Thymidylate Synthase Research Articles

Enhanced anticancer effect of thymidylate synthase dimer disrupters by promoting intracellular accumulation

IntroductionThymidylate synthase (TS) plays a crucial role in cellular growth, proliferation, DNA synthesis, and repair, thus gaining attention for targeted therapies in cancer. TS overexpression and the altered pharmacokinetics of anti-TS drugs are among the most prominent causes of cellular resistance. Decreased drug influx and/or efficient efflux result in reduced drug access to the intracellular targets.ResultsIn this study, we have evaluated and demonstrated the increased cytotoxic efficacy of novel TS dimer disrupters (Ddis) in the presence of specific inhibitors of drug efflux protein pumps in ovarian and colon cancer cells, suggesting that these compounds are substrates of the cellular drug extruders. A second strategy adopted to favor intracellular accumulation was to employ, as a drug delivery system, a molecular tool able to help less lipophilic compounds to cross the cell membrane. The Ddis were delivered through the SAINT-Protein transfection agent. The observed cell-killing effects agreed with the reduction of TS protein level and cell cycle perturbation.ConclusionOverall, this preclinical study suggests that the innovative TS dimer disrupters can be optimized by increasing their intracellular accumulation by both inhibiting their outflow and/or enhancing cellular uptake.

The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease soon to become the second leading cause of cancer deaths in the US. Beside surgery, current therapies have narrow clinical benefits with systemic toxicities. FOLFIRINOX is the current standard of care, one component of which is 5- Fluorouracil (5-FU), which causes serious gastrointestinal and hematopoietic toxicities and is vulnerable to resistance mechanisms. Recently, we have developed polymeric fluoropyrimidines (F10, CF10) which unlike 5-FU, are, in principle, completely converted to the thymidylate synthase inhibitory metabolite FdUMP, without generating appreciable levels of ribonucleotides that cause systemic toxicities while displaying much stronger anti-cancer activity. Here, we confirm the potency of CF10 and investigate enhancement of its efficacy through combination with inhibitors in vitro targeting replication stress, a hallmark of PDAC cells. CF10 is 308-times more potent as a single agent than 5-FU and was effective in the nM range in primary patient derived models. Further, we find that activity of CF10, but not 5-FU, is enhanced through combination with inhibitors of ATR and Wee1 that regulate the S and G2 DNA damage checkpoints and can be reversed by addition of dNTPs indicative of CF10 acting, at least in part, through inducing replication stress. Our results indicate CF10 has the potential to supersede the established benefit of 5-FU in PDAC treatment and indicate novel combination approaches that should be validated in vivo and may be beneficial in established regimens that include 5-FU.

NOD2 reduces the chemoresistance of melanoma by inhibiting the TYMS/PLK1 signaling axis

AbstractNucleotide-binding oligomerization domain 2 (NOD2) is an immune sensor crucial for eliciting the innate immune responses. Nevertheless, discrepancies exist regarding the effect of NOD2 on different types of cancer. This study aimed to investigate these function of NOD2 in melanoma and its underlying mechanisms. We have validated the tumor suppressor effect of NOD2 in melanoma. NOD2 inhibited the proliferation of melanoma cells, hindering their migration and invasion while promoting the onset of apoptosis. Our study showed that NOD2 expression is closely related to pyrimidine and folate metabolism. NOD2 inhibits thymidylate synthase (TYMS) expression by promoting K48-type ubiquitination modification of TYMS, thereby decreasing the resistance of melanoma cells to 5-fluorouracil (5-FU) and capecitabine (CAP). TYMS was identified to form a complex with Polo-like Kinase 1 (PLK1) and activate the PLK1 signaling pathway. Furthermore, we revealed that the combination of the PLK1 inhibitor volasertib (BI6727) with 5-FU or CAP had a synergistic effect repressing the proliferation, migration, and autophagy of melanoma cells. Overall, our research highlights the protective role of NOD2 in melanoma and suggests that targeting NOD2 and the TYMS/PLK1 signaling axis is a high-profile therapy that could be a prospect for melanoma treatment.

