Floxuridine

Loss of Uracil DNA Glycosylase Selectively Re-sensitizes p53 Mutant and Deficient Cells to 5-FdU

Abstract
Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Since p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5- FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53 mutated or deficient cells are highly resistant to 5-FdU. UDG depletion re-sensitizes 5-FdU in p53 mutant and deficient cells, whereas p53 wild-type cells are not affected under similar conditions. Utilizing paired HCT116 p53 wild-type (WT) and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. Additionally, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG expressing cells, but dramatically elevated in UDG depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53.This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53.

Introduction
Thymidylate synthase (TS) is a key enzyme that catalyzes the only means for de novo synthesis of deoxythymidine monophosphate (dTMP) [1]. TS utilizes 5,10- methylenetetrahydrofolate (5,10-CH2THF) as the methyl-group donor and catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to dTMP [1]. dTMP is subsequently phosphorylated to deoxythymidine triphosphate (dTTP), a critical precursor for DNA replication and repair. As TS contains binding sites for the substrate nucleotide (dUMP) and the cofactor folate (5,10-CH2THF), two structurally different classes of inhibitors, nucleotide or folate analogues block the activity of TS [2]. The class of fluoropyrimidines including 5- fluorouracil (5-FU) and floxuridine (5-FdU) target the nucleotide binding site, whereas the antifolates such as pemetrexed target the folate binding site of TS.Fluoropyrimidines are widely used in the treatment of various types of malignancies for their broad antitumor activity. Once taken into cells, fluoropyrimidines can be metabolized into fluorodeoxyuridine monophosphate (FdUMP) and fluorodeoxyuridine triphosphate (FdUTP) [2- 5]. The metabolite FdUMP inhibits TS by forming a stable ternary complex with TS and CH2THF [6-8], which ultimately leads to the depletion of dTTP and accumulation of deoxyuridine triphosphate (dUTP). The resulting imbalance of deoxynucleotide pools favors the utilization of dUTP and FdUTP during DNA replication and leads to the accumulation of both uracil and 5-FU in DNA [2-5]. Multi-targeted antifolates such as pemetrexed have been approved as components of first-line therapy in combination with cisplatin for the treatment of advanced non-small cell lung cancer [9]. Pemetrexed inhibits several folate-dependent enzymes, however TS is its predominant target [10-13]. Administration of pemetrexed leads to a globalreduction in nucleotide synthesis as well as accumulation of dUTP [14]. As a result, dUTP is used in DNA synthesis in place of dTTP, generating uracil mis-incorporation into DNA [15].

Mis-incorporated uracil and 5-FU are both primarily recognized and repaired by the uracil DNA glycosylase (UDG) initiated base excision repair (BER) pathway [16]. Although incorporation of uracil and 5-FU into DNA is well documented as a consequence of exposure to TS inhibitors [15], the impact of the downstream repair pathway directed by UDG on cell survival is not consistent. It has been hypothesized that thymine-less futile cycles of uracil mis- incorporation, excision by UDG, and further dUTP re-insertion result in DNA strand breaks and cell death [17]. If thymine-less cell death was dependent on UDG mediated removal of uracil and 5-FU, one would expect a correlation between the cytotoxicity of TS inhibitors and UDG expression. However, the majority of studies reported that neither overexpression, nor inhibition of UDG affected the sensitivity to TS inhibitors in human, mouse, or chicken DT40 cells [16, 18- 23]. In contrast, recently both our and the Karnitz group observed that loss of UDG highly potentiated the cytotoxicity of 5-FdU in several cancer cell lines, indicating that uracil and 5-FU incorporation played a key role in cell killing [24, 25].As the mediators of cell killing due to persistent uracil and 5-FU lesions in DNA are not clear, we assessed the likely pathways and noted that one of the major differences in these disparate findings is that cancer cells bearing p53 mutations were used in our and Karnitz’s experimental system, whereas non-transformed or p53 WT cancer cells were used in the majorities of others [16, 18-20, 22]. Mutation of TP53 is the most frequently observed gene alteration in cancers [26]. Mutations in p53 have been shown to influence cellular response to chemotherapeutic agents such as cisplatin, etoposide, and 5-FU [27, 28]. Notably, substantial evidence reveals that loss of p53, or p53 mutations, are linked to resistance to 5-FU due toinability to activate apoptosis pathway. For example, a study using isogenic cell systems demonstrated that deletion of p53 from a p53 WT colon cancer cell line (HCT116) rendered cells remarkably resistant to apoptosis induced by 5-FU [29].

