Search Results (14)

DNA polymerase epsilon (POLe) is the leading strand replicative polymerase. POLe mutations located primarily in the proofreading domain cause replication errors and increase mutation burden in cancer cells. Consequently, POLe has been classified as a cancer driver gene. Certain POLe frameshift mutations that affect the proofreading domain are purified in cancer cells, but point mutations in other domains have also been reported. Here we use an artificial intelligence algorithm to determine what other mutations co-occur with POLe mutations in colorectal cancers. We partitioned POLe mutations into driver, passenger, and WT (no mutation), then assessed mutations in other genes in these three groups. We found that a driver POLe mutation is not likely to associate with driver mutations in other genes. Thus, driver mutations in colorectal cancers appear to purify in a manner that is independent of POLe. Mutations that affect POLe function do not necessarily increase the frequency of driver mutations in other genes. Structural analysis shows that many POLe driver mutations affect coordination of the Mg²⁺ ion in the active site. Our data show that the accumulation of colorectal cancer mutations is driven by complex factors. Full article

Colorectal cancer (CRC) is the third most common malignancy worldwide and the second leading cause of cancer-related mortality in the United States. The incidence of early-onset colorectal cancer (EOCRC) has been increasing over the past several decades. While the etiologies for this rising incidence remain unclear, genetic factors likely play an important role. DNA polymerase epsilon (POLE) mutations occur at a higher rate than average-onset colorectal cancer (AOCRC). DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is a promising target for immune checkpoint inhibitors due to its association with various human malignancies, including colorectal cancer. EOCRC remains a major area of focus, and POLE mutations leading to the high-TMB subtype constitute a potential therapeutic target. Full article

Background: Endometrial cancer (EC) incidence and mortality have been steadily rising globally over recent decades. The introduction of advanced molecular technologies, such as next-generation sequencing (NGS) alongside the FIGO 2023 classification, presents opportunities for refined diagnostics and risk stratification. This study aimed to analyze differences in EC classification among oncology centers in southeastern Poland. Methods: Data were collected from 461 consecutive patients newly diagnosed with EC between 2022 and 2024 at four major oncology centers in southeastern Poland. Molecular and immunohistochemical (IHC) analyses were conducted on formalin-fixed paraffin-embedded (FFPE) tissues to identify key markers, including POLE mutations, MSI-H, and p53 status. Results: The application of the FIGO 2023 staging system revealed statistically significant inter-center differences, with Centers 1 and 4 diagnosing a higher proportion of early-stage cases. The most prevalent subtype was NSMP, observed in 51% of cases. MSI-H occurred in 13–36% of patients, depending on the center. p53 mutations ranged from 9% to 26%. POLE mutations were identified in 4% of patients overall. Significant variations in the molecular subtype distribution across centers highlight potential differences in diagnostic access or tumor biology. Conclusions: The findings demonstrate regional differences in EC staging and molecular profiles in Poland, potentially reflecting disparities in diagnostic resources, methodologies, or tumor characteristics. Addressing these variations through standardized diagnostic protocols and equitable access to molecular tools is critical for optimizing patient outcomes. Future research should focus on evaluating the impact of molecular markers on therapy response and prognosis to guide personalized treatment strategies. Full article

The incidence of infections caused by Candida species, specifically by drug-resistant isolates, is a major health concern as they can disseminate to and colonize most vital organs, enhancing morbidity and mortality. Several molecular mechanisms have been reported to be involved in drug resistance. These are mostly drug- and isolate-specific. Here, we characterized three different genetically modified strains of C. albicans that were multi-drug-resistant (MDR) and deciphered a uniform mechanism responsible for resistance. DNA polymerase epsilon (Polε) is a leading strand-specific polymerase consisting of four subunits, namely, Pol2, Dpb2, Dpb3, and Dpb4. The deletion of one or both of the Dpb3 and Dpb4 subunits in C. albicans rendered multi-drug resistance. A detailed characterization of these strains revealed that acquired mutagenesis, drug efflux pumps, and other known mechanisms did not play a significant role because the complemented strain showed drug sensitivity. More importantly, the function of heat shock protein 90 (Hsp90) in these knockout strains is critical for reducing susceptibility to several antifungal drugs. Cell wall deformity and composition in these strains can add to such a phenotype. The inhibition of Hsp90 function by geldanamycin and tricostatin A sensitized the MDR strains to antifungals. Considering our earlier research and this report, we suggest that replication stress induces Hsp90 expression and activity in order to orchestrate a cellular stress response circuit and thus develop fungal drug resistance. Thus, Hsp90 is an important drug target for use in combinatorial therapy. Full article

