (Fig. 4D). In comparison Albcre;mT/mg;p53 Δ/Δ mice displayed a clearly blunted response to 5-FU when compared to vehicle-treated controls (vehicle compared to 5-FU-treated tumor doubling-times were 8.83 vs. 8.93 days, respectively). Furthermore, tumors from 5-FU-treated Albcre;mT/ mg;p53 Δ/+ mice exhibited increased levels of apoptosis and decreased levels of mitosis when compared to tumors from 5-FU-treated Albcre;mT/mg;p53 Δ/Δ mice (Fig. 4E). To eliminate the possibility of hepatotoxicity affecting the outcomes we analyzed liver sections from both 5-FU-treated and -untreated mice and found no differences in tissue histology, or any change in body weight (Fig. S4). Taken together these results strongly implicate p53 in controlling liver catabolism and therapeutic efficacy of 5-FU.

Repression of DPYD by p53 is a consequence of thymidylate synthase inhibition and thymidine deficiency.
To better our understanding of the upstream signaling events behind the p53-dependent repression of DPYD by p53 we asked whether this was specifically related to the activity of 5-FU or just simply part of the cellular DNA damage response (DDR). Treating HCT-116 cells with different chemotherapeutic agents indicated that the repression of DPYD was not a result of DNA damage per se since treatments with the Topoisomerase-I and -II poisons irinotecan (CPT-11) and etoposide respectively did not reveal a p53-dependent inhibition of DPYD expression (Fig. 5A). Both irinotecan and etoposide caused induction of DPYD expression in a somewhat  15 . We hypothesized that the effect of 5-FU to repress DPYD may stem from TS targeting and the resulting depletion of thymidine pools that may trigger p53 activation. To address this specifically, we treated HCT-116-WT and HCT116 TP53 −/− cells with 5-FU and Tomudex (Raltitrexed), a specific inhibitor of TS. 5-FU forms an inactive ternary complex with TS, which causes a slight upward shift in the TS band signifying TS inhibition 37 , whereas TS inhibitors such as Tomudex, Pemetrexed (PX; Alimta ® ) and methotrexate relieve feed-back inhibition of TS mRNA translation causing modest increases in TS protein expression [37][38][39][40] . We found a p53-and dose-dependent repression of DPYD expression following 5-FU and Tomudex-treatment (Fig. 5B). As an alternative approach to block TS, we employed the TS-inhibitor methotrexate, not a direct inhibitor per se and direct siRNA targeting TS. Treatment of H460 cells with methotrexate and with TS siRNA generated similar results of p53-dependent DPYD repression as seen with 5-FU and Tomudex (Fig. 5C). The results were further expanded to Pemetrexed (PX; Alimta ® ) and Fluorodeoxyuridine (FdU; floxuridine) that also selectively act on TS (Fig. 5D). As data using five (5) different inhibitors yielded similar results with respect to p53-dependent inhibition of DPYD expression, we sought to determine whether thymidine supplementation could overcome the p53-mediated repression of DPYD. As expected addition of exogenous thymidine restored DPYD protein expression following targeting of TS with siRNA (Fig. 5E). As a control experiment to directly verify the impact of siRNA-mediated targeting of TS we analyzed changes in intracellular nucleotide pools. Inhibition of TS is known to cause reduced conversion of dUMP to dTMP and as a result compensatory increased conversion of dUMP to dUTP 15 . We found the levels of free dUTP increase in cells subjected to siRNA-targeting of TS and 5-FU (Fig. 5F). Interestingly, targeting of TS by siRNA was approximately three (3) times more effective than 5-FU to generate increased levels of cellular dUTP, indicating the functional relevance of the TS-inhibition approach. Taken together the data suggest that following efficient TS-inhibition, p53 may specifically repress the expression of DPYD protein and downregulate pyrimidine catabolism.

p53-dependent repression of DPYD is dependent on ATM and DNA-PK signaling following TS inhibition.
