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Dysregulation of human DNA polymerase delta (Polδ) subunits is associated with genome instability and pathological disorders. Genome databases suggest the expression of several spliced variants of subunits which may alter Polδ function. Here, we analyzed the protein‐encoding variants of the Polδ subunit p12 and their association with cancer. p12 isoform‐2 (p12*) encodes a 79 aa protein with a C‐terminal tail distinct from the previously characterized p12. Like p12, p12* dimerizes and interacts with p125 and p50 subunits and is thus an integral component of Polδ. Further, we observed dysregulated p12* expression in low‐grade glioma, renal, thyroid, and pancreatic carcinomas. This study identifies a previously unrecognized Polδ complex and highlights a possible regulatory role of p12 variants in cellular phenotypes. [ABSTRACT FROM AUTHOR]

Background The resistant and aggressive nature of triple-negative breast cancer (TNBC) renders it mostly incurable even following extensive multimodal treatment. Therefore, more studies are required to understand the underlying molecular mechanisms of its pathogenesis. SIRT1 is a class III histone deacetylase NAD + -dependent enzyme that is interlinked in tumor progression, apoptosis, metastasis, and other mechanisms of tumorigenesis, while DNA polymerase delta 1 (POLD1) functions as a gene coding for p125, which plays an important role in genome stability and DNA replication. Objective We aimed to investigate the downstream signaling pathway of EX-527, a potent and selective SIRT1 inhibitor, in MDA-MB-231 breast cancer cell lines, and the crosstalk between SIRT1 and POLD1, which is essential for the activities of polymerase δ. Methods The antiproliferative and apoptotic effects of EX-527 on MDA-MB-231 cells were assessed by MTT and annexin V/PI double staining assays. Migration and invasion activity of MDA-MB-231 cells were assessed by wound-healing scratch and transwell assays. Protein expressions were examined using Western Blot analysis. Results MDA-MB-231 cells treatment with IC50 values of 45.3 μM EX-527 significantly suppressed cell proliferation and induced apoptosis by down-regulating SIRT1. Also, it significantly repressed migration and invasion of MDA-MB-231 cells as evaluated by wound healing and transwell invasion assays. Western blot results showed that decreased expression of SIRT1 is positively correlated with expression of p53 along with down-regulating POLD1. Conclusion SIRT1 could have an oncogenic role in breast cancer development and progression via activating POLD1. These conclusions present new insights into the underlying mechanisms of TNBC. [ABSTRACT FROM AUTHOR]

Abstract Background Prostate cancer (PCa) is the second leading cause of tumor-related mortality in men. Metastasis from advanced tumors is the primary cause of death among patients. Identifying novel and effective biomarkers is essential for understanding the mechanisms of metastasis in PCa patients and developing successful interventions. Methods Using the GSE8511 and GSE27616 data sets, 21 metastasis-related genes were identified through the weighted gene co-expression network analysis (WGCNA) method. Subsequent functional analysis of these genes was conducted on the gene set cancer analysis (GSCA) website. Cluster analysis was utilized to explore the relationship between these genes, immune infiltration in PCa, and the efficacy of targeted drug IC50 scores. Machine learning algorithms were then employed to construct diagnostic and prognostic models, assessing their predictive accuracy. Additionally, multivariate COX regression analysis highlighted the significant role of POLD1 and examined its association with DNA methylation. Finally, molecular docking and immunohistochemistry experiments were carried out to assess the binding affinity of POLD1 to PCa drugs and its impact on PCa prognosis. Results The study identified 21 metastasis-related genes using the WGCNA method, which were found to be associated with DNA damage, hormone AR activation, and inhibition of the RTK pathway. Cluster analysis confirmed a significant correlation between these genes and PCa metastasis, particularly in the context of immunotherapy and targeted therapy drugs. A diagnostic model combining multiple machine learning algorithms showed strong predictive capabilities for PCa diagnosis, while a transfer model using the LASSO algorithm also yielded promising results. POLD1 emerged as a key prognostic gene among the metastatic genes, showing associations with DNA methylation. Molecular docking experiments supported its high affinity with PCa-targeted drugs. Immunohistochemistry experiments further validated that increased POLD1 expression is linked to poor prognosis in PCa patients. Conclusions The developed diagnostic and metastasis models provide substantial value for patients with prostate cancer. The discovery of POLD1 as a novel biomarker related to prostate cancer metastasis offers a promising avenue for enhancing treatment of prostate cancer metastasis.