Dedifferentiated endometrioid adenocarcinoma is characterised by the coexistence of an undifferentiated carcinoma and a low-grade endometrioid adenocarcinoma. The low-grade component in this subtype of endometrial carcinoma is Grade 1 or 2 according to the Federation of Gynaecology and Obstetrics (FIGO) grading system. The coexistence of low-grade endometrial carcinoma and solid undifferentiated carcinoma can cause diagnostic problems on histological examination. In fact, this combination can often be mistaken for a more common Grade 2 or Grade 3 endometrial carcinoma. Therefore, this subtype of uterine carcinoma can often go under-recognised. An accurate diagnosis of dedifferentiated endometrial carcinoma is mandatory because of its poorer prognosis compared to Grade 3 endometrial carcinoma, with a solid undifferentiated component that can amount to as much as 20% of the entire tumour. The aim of this review is to provide clinical, immunohistochemical, and molecular data to aid with making an accurate histological diagnosis and to establish whether there are any findings which could have an impact on the prognosis or therapeutic implications of this rare and aggressive uterine neoplasm. Full article

Assessing survival risk in patients with high-grade endometrial carcinomas has remained challenging. We aimed to investigate the distribution of molecular subtypes and assess their prognostic role in a large cohort of 355 patients with high-grade endometrial carcinoma. Molecular classification was determined using DNA polymerase epsilon (POLE) sequencing as well as immunohistochemical staining for p53 and mismatch repair (MMR) proteins. Endometrial carcinomas were stratified into four subtypes: POLE ultramutated, MMR-deficient, non-specific molecular profile (NSMP), and p53-mutant. This study included 177 and 178 patients with endometrioid and non-endometrioid carcinomas, respectively. Forty-two patients (11.8%) were categorized as POLE ultramutated, 106 (29.9%) as MMR-deficient, 128 (36.1%) as p53-mutant, and 79 (22.2%) as NSMP. Patients of different molecular subtypes had distinct survival times; molecular classification, but not histotype, was significantly associated with survival outcomes. When incorporating molecular classification into the stratification model, 52 patients (15.5%) switched risk groups, with 40 (11.9%) shifting to a lower risk for having a POLE mutation and 12 (3.6%) shifting to a higher risk owing to p53-mutant status. Molecular classification may provide more accurate prognostic information among patients with high-grade endometrial carcinomas and improve their stratification for purposes of clinical management. Full article

With advances in next generation sequencing (NGS) technologies, efforts have been made to develop personalized medicine, targeting the specific genetic makeup of an individual. Somatic or germline DNA Polymerase epsilon (PolE) mutations cause ultramutated (>100 mutations/Mb) cancer. In contrast to mismatch repair-deficient hypermutated (>10 mutations/Mb) cancer, PolE-associated cancer is primarily microsatellite stable (MSS) In this article, we provide a comprehensive review of this PolE-associated ultramutated tumor. We describe its molecular characteristics, including the mutation sites and mutation signature of this type of tumor and the mechanism of its ultramutagenesis. We discuss its good clinical prognosis and elucidate the mechanism for enhanced immunogenicity with a high tumor mutation burden, increased neoantigen load, and enriched tumor-infiltrating lymphocytes. We also provide the rationale for immune checkpoint inhibitors in PolE-mutated tumors. Full article

In metastatic colorectal cancer (mCRC), remarkable advances have been achieved with immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 and CTLA-4, only in a small subset of tumours (4–5%), harbouring a deficient mismatch repair system (dMMR)/microsatellite instability–high (MSI-H) or mutations in the catalytic subunit of polymerase epsilon (POLE). Within this framework, several combination strategies have been investigated to sensitize proficient mismatch repair (pMMR)/microsatellite stable (MSS) mCRC to ICIs, with disappointing results so far. However, at the last ESMO meeting, two phase II trials AtezoTRIBE and MAYA provided promising results in this field. In the comparative AtezoTRIBE trial, the addition of atezolizumab to FOLFOXIRI (5-fluoruracil, oxaliplatin and irinotecan) and bevacizumab led to a significant advantage in terms of progression free survival (PFS) in a population of untreated mCRC patients, not selected according to MMR/MSI status. In the single-arm MAYA trial, immune priming with temozolomide in pMMR/MSS chemo-resistant mCRC patients with silencing of O6-methylguanine-DNA methyltransferase (MGMT) allowed reporting signals of sensitivity to the subsequent therapy with nivolumab and a low dose of ipilimumab in some patients. Here, we discuss the rationale, results, criticisms and research perspectives opened by these two studies. Full article