242 (PB230): Targeting Thymidylate Synthase mRNA regulatory element with a novel family of Berberine derivatives

242 (PB230): Targeting Thymidylate Synthase mRNA regulatory element with a novel family of Berberine derivatives

Genomic and phenotypic polymorphism of Clostridium botulinum Group II strain Beluga through laboratory domestication

Laboratory domestication is the result of genetic and physiological changes of organisms acquired during numerous passages in vitro. This phenomenon has been observed in bacteria as well as in higher organisms. In an effort to understand the impact of laboratory domestication on the foodborne pathogen Clostridium botulinum and related microbial food safety research, we investigated multiple spore stocks of C. botulinum Group II Beluga from our collection, as that is a widely applied model strain used in laboratories over decades. An acquired nutrient auxotrophy was confirmed as thymidine dependency using phenotypic microarrays. In parallel, whole-genome re-sequencing of all stocks revealed a mutation in thyA encoding thymidylate synthase essential for de-novo synthesis of dTMP from dUMP in the auxotrophic stocks. A thyA-deficient Beluga variant stock was successfully complemented by introducing an intact variant of thyA and thymidine prototrophy was restored, indicating that the thymidine auxotrophy was solely due to the presence of a SNP in thyA. Our data suggested that this mutation, deleterious under nutrient-poor growth conditions in a chemically defined medium, has been present and maintained in laboratory stocks for nearly 30 years. Yet, the mutation remained unidentified since receiving the strain, most likely due to routine use of culture conditions optimized for growth performance. This work pinpoints the need for careful monitoring of model strains extensively used in laboratory settings at both phenotypic and genomic level. In applications like food safety challenge tests, compromised strains could cause incorrect predictions and thereby have deleterious consequences. To mitigate the risk of acquiring mutations, we recommend keeping passage numbers of laboratory strains low and to avoid single-colony passaging. In addition, relevant strains should be subjected to regular WGS checks and physiological validation to exclude DNA mutations with potential negative impacts on research data integrity and reproducibility.

Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil.

Ribosome quality control (RQC) resolves collided ribosomes, thus preventing their cytotoxic effects. The chemotherapeutic agent 5-Fluorouracil (5FU) is best known for its misincorporation into DNA and inhibition of thymidylate synthase. However, while a major determinant of 5FU's anticancer activity is its misincorporation into RNAs, the mechanisms by which cancer cells overcome the RNA-dependent 5FU toxicity remain ill-defined. Here, we report a role for RQC in mitigating the cytotoxic effects of 5FU. We show that 5FU treatment results in rapid induction of the mTOR signalling pathway, enhanced rate of mRNA translation initiation, and increased ribosome collisions. Consistently, a defective RQC exacerbates the 5FU-induced cell death, which is mitigated by blocking mTOR pathway or mRNA translation initiation. Furthermore, 5FU treatment enhances the expression of the key RQC factors ZNF598 and GIGYF2 via an mTOR-dependent post-translational mechanism. This adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment.

An RNA damage response network mediates the lethality of 5-FU in colorectal cancer

5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.

Design, synthesis and biological evaluation of novel curcumin-fluorouracil hybrids as potential anti-cancer agents

The latest global cancer data statistics report shows that cancer poses a serious threat to human life and health; The number of new cancer and death cases worldwide is severe. Molecular hybridization is considered an effective strategy for developing new anti-cancer drugs. Curcumin (Cur) is a natural active compound containing Michael receptors that target thioredoxin reductase (TrxR). Fluorouracil (5-FU) is the first anti-metabolic drug synthesized based on certain assumptions for tumor treatment, acting on thymidylate synthase (TS). This study synthesized a series of novel hybrid derivatives of Cur and 5-FU, and evaluated their anti-tumor cell proliferation effects. Several compounds with good cytotoxic activity against tumor cells were discovered; and they exhibited high selectivity towards A549 cells, compared to normal THLE cells. Among them, the hybrid derivative F-4 has the best anti-proliferative activity in tumor cells. F-4 can target TrxR, increase reactive oxygen species levels in tumor cells, and lead to tumor cell apoptosis, which may be related to the Michael receptor structure in the chemical structure of F-4; F-4 can also target TS, leading to cell cycle arrest in G0/G1 phase, which may be related to the 5-FU structure in the chemical structure of F-4. Moreover, F-4 can effectively exert anti-tumor activity in mice, significantly reduce tumor volume and weight, and has low toxic side effects. These results indicate that Cur-5-FU hybrid derivative F-4 is a novel lead compound with in vivo anti-tumor activity and minimal side effects, which deserves further investigation.