In addition, 5-FU resistance was also described in a variety of p53 mutated cancer cells, including colon, bladder, pancreatic, and gastric cancer [30-33]. However, few studies have reported on the link of p53 status with the response to other TS inhibitors such as 5-FdU.Given the divergent cell models with different p53 status used in our and other studies, the following questions remain unanswered: 1) does loss or mutation of p53 render cells resistant to 5-FdU, and 2) is the potentiated cytotoxicity of 5-FdU after UDG depletion reliant upon p53 status? To gain insight into these questions, we tested the impact of UDG depletion on 5-FdU cytotoxicity in a number of cancer cell lines with differing p53 status. We found that, in general, loss or mutation of p53 remarkably reduced the sensitivity to 5-FdU, and depletion of UDG selectively re-sensitized p53 deficient or mutated cancer cells to 5-FdU. In order to understand the underlying mechanism contributes to the distinct response after UDG depletion, we utilized paired HCT116 cell lines with, or without, deletion of the TP53 gene, and observed that loss of UDG selectively re-sensitized HCT116 cells with p53 deletion. This re-sensitization was also observed with pemetrexed, but to a lesser extent with 5-FU, which mainly causes damage in RNA [21, 34-37]. In the presence of wild type p53, 5-FdU treatment induced activation of the apoptosis pathway in both UDG competent, or UDG depleted cells at comparable levels. However, in the absence of wild type p53, apoptosis activation was compromised in UDG expressing cells and dramatically elevated in UDG depleted cells. Collectively, these findings suggest that loss, or mutation, of p53 is associated with 5-FdU resistance, and UDG depletioncan significantly restore sensitivity, indicating that UDG may serve as a therapeutic target to improve the clinical effectiveness of 5-FdU.Cell lines and drugs. HCT116 p53 KO cells were a gift from Dr. Guangbin Luo (Department of Genetics, Case Western Reserve University, Cleveland, OH). Other cancer cell lines were purchased from American Type Culture Collection.

Details of the cell lines used in this study are listed in Table 1. All cells were maintained in DMEM (Corning 15-017-CV) supplemented with 10% dialyzed fetal bovine serum, 2mM L-glutamine, 1% MEM NEAA, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. 5-FdU and 5-FU were purchased from Sigma-Aldrich, dissolved respectively in Milli-Q water and DMSO, and stored as a 10 mM stock at -80°C. Pemetrexed was purchased from LC laboratories, and prepared fresh for each experiment by dissolving in Milli-Q water.Lentiviral shRNA knockdown. p53 or UDG knockdown was achieved via shRNA transduction. Lentiviral vectors LV-THM-shp53 (which also expresses a GFP reporter) or LV- Bleo-shp53 to perform p53 KD in WT HCT116 cells were obtained from Dr. Mark Jackson’s laboratory at Case Western Reserve University, Cleveland, OH [38]. Lentiviral vector targeting GFP (sh-GFP) was used as control. UDG shRNA vectors (shUDG: NM_003362.2-656s21c1, shUDG-2: NM_003362.2-758s21c1, and shUDG-3: NM_003362.2-893s21c1) were purchased from Sigma, and a scramble targeting shRNA vector (Sigma) was used as paired control. The lentiviral production and infection were performed as previously described [24]. Cells stablyinfected with LV-THM-p53 were isolated by cell sorting on the basis of their GFP expression. Cells stably infected with LV-Bleo-p53 were selected with zeocin (Sigma). Selection of positive UDG KD cells was assessed with puromycin (Sigma).Clonogenic survival assay. As described previously [24], cancer cells (200-300 cells/well) were seeded in 6-well culture dishes and allowed to adhere overnight. For 5-FdU, cells were treated for 24 h, then gently washed with PBS once, and incubated with fresh media for at least 10 days to allow individual colonies to form. For 5-FU or pemetrexed, cells were treated continuously for at least 10 days to form colonies. After 10-18 days, the plates were stained with methylene blue.

