FTD Incorporation Research Articles

Effects and mechanisms of trifluridine alone or in combination with cryptotanshinone in inhibiting malignant biological behavior of gastric cancer

ABSTRACT Background The incidence of gastric cancer (GC) ranks fourth among all malignant tumors worldwide, and the fatality rate ranks second among all malignant tumors. Several Chinese traditional medicines have been used in the treatment of advanced gastric cancer. This study aims to investigate the effect of combinational use of natural product cryptotanshinone (CTS) with anti-cancer drug trifluorothymidine (FTD) in GC. Methods Cell Counting Kit-8 assay was used to detect the inhibitory effect of the combinational or separate use of FTD and CTS on the growth of HGC-27 and AGS GC cells. The combined index of FTD and CTS was calculated using CompuSyn software. To understand the mechanism, we applied flow cytometry to study the cell cycle and cell apoptosis after treatment. We also investigated the amount of FTD incorporated into the DNA by immunofluorescence assay. The expression of relevant proteins was monitored using western blot. Furthermore, the effect of using TAS-102 in combination with CTS was studied in xenograft tumor nude mice model. Results FTD and CTS inhibited the growth of GC cells in a dose-dependent manner, respectively. They both exhibited low to sub-micromolar potency in HGC-27 and AGS cells. The combination of FTD and CTS showed synergistic anticancer effect in HGC-27 cells and AGS cells. Our mechanism studies indicate that FTD could block HGC-27 cells at G2/M phase, while CTS could block HGC-27 cells at G1/G0 phase, while FTD combined with CTS could mainly block HGC-27 cells at G2 phase. FTD in combination with CTS significantly increased the apoptosis of HGC-27 cells. We observed that CTS treatment increased the incorporation of FTD into the DNA HGC-27 cell. FTD treatment activated STAT3 phosphorylation in HGC-27 cells, while CTS treatment down-regulated the concentration of p-STAT3. Interestingly, the combination of CTS and FTD reduced STAT3 phosphorylation induced by FTD. In the in vivo experiments, we observed that the combination of TAS-102 with CTS was significantly more potent than TAS-102 on tumor growth inhibition. Conclusions FTD combined with CTS has a synergistic anti-gastric cancer effect as shown by in vitro and in vivo experiments, and the combined treatment of FTD and CTS will be a promising treatment option for advanced gastric cancer.

Theoretical Computational Analysis Predicts Interaction Changes Due to Differences of a Single Molecule in DNA

Theoretical methods, such as molecular mechanics and molecular dynamics, are very useful in understanding differences in interactions at the single molecule level. In the life sciences, small conformational changes, including substituent modifications, often have a significant impact on function in vivo. Changes in binding interactions between nucleic acid molecules and binding proteins are a prime example. In this study, we propose a strategy to predict the complex structure of DNA-binding proteins with arbitrary DNA and analyze the differences in their interactions. We tested the utility of our strategy using the anticancer drug trifluoro-thymidine (FTD), which exerts its pharmacological effect by incorporation into DNA, and confirmed that the binding affinity of the BCL-2-associated X sequence to the p53 tetramer is increased by FTD incorporation. On the contrary, in p53-binding sequences extracted from FTD-resistant cells, the binding affinity of DNA containing FTD was found to be greatly reduced compared to normal DNA. This suggests that thymidine randomly substituted for FTD in resistant cells may acquire resistance by entering a position that inhibits binding to DNA-binding proteins. We believe that this is a versatile procedure that can also take energetics into account and will increase the importance of computational science in the life sciences.

Direct observation of DNA alterations induced by a DNA disruptor

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited. In this study, we utilized a single-molecule electrical detection method for the direct observation of DNA alterations on transcription factor binding motifs upon exposure to a nucleic acid analogue, trifluridine (FTD), and evaluated the effects of the DNA alteration on transcriptional activity in cancer cell line cells. We found ~ 10% FTD incorporation at the transcription factor p53 binding regions in cancer cells exposed to FTD for 5 months. Additionally, through single-molecule analysis of p53-enriched DNA, we found that the FTD incorporation at the p53 DNA binding regions led to less binding, likely due to weaken the binding of p53. This work suggests that single-molecule detection of DNA sequence alterations is a useful methodology for understanding DNA sequence alterations.