P13-19 Development of a thymidylate synthase assay to investigate a genotoxicity risk arising from adverse secondary pharmacology and influence drug design

P13-19 Development of a thymidylate synthase assay to investigate a genotoxicity risk arising from adverse secondary pharmacology and influence drug design

Synthesis and anticancer evaluation of diaryl pyrido[2,3-d]pyrimidine /alkyl substituted pyrido[2,3-d]pyrimidine derivatives as thymidylate synthase inhibitors

Worldwide, colorectal cancer (CRC) is the third most common type of cancer and the second most common cause of cancer-related deaths. Thymidylate synthase (TS) is a crucial component of DNA biosynthesis and has drawn interest as an essential target for cancer treatment. In the current work, we have designed and synthesized twenty-eight new diaryl-based pyrido[2,3-d]pyrimidine/alkyl-substituted pyrido[2,3-d]pyrimidine derivatives and evaluated their anticancer activity against the HCT 116, MCF-7, Hep G2, and PC-3 cell lines cell lines. Additionally, we have carried out TS inhibitory activity and in silico studies for compounds 1n and 2j. All the synthesized compounds exhibited good anticancer activity, but among them, compounds 1n and 2j showed excellent anticancer activity, having IC50 values of 1.98 ± 0.69, 2.18 ± 0.93, 4.04 ± 1.06, and 4.18 ± 1.87 µM; and 1.48 ± 0.86, 3.18 ± 0.79, 3.44 ± 1.51, and 5.18 ± 1.85 µM, against the HCT 116, MCF-7, Hep G2, and PC-3 cell lines respectively with control raltitrexed (IC50 1.07 ± 1.08, 1.98 ± 0.72, 1.34 ± 1.0, and 3.09 ± 0.96 µM, respectively) and hTS inhibitory activity with IC50 values of 20.47 ± 1.09 and 13.48 ± 0.96 nM with control raltitrexed (IC50 14.95 ± 1.01 nM). Further, the mechanism of inhibition was revealed by molecular docking, which showed the binding pattern of 1n and 2j to the catalytic site of TS with docking scores of -10.6 and − 9.5 kcal/mol, respectively, with reference raltitrexed (-9.4 kcal/mol). Additionally, the assessment of physicochemical, biochemical, structural, and toxicological characteristics were also in the acceptable range for these compounds. Based on the anticancer activity of compounds, SAR was also performed for lead optimization.

LncRNA BC200 promotes the development of EBV-associated nasopharyngeal carcinoma by competitively binding to miR-6834-5p to upregulate TYMS expression

Nasopharyngeal carcinoma (NPC) is closely related to Epstein–Barr virus (EBV) infection. Long noncoding RNAs (lncRNAs) play important roles in cancers. However, the molecular mechanism underlying the roles of lncRNAs in EBV-associated NPC remains largely unclear. In this study, we confirmed that the expression of the lncRNA brain cytoplasmic 200 (BC200) was significantly increased in EBV-infected NPC cells and tissues. BC200 facilitated the growth and migration of NPC cells, suggesting that it participated in NPC progression by functioning as an oncogene. Mechanistically, BC200 was found to act as a ceRNA by sponging and inhibiting miR-6834-5p. Thymidylate synthetase (TYMS), whose high expression was reported to be an independent indicator of poor prognosis in NPC via an unknown mechanism, was identified as a target gene of miR-6834-5p in the present study. BC200 upregulated TYMS expression in a manner that depends on miR-6834-5p. TYMS was abnormally upregulated in EBV-positive NPC cells and tissues, and its ectopic expression contributed to the proliferation and migration of NPC cells. This study highlights the role of lncRNA BC200, which is upregulated by EBV, in promoting the development of NPC, suggesting that BC200-mediated ceRNA network may be valuable biomarkers for the diagnosis and treatment of EBV-associated NPC.

Complete genome of the new bacilliform virus that causes Milky Hemolymph Syndrome in Chionoecetes bairdi (Rathbun, 1924)