Colonies containing ≥50 cells were counted. The percentage of survival was determined relative to untreated control averaged over 3 independent experiments.Western blots and qPCR. Western blots were performed as previously described [39]. Twenty microgram of protein was loaded on SDS-polyacrylamide gel. The following antibodies were used to detect proteins on the membrane: α-Tubulin (Calbiochem); GAPDH (Santa Cruz Biotechnology); UDG (FL-313) (Santa Cruz Biotechnology); cleaved PARP (Asp214)(19F4) (Cell Signaling); cleaved caspase 3 (Cell Signaling); p53 (FL-393) (Santa Cruz Biotechnology); and p21 (Santa Cruz Biotechnology). For quantitative RT-PCR, total RNA from cells was extracted by using RNeasy Plus Mini Kit (Qiagen). cDNA synthesis was performed by using SuperScript III First Strand Kit (Life Technologies). Q-PCR was achieved with validated TaqMAN MGB FAMTM dye labeled probes (Applied Biosystems) for nuclear UDG on an ABI 7500 Fast Real-time PCR System (Applied Biosystems). β-Actin was used as an endogenous control, and relative gene expression was calculated as 2−ΔΔCt.Flow cytometric assay of apoptosis. Cells were seeded in 6-well tissue culture plates (1.5X105 cells/well) and allowed to attach overnight. Cells were then treated with 25 nM 5-FdU for 24 h, washed twice with PBS, replenished with drug-free medium at 48, 72, and 96 h. After recovery, the cells floating in the medium were collected. The adherent cells were trypsinized, pelleted, washed in ice-cold PBS, and resuspended in 1X Binding Buffer according to the manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen). Cells were then stained with FITC Annexin V and PI for 15 minutes at room temperature in the dark. Annexin V- FITC detects translocation of phosphatidylinositol from the inner to the out of cell membrane during early apoptosis, and PI can enter the cells in late apoptosis or necrosis. Untreated cells were used as control for the double staining. The cells were analyzed immediately after staining using a Attune NXT instrument and FlowJo software. For each measurement, at least 20,000 cells were counted. Statistical significance between two treatment groups was analyzed using unpaired 2-tailed student’s t-test. Significance was assigned for a P-value < 0.05. Standard software GraphPad Prism (San Diego, CA, USA) and Excel 2013 (Microsoft Corp., Redmond, WA) were used for all statistical analysis. Results Given that p53 mutations or deficiencies are frequently observed in cancers, and studies have demonstrated that mutations of p53 reduce 5-FU cytotoxicity [29-33]. To understand whether these mutations also alter the response to 5-FdU, a panel of human cancer cell lines from colon, lung, ovarian, skin, and endometrium with intrinsically differing p53 status were utilized in this study. The p53 status of each cell line is listed in Table 1. To determine p53 protein functionality in p53 WT and p53 mutant (Mut) or deficient cancer cell lines, we assessed p53 levels and expression of p21, a widely accepted initiator of p53 activated signaling [40], 24 hours after administration of 8Gy gamma-irradiation. All the p53 WT cancer cell lines used in this work induced p21 expression after irradiation, indicating functional p53 in these cell lines (Supplemental figure 1). In order to establish the relationship between p53 status and 5-FdU sensitivity, we evaluated the cytotoxicity of 5-FdU in these cell lines by clonogenic survival assay. As shown in Figure 1A, the cell lines tested displayed a spectrum of 5-FdU sensitivities with IC50 values ranging from 1.32 ± 0.33 to 269.55 ± 0.73 nM for A2780 and H1299 lines, respectively. Importantly, we observed that, in general, cell lines with p53 mutation or deficiency (Figure 1A, solid lines) were significantly more resistant to 5-FdU than p53 WT cells (Figure 1A, dashed lines), with the exception of A375 which has wild-type p53 but an IC50 of 110.81 ±1.80 nM. In addition, except for A375, the IC50 values for the p53 WT cancer lines clustered together at a lower dose range (<10 nM), whereas p53 mutant or deficient lines clustered at a higher range (>100 nM) (Figure 1B).