Recently, guidelines for endometrial cancer (EC) were released that guide treatment decisions according to the tumors’ molecular profiles. To date, no real-world data regarding the clinical feasibility of molecular profiling have been released. This retrospective, monocentric study investigated the clinical feasibility of molecular profiling and its potential impact on treatment decisions. Tumor specimens underwent molecular profiling (testing for genetic alterations, (immune-)histological examination of lymphovascular space invasion (LVSI), and L1CAM) as part of the clinical routine and were classified according to the European Society for Medical Oncology (ESMO) classification system and to an integrated molecular risk stratification. Shifts between risk groups and potential treatment alterations are described. A total of 60 cases were included, of which twelve were excluded (20%), and eight of the remaining 48 were not characterized (drop-out rate of 16.7%). Molecular profiling revealed 4, 6, 25, and 5 patients with DNA polymerase-epsilon mutation, microsatellite instability, no specific molecular profile, and TP53 mutation, respectively. Three patients had substantial LVSI, and four patients showed high L1CAM expression. Molecular profiling took a median of 18.5 days. Substantial shifts occurred between the classification systems: four patients were upstaged, and 19 patients were downstaged. Molecular profiling of EC specimens is feasible in a daily routine, and new risk classification systems will change treatment decisions substantially. Full article

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks. Full article

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5’ terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs). Full article

Background: Metastatic CRC (mCRC) is a molecular heterogeneous disease. The aim of this review is to give an overview of molecular-driven treatment of mCRC patients. Methods: A review of clinical trials, retrospective studies and case reports was performed regarding molecular biomarkers with therapeutic implications. Results: RAS wild-type status was confirmed as being crucial for anti-epidermal growth factor receptor (EGFR) monoclonal antibodies and for rechallenge strategy. Antiangiogenic therapies improve survival in first- and second-line settings, irrespective of RAS status, while tyrosine kinase inhibitors (TKIs) remain promising in refractory mCRC. Promising results emerged from anti-HER2 drugs trials in HER2-positive mCRC. Target inhibitors were successful for BRAF^V600E mutant mCRC patients, while immunotherapy was successful for microsatellite instability-high/defective mismatch repair (MSI-H/dMMR) or DNA polymerase epsilon catalytic subunit (POLE-1) mutant patients. Data are still lacking on NTRK, RET, MGMT, and TGF-β, which require further research. Conclusion: Several molecular biomarkers have been identified for the tailored treatment of mCRC patients and multiple efforts are currently ongoing to increase the therapeutic options. In the era of precision medicine, molecular-biology-driven treatment is the key to impro patient selection and patient outcomes. Further research and large phase III trials are required to ameliorate the therapeutic management of these patients. Full article

Due to their sedentary lifestyle, plants are constantly exposed to different stress stimuli. Stress comes in variety of forms where factors like radiation, free radicals, “replication errors, polymerase slippage”, and chemical mutagens result in genotoxic or cytotoxic damage. In order to face “the base oxidation or DNA replication stress”, plants have developed many sophisticated mechanisms. One of them is the DNA mismatch repair (MMR) pathway. The main part of the MMR is the MutS homologue (MSH) protein family. The genome of Arabidopsis thaliana encodes at least seven homologues of the MSH family: AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6, and AtMSH7. Despite their importance, the functions of AtMSH homologs have not been investigated. In this work, bioinformatics tools were used to obtain a better understanding of MSH-mediated DNA repair mechanisms in Arabidopsis thaliana and to understand the additional biological roles of AtMSH family members. In silico analysis, including phylogeny tracking, prediction of 3D structure, interactome analysis, and docking site prediction, suggested interactions with proteins were important for physiological development of A. thaliana. The MSH homologs extensively interacted with both TIL1 and TIL2 (DNA polymerase epsilon catalytic subunit), proteins involved in cell fate determination during plant embryogenesis and involved in flowering time repression. Additionally, interactions with the RECQ protein family (helicase enzymes) and proteins of nucleotide excision repair pathway were detected. Taken together, the results presented here confirm the important role of AtMSH proteins in mismatch repair and suggest important new physiological roles. Full article

The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication. Full article