Protumorigenic Role of Elevated Levels of DNA Polymerase Epsilon Predicts an Immune-Suppressive Microenvironment in Clear Cell Renal Cell Carcinoma

Increasing evidence indicates that DNA polymerase epsilon (POLE), which mediates DNA damage repair, is significantly associated with tumor prognosis. This study aimed to analyze POLE expression in tumor samples and its prognostic value for patients with clear cell renal cell carcinoma (ccRCC). We found significantly elevated POLE expression in ccRCC tissues compared with normal tissues of multiple independent cohorts. The POLE expression levels of 523 patients with ccRCC (The Cancer Genome Atlas RNA-seq data) and 179 patients with ccRCC with immunohistochemical data (Fudan University Shanghai Cancer Center) were analyzed to investigate the prognostic implications of POLE expression. Cox regression analyses were implemented to explore the effect of POLE expression on the prognosis of pan-cancer. These findings revealed that elevated POLE expression levels significantly correlated with shorter overall survival (p < 0.001, n = 701) of patients with ccRCC. These data indicate that POLE expression may serve as a prognostic biomarker for cancers. Although POLE mutations were not significantly associated with survival benefits conferred upon patients with ccRCC, a CD4+ T cell-regulated immune microenvironment was significantly activated. Moreover, we found that POLE expression in cancers significantly correlated with an immunosuppressive tumor microenvironment, higher intratumoral heterogeneity, and expression of immune checkpoint genes PDCD1, CTLA4, and CD86, possibly mediated via the JAK/STAT and Notch signaling pathways. In conclusion, the present study is the first to our knowledge to indicate that elevated POLE expression is significantly associated with poor survival and an immune-suppressive tumor microenvironment in ccRCC. These findings suggest that POLE can serve as a biomarker for guiding molecular diagnosis and facilitating the development of novel individual therapeutic strategies for patients with advanced ccRCC.

Activation of oxidative phosphorylation in TP53-inactive endometrial carcinomas with a poor prognosis

ObjectiveWe aimed to identify pathways for potential therapeutic targets by conducting molecular profiling of endometrial carcinomas in patients with poor prognosis.MethodsThe classification of endometrial carcinomas has undergone a paradigm shift with the advent of next generation sequencing based molecular profiling. Although this emerging classification reflects poor prognosis in patients with endometrial carcinoma, knowledge of affected biological pathways is still lacking. In this study, 85 patients with endometrial carcinomas at the Shizuoka Cancer Center were evaluated from January 2014 to March 2019 and classified based on The Cancer Genome Atlas subgroups. The accumulation of germline and somatic mutations was determined using next generation sequencing. Gene expression profiling was used to determine the effect of TP53 inactivation on the recurrence of endometrial carcinoma. Additionally, the biological pathways associated with TP53 inactivation were estimated by pathway analysis based on gene expression.ResultsBased on The Cancer Genome Atlas classification, the ratio of polymerase-epsilon to copy number-high subgroups and the frequency of PTEN and TP53 mutations differed in patients, and mutations of ARHGAP35 observed in normal endometrium were accumulated in the polymerase-epsilon and microsatellite instability subgroups. We revealed that copy number-high reflects TP53 inactivation in endometrial carcinomas, and that TP53-inactive tumors with or without TP53 mutations have poor prognosis. Furthermore, overexpression of aurora kinase A and activation of oxidative phosphorylation were found in TP53-inactivated endometrial carcinomas, suggesting that the PI3K/mTOR and autophagy pathways are potential drug targets.DiscussionOur analysis revealed a relationship between pathways involved in oxidative phosphorylation and poor prognosis and provides insight into potential drug targets.

Detection and characterization of replication origins defined by DNA polymerase epsilon

Human origins of replication (ORI) are recognized by the cellular machinery through mechanisms which are still poorly understood. The process of DNA replication is highly conserved, but the location of ORI may vary considerably from one round of replication to the next, and depends on many factors, making them difficult to map in the genome. We investigated the possibility that at genomic scale, the mutator phenotype associated with DNA polymerase epsilon; exonuclease domain mutation could identify genomic positions of replication origins. Here we report the genome-wide localization of DNA replication origins in POLE-mutated tumors using whole genome sequencing data from The Cancer Genome Atlas project. We show that mutation-based detection of replication origins allows to identify constitutive origins shared between various individuals. We developed a novel method to compare two or more sets of genomic coordinates based on Smith-Waterman-like dynamic programming, which we used to compare replication origin positions obtained using multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

An in-silico study of Polymerase Epsilon catalytic subunit proteins in Arabidopsis thaliana

Arabidopsis thaliana genome encodes two POLE2 homologs known as polymerase epsilon catalytic subunit A (POLE2A) and polymerase epsilon catalytic subunit B (POLE2B). They play a very important role in DNA repair mechanisms. In this study, bioinformatics tools were used to understand DNA repair mechanisms in A. thaliana in which POLE2A and POLE2B proteins are involved. Through interactome analysis of POLE2A and POLE2B homolog proteins in A. thaliana, their additional roles in DNA repair were explored. The most important proteins that are participating in DNA repairs, like MSH2, MSH5, PCNA1, PCNA2, PRL, and CDC45 were identified as interactors of both POLE2A and POLE2B. The three-dimensional structure of POLE2 proteins was identified to decipher the complexity of NER, GG-NER, MMR, TFIIH, and TC-NER repair mechanisms through the identification of docking sites. The interaction complex of POLE2A and POLE2B with six proteins was confirmed and found to have a significant role in DNA repair processes and UV-B tolerance. The interactome analysis of POLE2A and POLE2B performed here once again confirms the complexity of the DNA repair mechanism in plants.