These observations are consistent with the hypothesis that p53 mutation or deficiency is associated with resistance to 5-FdU.Previously, the discordant findings on sensitization to 5-FdU following UDG depletion were reported using cell models with differing p53 status [16, 18-25]. To understand whether the divergent responses could be attributed to p53 status, we explored whether UDG depletion could sensitize p53 mutant or deficient cancer cells to 5-FdU differentially. For these experiments, we used shRNA to deplete UDG in various cancer cells lines with differing p53 status, as listed in Table 1. UDG stable knockdown was evaluated by western blot (Figure 2A and B, insert). Based on a clonogenic survival assay, we observed that UDG depletion selectively sensitized cells with p53 mutation or deficiency to 5-FdU exposure (Figure 2A). However, in p53 WT cell lines, UDG depletion did not alter the cytotoxicity of 5-FdU (Figure 2B). Collectively, these results demonstrate that UDG depletion re-sensitizes p53 mutant or deficient cancer cells, providing a novel therapeutic target for patients with p53 mutant tumors.Since many studies have identified gain of various functions for specific p53 mutated proteins [41, 42], we next asked whether loss of wild-type p53 protein expression can alter the response to 5-FdU. To address this, we utilized paired HCT116 colon cancer cell lines with or without genetic TP53 deletion and tested their sensitivity to 5-FdU, and the loss of p53 expression was evaluated by western blot (Figure 3A). Using a clonogenic survival assay, we demonstrated that p53 KO cells were more resistant to 5-FdU than p53 WT cells (Figure 3B). Knockdown of p53 by shRNA recapitulates the resistance observed in p53 KO cells (Figure 3B), indicating that p53 status is a key mediator of the response of HCT116 cells to 5-FdU.

To understand whether loss of p53 protein will affect the response to 5-FdU after UDG depletion, we knocked down UDG by shRNA in both HCT116 p53 WT and p53 KO cells. UDG knockdown levels were shown to be greater than 90% as evaluated by western blot and Q-PCR (Figure 3C, D). In agreement with our data using p53 mutant cells, UDG depletion greatly enhanced cytotoxicity of 5-FdU in p53 KO cells but did not significantly affect p53 WT cells (Figure 3E, F), indicating that p53 is involved in regulating the response to 5-FdU following UDG depletion. To exclude the off-target effect of a single shRNA, we also utilized two other shRNAs that target UDG in HCT116 p53 WT and p53 KO cells and observed similar effect (Supplemental figure 2). In addition, depletion of UDG also potentiated 5-FdU cytotoxicity in two HCT116 cancer cells with different shRNAs targeted to p53 (Figure 4A-E). Collectively, these results confirm that loss of p53 protein renders cells resistant to 5-FdU, and UDG depletion selectively re-sensitizes p53 KO and KD cells to 5-FdU.Although all TS inhibitors have the ability to block TS, disrupting DNA replication and leading to uracil incorporation into DNA, differences among distinct TS inhibitors have been reported in terms of their other metabolism mediated cytotoxic pathways [2]. For example, pemetrexed polyglutamate derivatives also demonstrate inhibitory activity for other folate- dependent enzymes such as glycinamide ribonucleotide, but to a lesser extent [10-13]. Moreover, unlike 5-FdU, which mainly exerts its cytotoxicity due to effects at the DNA level [24], studies have revealed that the cytotoxicity of 5-FU is primarily RNA-mediated, as 5-FU is metabolized to fluorouridine triphosphate (FUTP) which affects multiple RNA processes following its incorporation into RNAs [21, 34-37]. In order to address the question of whether p53 status isresponsible for differences in sensitivity to other TS inhibitors, including pemetrexed and 5-FU, in UDG depleted cells, we evaluated cell viability following drug exposure in UDG depleted p53 WT and p53 KO cancer cells.