A decrease in the thymidine pools can result in nucleotide imbalance and cause DNA damage that in turn may lead to p53 activation 41 . We hypothesized that blocking upstream DDR kinases that impact on p53 stabilization potentially could relieve inhibition of DPYD repression. We treated HCT-116 TP53 WT or TP53 −/− cells with inhibitors of ATM (KU-55933) or DNA-PK (NU7026) in the context of 5-FU-induced damage. We observed that in wild-type p53-expressing cells the 5-FU induced repression of DPYD was alleviated when cells were co-treated with the kinase inhibitors (Fig. 6A). By contrast, in TP53 −/− cells no change in the expression of DPYD was observed following co-treatment with KU-55933 or NU7026. We extended our analysis to include siRNA to TS (TSsi) and Tomudex, (Fig. 6B,C). The results indicate that following more selective targeting of TS, p53-dependent inhibition of DPYD expression requires functional DNA-PK and ATM. Despite some observed discrepancies between DNA damaging chemotherapeutics such as etoposide, CPT-11 and 5-FU in the triggering p53-dependent repression of DPYD (Fig. 5F), our data indicate that key DNA-damage signaling kinases such as ATM and DNA-PK are required to signal p53-dependent repression of DPYD expression following TS inhibition. In order to functionally validate how DPYD influences the cancer cell intrinsic response to TS inhibitory drugs we targeted DPYD expression in p53-null cancer cells with siRNA and subjected them to treatment with Tomudex and 5-FU. Knockdown of DPYD in HCT-116-p53 −/− cells significantly sensitized them to the toxic effects of 5-FU and Tomudex (Fig. 6D). Thus, it appears that elevated levels of DPYD may contribute to a resistance phenotype to TS inhibitors observed in cancers that have lost functional p53. Furthermore, this may indicate that DPYD can confer drug resistance to TS inhibitory drugs that is independent of systemic catabolism and reduced bioavailability.
DPYD mRNA expression correlates with poor disease free survival in colorectal cancer. We analyzed whether tumor DPYD mRNA expression could predict poor outcome in colorectal cancer patients treated with chemotherapy. Analysis of the GSE14333 data-set 42 in 226 colorectal cancer patients comprised mainly of Duke Stage B and C cancers revealed that high DPYD mRNA expression correlates with poor disease free survival as compared to those expressing low DPYD mRNA levels (Fig. 6E and Supplemental Table 2). Analysis of a TCGA cohort for correlation between TP53 and DPYD expression revealed a strong inverse trend, where higher TP53 expression showed lower DPYD expression along with poorer survival rate in patients having TP53 Low /DPYD High vs TP53 High /DPYD Low (Fig. S5). However, these analyses did not reach statistical significance.
Taken together, our data suggests that an imbalance in the cellular nucleotide pool resulting from reduced levels of thymidine required for the synthesis of DNA triggers p53-dependent inhibition of the key rate-limiting enzyme of pyrimidine catabolism DPYD which in turn reduces 5-FU catabolism (Fig. 7).

Discussion
We show for the first time that the p53 tumor suppressor protein controls 5-FU catabolism by repressing the expression of the key rate limiting enzyme in pyrimidine degradation DPYD. p53 has a well-documented function in the cell death response following 5-FU treatment in pre-clinical experiments in vitro and in vivo 19,43 . In clinical settings TP53 mutation status has been correlated with survival following 5-FU-based chemotherapy [17][18][19] . However, it has been difficult to identify key genes downstream of p53 that predict the 5-FU response in patients in vivo and TP53 mutation status may not necessarily be a predictor of the response to other chemotherapy agents. This may partly be due to the complexity of the mechanism of action of 5-FU that ranges from interference with both DNA and RNA synthesis through direct incorporation in such polynucleotide strands and subsequent repair thereof and inhibition of dTTP production through targeting of TS 44 .
A critical determinant of the efficacy of 5-FU treatment directly relates to the expression of key metabolic genes that are responsible for the biotransformation of the drug. DPYD is an important catabolic enzyme that limits the bioavailability of 5-FU to therapeutically relevant anabolic pathways and has been linked to 5-FU efficacy and toxicity 45 . We show that high expression levels of DPYD is linked to poor outcomes in colorectal cancer. Interestingly, the link we have uncovered between TP53 and DPYD suggests that expression of both of these genes may serve as markers in determining the response to 5-FU. Furthermore, observations in humanized TP53 knock-in mice and patient samples suggest that polymorphisms in the TP53 gene can influence the inhibition of DPYD expression. Based on our data, the capability of the P72 TP53 allele, which is a more active transcriptional variant of TP53, in repressing DPYD expression over the R72 TP53 allele suggest that p53 may alter the 5-FU drug sensitivity heterogeneously in the general population through its impact on 5-FU catabolism of normal tissues such as the liver (Fig. 2C,D). The tumor response observed in our syngeneic mouse model clearly demonstrates the importance of systemic effects of this interaction (Fig. 4D). However, it should be noted that the syngeneic tumors in our study expressed low levels of DPYD in comparison to liver tissue (data not shown). Evidence suggests that some tumors express high levels of DPYD compared to surrounding normal tissues as something that may in particular be true for colorectal cancers that metastasize to the liver 46 . Overexpression of TS and DPYD is seen frequently in colorectal tumors 47,48 . It is generally known in the clinical setting that tumors with low DPYD expression, low TS expression and that are wild-type for TP53 show a favorable response rate following treatment with 5-FU 49 . Subsequently, loss of functional p53-signaling in colorectal cancer, a typical late-stage event in the disease, may fail to suppress DPYD expression and add another level of complexity to 5-FU treatment by contributing to drug resistance. This idea is supported by the DPYD gene expression profile in p53 WT cell lines (Fig. 3) as well as in advanced stage colorectal tumor patients where higher expression of DPYD predicts poor disease-free survival (Fig. 6E and Fig. S5). It remains to be seen to what extent tumor levels of DPYD can make a significant contribution to 5-FU catabolism in vivo.