Theoretical analyses and experimental validation of the effects caused by the fluorinated substituent modification of DNA

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH–{rm{pi }}, lone pair (LP)–{rm{pi }}, and {rm{pi }}–{rm{pi }} interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

ChIP-seq Analysis to Explore DNA Replication Profile in Trifluridine-treated Human Colorectal Cancer Cells In Vitro.

Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil that has been approved for the treatment of metastatic colorectal cancer. In this study, a comprehensive analysis of DNA replication profile in FTD-treated colon cancer cells was performed. HCT-116 cells were exposed to BrdU or FTD and subjected to DNA immunoprecipitation. Immunoprecipitated DNA was sequenced; the density of aligned reads along the genome was calculated. Peak finding, gene ontology, and motif analysis were performed using MACS, GREAT, and MEME, respectively. We identified 6,043 and 5,080 high-confidence FTD and BrdU peaks in HCT-116 cells, respectively. Of 6,043 FTD peaks, 2,911 peaks were uncommon to BrdU. We observed that FTD was preferentially incorporated into genomic regions containing simple repeats, CpG islands, and gene bodies. Conserved motifs in FTD peaks contained dinucleotide repeats such as (GT)n. Global FTD incorporation patterns delineated FTD, preferentially incorporating loci in cancer cells.

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.

Effective Sequential Combined Chemotherapy with Trifluridine/Tipiracil and Regorafenib in Human Colorectal Cancer Cells.

Salvage chemotherapy for refractory metastatic colorectal cancer using trifluridine/tipiracil (FTD/TPI) and regorafenib has shown survival benefits. We evaluated the antitumor effects of FTD or FTD/TPI combined with regorafenib in vitro and in vivo. SW620, HCT 116, and HT-29 human colorectal cancer cell lines were treated with FTD and regorafenib simultaneously and sequentially. Cell death, incorporation of FTD into DNA, and molecules related to FTD and regorafenib-associated cell death were investigated. The antitumor effects of FTD combined with regorafenib in SW620 and COLO205 xenografts were also evaluated. Cell death was greater after sequential treatment with FTD followed by regorafenib in SW620 cells, but not in HCT 116 and HT-29 cells, than after treatment with FTD alone, which was attributable to thymidylate synthase reduction with the induction of apoptosis. In contrast, simultaneous and sequential exposure to regorafenib followed by FTD, but not FTD alone, attenuated the cell death effect. Furthermore, combined FTD/TPI treatment followed by regorafenib had greater antitumor activity than either monotherapy in SW620 and COLO205 xenograft models. Treatment results following regorafenib administration subsequent to FTD or FTD/TPI suggest that sequential therapy with FTD/TPI prior to regorafenib may be effective in a clinical setting.