Similar to our observations with 5-FdU, no significant survival differences were found between UDG expressing and UDG depleted cells in the presence of p53 (Figure 5A, B). However, in the absence of p53, UDG depletion sensitized cells to pemetrexed (Figure 5C), while loss of UDG only moderately sensitized cells to 5-FU at high concentrations (Figure 5D), reaffirming that the primary cytotoxic effect of 5-FU depends on RNA incorporation. Together, these results indicate that UDG depletion also sensitizes cells without p53 to other TS inhibitors, mainly through generation of DNA damage. To understand whether 5-FdU resistance observed in p53 KO cells is due to a failure to activate cell death pathways, we monitored cell death progression by Annexin V and propidium iodide (PI) staining. Cells were exposed to 5-FdU for 24 h, washed with PBS, and then allowed to recover in drug free medium for a total of 48, 72, and 96 h (Figure 6A). In cells with wild-type p53, 5-FdU caused significant cell death (Annexin V and PI positive) at 48 h which was retained at 72 h and 96 h in both UDG expressing and UDG depleted cells (Figure 6B, C). However, in the absence of p53, cell death caused by 5-FdU was significantly lower in UDG expressing cells, while in UDG depleted cells, cell death was detected at 24 h and significantly elevated at 48 to 96 h (Figure 6B, C). These data suggest that 5-FdU induced cell death is dependent upon p53, supporting the observation that drug resistance can be observed as a result of abrogation of the p53 mediated cell death pathway. Importantly, UDG depletion significantly potentiates death of cells lacking wild type p53 activity through a p53 independent pathway.To further elucidate whether the cell death caused by 5-FdU is due to apoptosis, we examined expression of proteins involved in the activation of the apoptotic pathway. In wild-type p53 cells, we observed that p53 expression was induced at 24 h, and the induction remained for96 hours in both shSCR and shUDG cells following 5-FdU exposure (Figure 6D). The expression of cleaved PARP, a hallmark of apoptotic cell death, was induced at 48 h and persisted through 72 h and 96 h in p53 WT cells regardless of whether UDG was present or not (Figure 6D). In addition, cleaved caspase 3 was also detected in both UDG expressing or depleted p53 WT cells (Figure 6D). In the absence of p53, induction of cleaved PARP or caspase 3 was not readily detected in cells expressing UDG after 5-FdU exposure (Figure 6E), while both were robustly induced from 48 h to 96 h in cells depleted of UDG (Figure 6E). Taken together, our results suggest that 5-FdU induced apoptosis is mediated through p53, and the lack of apoptosis activation due to loss of p53 is responsible for the enhanced cell survival observed in p53 KO cells. However, in p53 KO cell with coincident UDG depletion, 5-FdU selectively activates a p53-independent apoptotic pathway through a mechanism which needs further investigation.

Discussion
In this study, we utilized multiple cancer cells bearing differing p53 status with or without UDG expression. We observed that loss of UDG selectively re-sensitized cancer cells with p53 mutation or deficiency to 5-FdU, but did not alter the response of p53 wild-type cells. These results demonstrate that UDG, through its function of removing uracil or 5-FU, plays a major role in the effect of 5-FdU on the response of cells lacking wild type p53 activity. Our findings resolve the unexplained discrepancy observed in a number of prior studies regarding the role of UDG in sensitivity to TS inhibitors. Prior studies revealed that either loss of UDG enhanced the cytotoxicity of 5-FdU or pemetrexed in cancer cells [24, 25], or overexpression or inhibition of UDG had no effect on the sensitivity of human or mouse cells to TS inhibition [16, 18-20, 22]. The difference, we propose, is dependent on p53 status. p53 plays a key role in determining the sensitivity of cells to 5-FU. A number of studies reported that enhanced 5-FU resistance has been observed in cells bearing TP53 deletion or mutations [29-33]. However, unlike other TS inhibitors, 5-FU exposure caused only slightly potentiated cytotoxicity at higher doses in UDG depleted, p53 KO cell lines. Recently, several studies have observed that the cytotoxicity of 5-FU is more dependent on its incorporation into RNA than its inhibition of TS, diminishing its effect on DNA [21, 34-37]. In addition, activation of p53 following 5-FU exposure has been identified as working through RNA mechanisms [43, 44]. Since UDG recognizes only DNA lesions, it is not surprising that depletion of UDG does not significantly alter cellular responses to agents that primarily affect RNA function. Together, this suggests that the increased cytotoxicity of 5-FdU and pemetrexed observed in UDG depleted cells is primarily due to uracil and 5-FU incorporation into DNA.