The genome of a new member of the Nimaviridae family has been sequenced. The Chionoecetes bairdi bacilliform virus (CbBV) causes Milky Hemolymph Syndrome (MHS) in Chionoecetes bairdi populations of the Pacific coast of Kamchatka. The CbBV genome is represented by double-stranded DNA with a length of 245,567 nucleotides containing 120 ORFs. Of these, 85 proteins had significant matches in the NCBI database, and 57 genes encoded capsid, envelope, tegument and nonstructural proteins. Comparative analysis of the genomes of CbBV and a number of representatives of the class nuclear arthropod large DNA viruses (NALDVs) made it possible to isolate 49 evolutionarily conserved orthologue core genes. Among them, 5 were multicopy genes, and 44 were single-copy genes. There were ancestral genes characteristic of all Naldaviricetes − per os infectivity complex genes, one DNA polymerase gene and one thymidylate synthase gene.Phylogenetic analysis of representatives of the Nimaviridae family revealed that the CbBV and Chionoecetes opilio bacilliform virus (CoBV) form an independent clade within the family separate from the clade containing WSSV strains. This is supported by data on the order and arrangement of genes in the genomes of nimaviruses that were identical within each clade but differed between them. In addition, a high identity of the genomes and proteomes of CbBV and CoBV (approximately 99%) was shown, and their identity with WSSV strains was no more than 33%.The data on the structure of the genome of the new virus that causes MHS in C. bairdi indicate that it belongs to the family Nimaviridae, genus Whispovirus. Thus, the CbBV infecting the commercially important species of Tanner crab in populations of the Pacific coast of Kamchatka is the second “wild” representative of replicating nimaviruses whose genome has been characterized after the CoBV that causes MHS in C. opilio in populations of the Sea of Japan. The discovery of a new member of the family that infects decapods indicates the prevalence of nimaviruses in marine ecosystems. The information obtained is important for understanding the evolution of representatives of the class of nuclear arthropod large DNA viruses. The discovery of a new nimavirus that causes MHS in Chionoecetes crabs, in contrast to the white spot syndrome (WSS) caused by WSSV strains, makes it relevant to identify two variants and possibly species within the family, namely, WSSV and Milky Hemolymph Syndrome virus (MHSV).

Sitagliptin synergizes 5-fluorouracil efficacy in colon cancer cells through MDR1-mediated flux impairment and down regulation of NFκB2 and p-AKT survival proteins.

5-fluorouracil (5-FU) is an inexpensive treatment for colon cancer; however, its efficacy is limited by chemoresistance. This study investigates the combination therapy approach of 5-FU with Sitagliptin (Sita), a diabetic drug with potential cancer-modulating effects. The combination was evaluated in vitro and in silico, focusing on the effects of Sita and 5-FU on colon cancer cells. The results showed that the addition of Sita significantly decreased the IC50 of 5-FU compared to 5-Fu monotherapy. The study also found that Sita and 5-FU interact synergistically, with a combination index below 1. Sita successfully lowered the 5-FU dosage reduction index, decreasing the expression of MDR1 mRNA and p-AKT and NFκB2 subunits p100/p52 protein. Molecular docking analyses confirmed Sita's antagonistic action on MDR1 and thymidylate synthase proteins. The study concludes that sitagliptin can target MDR1, increase apoptosis, and significantly reduce the expression of p-AKT and NFκB2 cell-survival proteins. These effects sensitize colon cancer cells to 5-FU. Repurposing sitagliptin may enhance the anticancer effects of 5-FU at lower dosages.

Mycobacterium tuberculosis Essential Gene Thymidylate Synthase Is Involved in Immune Modulation and Survival inside the Host.

A Mycobacterium tuberculosis essential gene, ThyX (Rv2754c), plays a key role in intermediate metabolism and respiration by catalyzing the formation of dTMP and tetrahydrofolate from dUMP and methylenetetrahydrofolate. ThyX is present in the M.tb complex and in M. smegmatis a nonpathogenic strain of Mycobacteria. In this study, we identified a novel function of ThyX, an enzyme with immune-modulating properties. We have shown that ThyX can activate the macrophages in the host toward M1 response. Overexpression of ThyX stimulates the production of nitrite oxide (NO) and induces apoptosis in macrophages; indeed both responses help the host to control growth of M.tb. ThyX was also discovered to play a role in the recombinant bacterium's ability to survive when it was subjected to oxidative and hypoxic stress by macrophages. These findings demonstrate the protein's functional importance in M.tb. Indeed these findings represent ThyX as a potential candidate for future research and show this as a therapeutic target.

Intersite communication in dimeric enzymes highlighted by structural and thermodynamic analysis of didansyltyrosine binding to thymidylate synthases

Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity.

A novel cocrystal of 5-fluorouracil with 2,2’-dipyridylamine: Synthesis, structural characterization, thermal analysis and in silico anticancer evaluation as a thymidylate synthase inhibitor

A new cocrystal containing 5-fluorouracil (5-FU) and 2,2’-dipyridylamine (dpya) was successfully prepared by a slow evaporation solution method and characterized using solid-state techniques such as FTIR, thermal analysis (DTA/TG) and single crystal X-ray diffraction. X-ray crystallography indicated the formation of a 2:1 5-FU:dpya cocrystal. The 5-FU and dpya molecules in the cocrystal were linked by N−H···O, N−H···N, C−H···O and C−H···F hydrogen bonds, forming a two-dimensional layers, further extended to a three dimensional supramolecular network via π-stacking interactions. Thermal analysis data showed that the cocrystal exhibited a lower thermal stability than 5-FU, but it is more stable than dpya. Molecular docking method was used to investigate in silico anticancer activity of the new co-crystal against a cancer target protein, human thymidylate synthase (TS). The free binding affinity of the cocrystal towards TS was notably higher than those of both reference ligands 5-FU and 5-fluorodeoxyuridine monophosphate (FdUMP), indicating the enzyme inhibition and anticancer potential of the cocrystal. Additionally, the molecular dynamics (MD) simulation and binding free energy studies confirmed the stability of the TS-cocrystal complex in a period of 50 ns.