Abstract 2886: ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro

Abstract Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is efficiently incorporated into the genomic DNA of tumor cells. However, the molecular mechanisms underlying its cytotoxic effects remain unclear. Therefore, the DNA replication profile in FTD-treated cells was comprehensively analyzed to explore the mode of FTD incorporation into the genomic DNA. Method: We generated the DNA replication profile in HCT-116 human colorectal cancer cells as follows: asynchronous cultures of HCT-116 were exposed to 5 μM 5-bromo-2′-deoxyuridine (BrdU), a non-cytotoxic control drug or FTD, for 4 h, harvested, and subjected to DNA immunoprecipitation using an anti-BrdU antibody. An input and immunoprecipitated DNA from each experiment were sequenced, and the sequence reads generated using Illumina sequencing were aligned uniquely to the human genome (hg19), allowing up to two mismatches using the BWA algorithm with default settings. The density of aligned reads along the genome was calculated and stored in a bigWig file for visualization in genome browsers. Peak finding and gene ontology analysis were performed using MACS and Genomic Regions Enrichment of Annotations Tool (GREAT). The 60-bp sequences surrounding the summits of the top 1000 peaks were analyzed for conserved motifs using the online program MEME. Results: In an antibody validation step, we observed significant FTD peaks over input DNA. We identified 6043 and 5080 high confident FTD and BrdU peaks in HCT-116 cells, respectively. Interestingly, 2911 of 6043 FTD peaks were uncommon to BrdU peaks, while 3232 peaks were common in FTD and BrdU. The FTD and BrdU peaks were not randomly distributed throughout the genome. Most FTD and BrdU peaks were ±5 kb or further away from transcription start sites. We identified the genes associated with distant peaks of FTD using GREAT. FTD peaks occurred near genes involved in extracellular matrix structural composition such as ACAN, BGN, CHAD, and COL12A1, while BrdU peaks were near genes involved in cysteine-type endopeptidase inhibitor activity such as ARRB1, BIRC7, LCN1, and CST1. Dinucleotide repeats such as TGTGTG were observed as conserved motifs in FTD-peak sequences. Conclusion: Global FTD incorporation patterns can delineate active replication loci, namely FTD preferentially incorporating loci, in human cancer cells. The DNA replication profile in FTD-treated cells differed from that in BrdU-treated cells. Thus, differences in the incorporation patterns of FTD and BrdU into the genomic DNA revealed the mechanisms of antitumor activities of FTD. Citation Format: Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2886.

Pre-exposure to Fluorouracil Increased Trifluridine Incorporation and Enhanced its Anti-tumor Effect for Colorectal Cancer.

Trifluridine/tipiracil (FTD/TPI) is used for metastatic colorectal cancer, that is refractory to 5-fluorouracil (5-FU)-based therapies. However, the impact of pre-exposure to 5-FU on the anti-cancer effect of FTD, which is a key component of FTD/TPI, is unclear. The incorporation into DNA and anti-cancer activity of FTD were analyzed in several cancer cell lines under response to FTD treatment with or without 5-FU pre-exposure. The volumes of tumors in xenografted nude mice were examined among groups that were either untreated or treated with S-1, FTD/TPI or FTD/TPI with pre-exposure to S-1. Pre-exposure to 5-FU significantly increased FTD incorporation into DNA and enhanced its anti-cancer effect for viability and proliferation of cancer cells. In the xenograft nude mouse model, the tumor volumes in the FTD/TPI-treated and S-1-pre-exposed group were lower than those in the FTD/TPI-only-treated group. Although both FTD dose and exposure time in the FTD/TPI-treated and S-1-pre-exposed mice were smaller than those in the FTD/TPI-only-treated mice, the incorporated FTD in the tumors in the former group was 86.5% of that in the latter group. Pre-exposure to 5-FU enhanced the incorporation into DNA and the anti-cancer effect of FTD in the context of colorectal cancer. Our data indicate the potential for a new sequential therapy using S-1 and FTD/TPI to improve prognosis of colorectal cancer.

Monitoring trifluridine incorporation in the peripheral blood mononuclear cells of colorectal cancer patients under trifluridine/tipiracil medication

Trifluridine/tipiracil (TFTD, TAS-102) is an orally administrated anti-cancer drug with efficacy validated for patients with metastatic colorectal cancer (mCRC). Trifluridine (FTD) is an active cytotoxic component of TFTD and mediates the anticancer effect via its incorporation into DNA. However, it has not been examined whether FTD is incorporated into the tissues of patients who received TFTD medication. By detecting FTD incorporation into DNA by a specific antibody, we successfully detected FTD in the bone marrow and spleen cells isolated from FTD-challenged mice as well as human peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-P and exposed to FTD in vitro. FTD was also detected in PBMCs isolated from mCRC patients who had administrated TFTD medication. Intriguingly, weekly evaluation of PBMCs from mCRC patients revealed the percentage of FTD-positive PBMCs increased and decreased in parallel with the administration and cessation of TFTD medication, respectively. To our knowledge, this is the first report to detect an active cytotoxic component of a chemotherapeutic drug in clinical specimens using a specific antibody. This technique may enable us to predict the clinical benefits or the adverse effects of TFTD in mCRC patients.