The present results illustrate that cells with p53 mutation or deficiency are significantly resistant to 5-FdU in comparison with p53 WT cells. It is clear that many different mutant p53s also acquire oncogenic functions that are distinct from the activities of wild-type p53 [41, 42]. Some p53 mutants provide enhanced resistance to apoptosis induced by a variety of treatments, including certain chemotherapeutic drugs [27, 45]. In particular, one study identified that p53 mutants activate expression of dUTPase [46], which has been related to the resistance to TS inhibitors [47-49]. Our results on a select group of p53 mutants as well as p53 KO cell lines revealed resistance to 5-FdU treatment. However, the p53 KO cell line is much less resistant to 5-FdU than other p53 mutant cell lines, suggesting the potential for enhanced resistance due to gained functions for certain p53 mutants. Our results demonstrated that inhibition of UDG selectively sensitized p53 mutant and deficient cancer cells to 5-FdU, but did not alter the response in p53 WT cells. Importantly, we have observed that apoptosis following 5-FdU is efficiently induced in the presence of p53 but highly compromised in cells lacking p53, indicating that the activation of the 5-FdU induced cell death pathway is dependent on p53. Further studies with different p53 WT cell lines also revealed cells highly sensitive to 5-FdU with IC50 values lower than 10 nM. One exception we observed was in the A375 melanoma cells line, which has a wild type TP53 gene. A375 was relatively insensitive to 5-FdU and had an IC50 of 110.81 ± 1.80 nM. Clearly, more knowledge is needed regarding the p53 mediated cell death pathway and how 5-FdU, with or without UDG, causes damage and triggers cell death. In response to 5-FdU, cells lacking wild-type p53, combined with UDG depletion, activate cell death in a p53 independent manner, which reverses chemoresistance and selectively re-sensitizes these cancer cells to 5-FdU.

The current findings of this paper focus on the role of p53 in apoptosis and shows that in the presence of p53, both UDG WT and UDG depleted cells activate apoptosis at similar levels, however, in the absence of p53, apoptosis induction is compromised in the UDG WT cells but significantly increased in UDG depleted cells. These data explain that p53 WT cells have 5-FdU IC50 values less than 10 nM, whereas p53 mutant or deficient cells have IC50 values higher than 100 nM. The result is consistent with a previous publication from Janet Houghton’s group [30] that cells with wild-type p53 displayed acute apoptosis, while cells with mutant p53 showed delayed or compromised apoptosis following fluoropyrimidine treatment. Based on these results, we propose the mechanism of cell death that: first, DNA damage due to loss of UDG enhanced the apoptosis in p53 mutant or deficient cancer cells, but did not change the apoptosis in p53 WT cells which has already been activated in the presence of wild-type p53 following 5- FdU induced stress. Second, loss of UDG increases levels of persistent uracil and 5-FU incorporation at the replication forks that without p53 results in early S phase arrest and disintegration of the replication fork progression, as we have shown previously [24]. Further support for this mechanism comes from a paper published recently by Swati Palit Deb’s lab [50] shown that lack of wild-type p53 increases DNA origin firing compared with p53 WT cells. In our system, more DNA origin firings would result in both more 5-FU and uracil incorporation and further disruption of these replication forks that improve the killing effect in p53 mutant or deficient cancer cells. Taken together, these results provide an explanation for the discordant findings in previous published data regarding the role of UDG in mediating the cytotoxicity of TS inhibitors and suggest that UDG is an attractive therapeutic target in cancer cells with p53 mutation or deficiency, Floxuridine to enhance their response to TS inhibitors.