DNA polymerase epsilon (POLE) is differentially expressed in the lymph node and brain metastases of patients with metastatic breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes to discover genes associated with brain metastasis in patients with metastatic breast cancer. We found that the DNA polymerase epsilon, catalytic subunit, encoded by POLE was among the genes whose expression was most different in the brain and lymph node metastases of patients with metastatic breast cancer as compared to primary tumors of the breast and normal breast tissues, respectively. POLE mRNA was present at higher quantities in brain metastatic tissues as compared to primary tumors of the breast. These data combined suggest some level of common origin for metastases that reside in the lymph nodes and colonize the brain.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis

Background Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Results We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. Conclusion These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

740 Biomarkers diverging between tumor mutation burden and microsatellite instability

BackgroundDNA repair is a critical process to maintain DNA integrity. It is conducted by distinct pathways of genes, many of whose alterations are thought to result in genomic instability and hypermutability, ultimately contributing to tumorigenesis. Tumor Mutation Burden (TMB) and Microsatellite Instability (MSI) are considered as efficacy biomarkers for immunotherapy.1 2 However, there has been little characterization of the association between DNA repair genes and TMB/MSI in cancer. This study aims to further understand DNA repair genes and evaluate the contribution of their alteration to TMB and MSI.MethodsWe systematically analyzed 282 DNA repair genes involved in 20 DNA repair pathways. These genes were evaluated for mutations based on 274 sequenced colorectal tumor samples from the TCGA database. The functional impacts of these mutations were analyzed, and only damaging mutations were used for the subsequent analysis. The most frequently mutated genes were identified. The association between the damaging mutations and TMB/MSI status was calculated for each gene, and the significant genes were subject to further pathway enrichment analysis. We also compared the gene expression between TMB high and low as well as between MSI-H and MSI-L/MSS for each gene based on their RNAseq data. The potential associations with TMB/MSI high phenotypes were evaluated.Results94 genes were identified to be significantly mutated in TMB high, including all of the 26 genes that were significant in MSI-H . The genes are enriched in multiple pathways, including Fanconi anemia, Base excision repair, and Mismatch repair. At the expression level, 28 genes are significantly downregulated in TMB high samples, while 35 genes in MSI-H, suggesting that the inactivation of these genes might be mediated by epigenetic abnormalities (figure 1). 10 genes, including POLE, were identified that are significantly mutated in TMB high samples as compared to MSI-H samples (table 1). Loss of function of these genes may result in an ultra-mutated phenotype. Contradicting the notion that POLE mutation is predominantly associated with MSS tumors and are mutually exclusive with the complete loss of MMR,4–6 we found about half of POLE-mutant samples (8/16) were MSI high, five of which had MMR mutations (figure 2).Abstract 740 Figure 1Venn diagram of significant genes associated with MSI-H and TMB-high, identified using expression changes and loss of function mutations.Abstract 740 Table 1Genes significantly mutated in TMB-high compared to MSI-H.Abstract 740 Figure 2MLH1 expressed significantly lower in MSI-H samplesConclusionsThe study investigated the association of DNA repair genes with TMB and MSI. We compared genes significantly altered in TMB high and MSI-H samples and identified genes pointing to a potential mechanism that induces ultra-mutation in a subset of cancer patients with intact MMR, which can serve as potential biomarkers for immunotherapy efficacy linked with high TMB.ReferencesChan TA, Yarchoan M, Jaffee E, et al. Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic. Ann Oncol 2019; 30(1):44–56.Chang L, Chang M, Chang HM, Chang F. Microsatellite Instability: A Predictive Biomarker for Cancer Immunotherapy. Appl Immunohistochem Mol Morphol 2018;26(2):e15–e21.Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012;487:330–337.Albertson TM, Ogawa M, Bugni JM, et al. DNA polymerase epsilon and delta proofreading suppress discrete mutator and cancer phenotypes in mice. Proc Natl Acad Sci U S A 2009;106:17101-17104.Church DN, Briggs SE, Palles C, et al. DNA polymerase epsilon and delta exonuclease domain mutations in endometrial cancer. Hum Mol Genet 2013;22:2820–2828.Valle L, Hernandez-Illan E, Bellido F, et al. New insights into POLE and POLD1 germline mutations in familial colorectal cancer and polyposis. Hum Mol Genet 2014;23:3506–3512.

DNA polymerase epsilon is a central coordinator of heterochromatin structure and function in Arabidopsis

BackgroundChromatin organizes the DNA molecule and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear.ResultsWe isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, release of heterochromatin silencing in POL2A deficient mutants paradoxically occurs in a chromatin context of increased level of these two repressive epigenetic marks. At the nuclear level, POL2A defect is associated with fragmentation of heterochromatin.ConclusionThese results indicate that POL2A is critical to secure both heterochromatin structure and function. We also reveal that unhindered replisome progression is required for the faithful propagation of DNA methylation through the cell cycle.