Abstract 2706: Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA

Abstract Trifluridine (FTD), known as main component of TAS-102, is beginning to be applied as anti-tumor agent due to the highly efficacious antitumor potency although there is little to distinguish FTD structure from thymidine structure. TAS-102 has first been used in Japan in clinical therapy as an oral agent. Some experimental results hypothesized that FTD pharmacological antitumor effect can be arisen by inhibition of thymidylate synthase and incorporation of FTD itself into DNA. One of them is the thermal denaturation experiments for DNA duplexes of containing some FTD or not, performed by J. C. Markley et al. Their experiments have shown that the DNA duplexes containing FTD are slightly less stable, with a melting temperature about three degrees lower than not containing duplexes. However, the change of thermodynamic structure and inter-nucleobase interaction changes are not yet known. In this presentation, we have performed molecular dynamics (MD) simulations and ab initio molecular orbital (MO) calculations to analyze the differences of thermodynamics structure and inter-nucleobase interaction between containing and not containing FTD. From these results, we sought to qualitatively understand the effects of FTD. At the first step, we have estimated the stabilization structures of same two DNA duplexes (containing FTD and not) used by J. C. Markley, with energy minimization. Then, a large differences were shown between these structures. The hydrogen bond breaking is observed between FTD and complementary Adenine base while the not containing DNA duplex keeps having a stabilized structure. The behavior means that the essential difference of intra-DNA interactions was arisen. At the second step, MD simulations were performed to analyze the distribution of distances between FTD and complementary base (i.e. thermally available conformations). With some conformations from MD sampling, the MO calculations were performed to discuss the difference of intra-base interactions. From our results, we found that thermodynamic conformational change of DNA duplex containing FTD occurs even if it is in internal body temperature, and the break hydrogen bonding between FTD and paired base was observed more frequently. In addition, strong interactions between FTD and nearby π-conjugate base were observed. These behaviors might be one of the factor of inducing unstabilization in DNA duplexes. Citation Format: Jun Koseki, Kenta Tsunekuni, Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2706.

Genetic variants of ATM and XRCC3 to predict efficacy of TAS-102 in patients with refractory metastatic colorectal cancer.

3579Background: TAS-102 is an oral combination of trifluridine (FTD) and tipiracil hydrochloride. Incorporation of tri-phosphorylated FTD into DNA is the main anti-tumor mechanism of action. We hyp...

The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2′-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

TAS-102, a novel antitumor agent: a review of the mechanism of action.

Inhibition of nucleoside metabolism is an important principle in cancer therapy as evidenced by the role of fluoropyrimidines, such as 5-fluorouracil (5-FU), and antifolates in the treatment of many cancers. TAS-102 is an oral combination therapy consisting of trifluridine (FTD), a thymidine-based nucleoside analog, plus tipiracil hydrochloride (TPI), a novel thymidine phosphorylase inhibitor that improves the bioavailability of FTD. TAS-102 has demonstrated efficacy in 5-FU-refractory patients based on a different mechanism of action and has been approved for the treatment of metastatic colorectal cancer in Japan. This review describes the mechanism of action of TAS-102, highlighting key differences between TAS-102 and 5-FU-based therapies. While both FTD and 5-FU inhibit thymidylate synthase (TS), a central enzyme in DNA synthesis, sufficient TS inhibition by FTD requires continuous infusion; therefore, it is not considered a clinically relevant mechanism with oral dosing. Instead, the primary cytotoxic mechanism with twice-daily oral dosing, the schedule used in TAS-102 clinical development, is DNA incorporation. FTD incorporation into DNA induces DNA dysfunction, including DNA strand breaks. Uracil-based analogs such as 5-FU may also be incorporated into DNA; however, they are immediately cleaved off by uracil-DNA glycosylases, reducing their ability to damage DNA. Moreover, the TPI component may enhance the durability of response to FTD. With its distinct mechanism of action and metabolism, TAS-102 is a promising treatment option for patients resistant to or intolerant of 5-FU-based fluoropyrimidines.

Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA.

Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

29 TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does not diminish therapeutic window in human tumor xenografts

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.

28 Characterization of the type of cell death induced by novel tambjamine analogs in lung cancer

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.

27 TAS-102 treatment results in high trifluridine incorporation into DNA with pyrimidine metabolic pathway markedly up-regulated in cancer

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models.

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients.

Abstract B87: Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer.

Abstract Background: Irinotecan (CPT-11) is a topoisomeraseI (TopoI) inhibitor, which is widely used for the treatment of metastatic colorectal cancer. CPT-11 inhibits DNA religation by TopoI, thus it stabilizes DNA-TopoI complexes and subsequently induces cytotoxicity. TAS-102, a novel oral antitumor agent, consisting of trifluorothymidine(FTD) and thymidine phosphorylase inhibitor, has reported promising clinical results in metastatic colorectal cancer. Several reports indicated that aberrant nucleoside incorporation into DNA, such as with cytarabine or gemcitabine, enhanced DNA-TopoI complexes. FTD, the effector of TAS-102, is a nucleoside which is efficiently incorporated and retained into DNA. Here, we evaluated the potency of TAS-102 in combination with CPT-11 in vitro and in vivo Materials and Methods: The combinational effect of FTD and SN-38 which is the active metabolite of CPT-11 was evaluated with several treatment schedules. Various colorectal cancer cell lines (SW48, SW620, LS174T and HCT116) were used in this assay. In one set, cells were washed after a 24 hour pre-exposure to FTD followed by a 48 hour exposure to SN-38 (FTD pre-exposure schedule); in the other set, cells were washed after a 6 hour pre-exposure to SN-38 followed by a 66 hour exposure to FTD (SN-38 pre-exposure schedule). A combinational intensity was estimated by using a combination index (CI) which indicates synergism (CI<0.8), additivity (0.8<CI<1.2), or antagonism (CI>1.2). Additionally, the antitumor effect of TAS-102 in combination with CPT-11 was investigated in a human colorectal cancer xenograft model. TAS-102 was orally administered for 14 days consecutively and CPT-11 was intravenously administered once a week to mice. Results: The combination of SN-38 and FTD showed a synergistic effect (SW48, SW620 and LS174T) or an additive effect (HCT116) in both the FTD pre-exposure schedule as well as the SN-38 pre-exposure schedule. Also, the enhancement of antitumor effect of TAS-102 in combination with CPT-11 was observed in vivo. Furthermore, these cell lines had various genotypes as regards KRAS, PI3CA and other DNA repair enzymes frequently detected in colorectal cancer, indicating that the combination's efficacy seemed to be independent of genetic status. Conclusion: TAS-102/CPT-11 represents a novel combination strategy for the treatment of advanced colorectal cancer. A synergistic effect was observed in both schedules tested, suggesting that various underlying mechanisms may exist. One potential mechanism is that FTD uptake or incorporation into DNA is increased by TopoI inhibition, and that FTD incorporation then further stabilizes the CPT-11 induced cleavable complex. However, the precise mechanism needs to be elucidated in the future. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B87. Citation Format: Keiji Ishida, Nozomu Tanaka, Sayaka Tsukioka, Kokoro Eshima, Akio Fujioka, Keisuke Yamamura, Satoko Ito, Kenichi Matsuo, Teruhiro Utsugi. Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B87.