Our data in tumor cell lines suggest that inhibition of TS by 5-FU, methotrexate, Tomudex or Pemetrexed causes p53-dependent repression of DPYD expression (Fig. 5D). We provide evidence that repression by p53 of DPYD expression is related to changes in the dUTP/dTTP ratio and signaling from ATM and DNA-PK following TS-inhibition unlike other DNA damaging agents. A potential compensatory response to the reduction in dTTP levels, as a result of TS-inhibition, could be to reduce catabolism of pyrimidines to salvage thymine and uracil necessary for dTTP synthesis and DNA replication recovery. Suppression of pyrimidine catabolism by p53 following nucleotide imbalance may be another way for the TP53 tumor suppressor to control the integrity of DNA synthesis by favoring nucleotide salvage of thymidine and prevent errors during replication. However, since blocking pyrimidine catabolism would also affect catabolism of 5-FU, this would in turn cause an increase in 5-FU bioavailability emphasizing the deleterious effects in wild-type p53-expressing cells. In line with this, siRNA targeting of DPYD in TP53-null cells sensitizes to chemotherapeutics that target TS such as Tomudex (Fig. 6D).
Our data suggest that inhibiting DPYD would work synergistically with TS inhibitors. In support of our observations, use of a clinical DPYD inhibitor gimeracil as a component of the oral S-1 (tegafur, gimeracil and oteracil) mix has already proved promising in many solid tumors and is approved in more than 50 countries but not in the US. Early indications of synergy between S-1 and TS inhibitors have been reported for 5-FU resistant tumors 50 . S-1 has been combined with HDAC inhibitors, which are known to downregulate TS expression 51 . Figure 7. Model depicting the repression of DPYD by p53 following thymidylate synthase (TS) inhibition, dTTP imbalance and DNA damage. P53 transcriptionally represses DPYD expression and negatively impacts the catabolism of pyrimidines following inhibition of TS. In extension, this results in an imbalance in the dTTP/ dUTP ratio and a need to salvage cellular levels of dTTP to maintain uninterrupted DNA replication and repair. However, in the presence of 5-FU this might cause increased cell death due to reduced catabolism and increased incorporation of the 5-FU metabolites FdUTP and FUTP into DNA and RNA respectively.
SCiEnTifiC RepoRts | 7: 9711 | DOI:10.1038/s41598-017-09859-x Moreover, targeting DPYD with the oral irreversible inhibitor eniluracil, has significantly improved PFS and OS of patients with metastatic breast cancer in comparison to patients who were refractory to capecitabine (oral 5-FU) 12 . Eniluracil also limited the frequency of hand-foot syndrome, a toxicity phenotype believed to result from metabolites of 5-FU catabolism. Considering that recent evidence indicates that DPYD may play a role in breast cancer metastasis 2 , it would be interesting to determine if a DPYD inhibitor might provide added benefit to patients by limiting toxicity as well as targeting tumors that undergo EMT.
In conclusion, our study provides the first evidence for a role of the tumor suppressor p53 protein in downregulating pyrimidine and 5-FU catabolism by repressing DPYD gene expression following TS inhibition. These effects are not observed with other DNA damaging chemotherapeutic drugs like the topoisomerase inhibitors etoposide or irinotecan. Further studies would need to evaluate the interplay between combined use of 5-FU and irinotecan as compared to 5-FU alone with regard to p53-dependent regulation of DPYD. The idea that mutant p53 could through derepression up-regulate DPYD as a resistance mechanism to 5-FU treatment is also a focus of future research as 5-FU is used in multiple consecutive regimens in the therapy of evolving colorectal tumors. Overall our data document a role for tumor suppressor p53 in controlling pyrimidine catabolism through DPYD, particularly following metabolic stress imposed by nucleotide imbalance, and signaling effects through DNA-PK and ATM. The findings have implications for the toxicity and efficacy of the cancer therapeutic 5-FU. Previous findings from our lab have demonstrated that monitoring 5-FU levels can minimize toxicity and improve outcomes 52 and thus this study can provide some avenues for future design of potentially more effective treatments or treatment monitoring.