Genotype and Allele Frequency of Methylenetetrahydrofolate Reductase 677CTmutation in Female Arabs residing in the United Arab Emirates

The MTHFR gene (Methylenetetrahydrofolate reductase), responsible for encoding the MTHFR enzyme, is vital for the body’s methylation processes, which are crucial for DNA synthesis, repair, and overall metabolic functions. Previous studies have shown that mutations in the MTHFR gene are associated with various diseases, including neural tube defects, male infertility, type II diabetes mellitus, cardiovascular diseases, and certain cancers. Additionally, abnormal methylation of the MTHFR gene can suppress other important genes such as methionine synthase, thymidylate synthase, choline kinase, and folate receptor genes. This suppression can result in a deficiency of the MTHFR enzyme, essential for proper cellular function, especially when a 677CT (C to T) transition occurs, leading to reduced enzyme activity. Despite the known implications of MTHFR gene mutations, no studies have probed these mutations in the female Arab population. To fill this gap, a pilot study was conducted to determine the prevalence of the MTHFR C677T gene mutation among 45 healthy Arab individuals in the United Arab Emirates. The study used the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method to detect the mutation. The study found that the mutated T allele had a 6% occurrence rate in the female Arab population. The genotype frequencies were 86.6% for the CC genotype, 13.3% for the CT genotype, and 0% for the TT genotype, indicating a relatively low prevalence of the MTHFR C677T polymorphism in Arab women. These findings provide preliminary data that can form the basis for further research on MTHFR gene mutations in this population, potentially enhancing the understanding and management of related health conditions. J MEDICINE 2024; 25: 141-148

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Intrinsic PARG inhibitor sensitivity is mimicked by TIMELESS haploinsufficiency and rescued by nucleoside supplementation.

A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analysed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork stalling, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential. Importantly nucleoside supplementation restores PARG inhibitor resistance despite the continued presence of PAR chains, indicating that sensitivity does not correlate with PAR levels. In addition, we show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances.

TYMS Knockdown Suppresses Cells Proliferation, Promotes Ferroptosis via Inhibits PI3K/Akt/mTOR Signaling Pathway Activation in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by a grim prognosis and numerous challenges. The objective of our study was to examine the role of thymidylate synthase (TYMS) in TNBC and its impact on ferroptosis. The expression of TYMS was analyzed in databases, along with its prognostic correlation. TYMS positive expression was identified through immunohistochemistry (IHC), while real-time quantitative PCR (qRTPCR) was employed to measure TYMS mRNA levels in various cell lines. Western blotting was utilized to assess protein expression. Cell proliferation, mobility, apoptosis, and reactive oxygen species (ROS) levels were evaluated using CCK8, wound scratch healing assay, transwell assay, and flow cytometry, respectively. Additionally, a tumor xenograft model was established in BALB/c nude mice for further investigation. Tumor volume and weight were measured, and histopathological analysis using hematoxylin and eosin (H&E) staining was conducted to assess tumor tissue changes. IHC staining was employed to detect the expression of Ki67 in tumor tissues. High expression of TYMS was observed in TNBC and was found to be correlated with poor prognosis in patients. Among various cell lines, TYMS expression was highest in BT549 cells. Knockdown of TYMS resulted in suppression of cell proliferation and mobility, as well as promotion of apoptosis. Furthermore, knockdown of TYMS led to increased accumulation of ROS and Fe2+ levels, along with upregulation of ACLS4 expression and downregulation of glutathione peroxidase 4 (GPX4) expression. In vivo studies showed that knockdown of TYMS inhibited tumor growth. Additionally, knockdown of TYMS was associated with inhibition of mTOR, p-PI3K, and p-Akt expression. Our research showed that the knockdown of TYMS suppressed the TNBC progression by inhibited cells proliferation via ferroptosis. Its underlying mechanism is related to the PI3K /Akt pathway. Our study provides a novel sight for the suppression effect of TYMS on TNBC.