Background: Prostate cancer (PCa) is the second leading cause of tumor-related mortality in men. Metastasis from advanced tumors is the primary cause of death among patients. Identifying novel and effective biomarkers is essential for understanding the mechanisms of metastasis in PCa patients and developing successful interventions. Methods: Using the GSE8511 and GSE27616 data sets, 21 metastasis-related genes were identified through the weighted gene co-expression network analysis (WGCNA) method. Subsequent functional analysis of these genes was conducted on the gene set cancer analysis (GSCA) website. Cluster analysis was utilized to explore the relationship between these genes, immune infiltration in PCa, and the efficacy of targeted drug IC50 scores. Machine learning algorithms were then employed to construct diagnostic and prognostic models, assessing their predictive accuracy. Additionally, multivariate COX regression analysis highlighted the significant role of POLD1 and examined its association with DNA methylation. Finally, molecular docking and immunohistochemistry experiments were carried out to assess the binding affinity of POLD1 to PCa drugs and its impact on PCa prognosis. Results: The study identified 21 metastasis-related genes using the WGCNA method, which were found to be associated with DNA damage, hormone AR activation, and inhibition of the RTK pathway. Cluster analysis confirmed a significant correlation between these genes and PCa metastasis, particularly in the context of immunotherapy and targeted therapy drugs. A diagnostic model combining multiple machine learning algorithms showed strong predictive capabilities for PCa diagnosis, while a transfer model using the LASSO algorithm also yielded promising results. POLD1 emerged as a key prognostic gene among the metastatic genes, showing associations with DNA methylation. Molecular docking experiments supported its high affinity with PCa-targeted drugs. Immunohistochemistry experiments further validated that increased POLD1 expression is linked to poor prognosis in PCa patients. Conclusions: The developed diagnostic and metastasis models provide substantial value for patients with prostate cancer. The discovery of POLD1 as a novel biomarker related to prostate cancer metastasis offers a promising avenue for enhancing treatment of prostate cancer metastasis. [ABSTRACT FROM AUTHOR]

Abstract Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy.

Background/Aim: The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways. Materials and Methods: The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines. Results: High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G1/S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines. Conclusion: POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G1/S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM. [ABSTRACT FROM AUTHOR]

Simple Summary: Germline mutations in the DNA polymerase delta 1 (POLD1) exonuclease domain cause DNA proofreading defects, tumor hypermutation, and predispose to hereditary colorectal and endometrial cancer. In this work, we used a multiprong approach to demonstrate that mutations that alter the amino acid charges in the DEDD motif of exonuclease domains could lead to proofreading deficiency. This novel mutation class, which is currently classified as a variant of uncertain significance, should be reclassified as likely pathogenic. Background: Mutations in the DNA polymerase delta 1 (POLD1) exonuclease domain cause DNA proofreading defects, hypermutation, hereditary colorectal and endometrial cancer, and are predictive of immunotherapy response. Exonuclease activity is carried out by two magnesium cations, bound to four highly conserved, negatively charged amino acids (AA) consisting of aspartic acid at amino acid position 316 (p.D316), glutamic acid at position 318 (p.E318), p.D402, and p.D515 (termed DEDD motif). Germline polymorphisms resulting in charge-discordant AA substitutions in the DEDD motif are classified as variants of uncertain significance (VUSs) by laboratories and thus would be considered clinically inactionable. We hypothesize this mutation class is clinically pathogenic. Methods: A review of clinical presentation was performed in our index patient with a POLD1(p.D402N) heterozygous proband with endometrial cancer. Implications of this mutation class were evaluated by a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)-guided systematic review, in silico analysis with orthogonal biochemical confirmation, and whole-exome and RNA sequencing analysis of the patient's tumor and engineered cell lines. Results: Our systematic review favored a Mendelian disease mutation class associated with endometrial and colorectal cancers. In silico analysis predicted defective protein function, confirmed by biochemical assay demonstrating loss of nuclease activity. A POLD1-specific mutational signature was found in both the patient's tumor and POLD1(p.D402N) overexpressing cell. Furthermore, paired whole-exome/transcriptome analysis of endometrial tumor demonstrated hypermutation and T cell-inflamed gene expression profile (GEP), which are joint predictive biomarkers for pembrolizumab. Our patient showed a deep, durable response to immune checkpoint inhibitor (ICI). Conclusion: Charge-discordant AA substitution in the DEDD motif of POLD1 is detrimental to DNA proofreading and should be reclassified as likely pathogenic and possibly predictive of ICI sensitivity. [ABSTRACT FROM AUTHOR]

Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy. [ABSTRACT FROM AUTHOR]

Aging has been considered as a risk factor in many diseases, thus, comprehensively understanding the cellular and molecular mechanisms of delayed aging is important. Here we investigated whether Krüppel‐like factor 14 (KLF14) is a suppressor of cellular senescence and aging. In our research, KLF14 levels significantly decreased not only in the lymphocytes of healthy people but also in the cells and tissues of mice with aging. We performed in vitro and in vivo experiments on cells and mice to reveal the function of KLF14 in aging. KLF14 deficiency facilitates cellular senescence and aging‐related pathologies in C57BL/6J mice, whereas KLF14 overexpression attenuates cellular senescence. Mechanistically, KLF14 delays aging by binding to the POLD1 promoter to positively regulate POLD1 expression. Remarkably, cellular senescence mediated by KLF14 downregulation could be alleviated by POLD1 expression. In addition, perhexiline, an agonist of KLF14, could delay cellular senescence and aging‐related pathologies in senescence‐accelerated P8 mice by inducing POLD1 expression, as perhexiline could enhance the effect of KLF14's transcription activation to POLD1 by elevating the binding level of KLF14 to the POLD1 promoter. Our data indicate that KLF14 might be a critical element in aging by upregulating POLD1 expression, indicating that the activation of KLF14 may delay aging and aging‐associated diseases. [ABSTRACT FROM AUTHOR]

Germline variations in the DNA polymerase genes, POLE and POLD1, can lead to a hereditary cancer syndrome that is characterized by frequent gastrointestinal polyposis and multiple primary malignant tumors. However, because of its rare occurrence, this disorder has not been extensively studied. In this report, we present the case of a 22-year-old female patient who had been diagnosed with gastrointestinal polyposis, breast fibroadenoma, multiple primary colorectal cancers, and glioblastoma (grade IV) within a span of 4 years. Next-generation sequencing analysis revealed a germline variant in POLD1 (c.1816C>A; p.L606M). In silico analysis using protein functional predicting software, including SIFT, Polyphen, GERP++, and CADD, further confirmed the pathogenicity of POLD1 p.L606M (classified as ACMG grade Class 4). In line with polymerase deficiency, both rectal cancer and glioblastoma tissues exhibited a high tumor mutation burden, with 16.9 muts/Mb and 347.1 muts/Mb, respectively. Interestingly, the patient has no family history of cancer, and gene examination of both parents confirms that this is a de novo germline variant. Therefore, molecular screening for POLD1 may be necessary for patients with such a cancer spectrum, regardless of their family history. [ABSTRACT FROM AUTHOR]

Introduction: The study aimed to determine the immunoexpression levels of polymerase delta 1 catalytic subunit (POLD1), a catalytic and proofreading subunit of DNA polymerase delta, in the sections of colorectal cancer (CRC), and to evaluate the significance of POLD1 as a potential prognostic factor in CRC. Material and methods: Paired, tumour and non-cancerous tissue samples of the large intestine distant to the neoplasm were collected from the postoperative material of 78 patients who underwent surgical resection of CRC tumours. Polymerase delta 1 catalytic subunit protein levels were determined using immunohistochemistry. Clinical, pathomorphological, and survival data of the patients were pooled. In addition, POLD1 mRNA expression levels of 599 CRC patients were extracted from The Cancer Genome Atlas (TCGA) datasets and subjected to statistical and survival analysis including the Kaplan-Meier method followed by the log-rank test. Results: Immunoexpression of POLD1 was found in the nuclei of the tumour cells and epithelial cells of unchanged intestinal mucosa. Polymerase delta 1 catalytic subunit immunoreactivity in the tumour was heterogenous, and the average immunoreactivity score was decreased in cancer cells when compared to the mucosa of matched sections of unchanged large intestine (p = 0.0259). However, POLD1 expression at the protein and mRNA levels did not associate with clinicopathological characteristics of the patients and their survival. Conclusions: Despite previous studies suggesting that POLD1 genetic alterations could be promising molecular biomarkers in CRC, our results do not support any prognostic significance of POLD1 expression in CRC. [ABSTRACT FROM AUTHOR]

In 20-30% of patients suspected of a familial colorectal cancer (CRC) syndrome, no underlying genetic cause is detected. Recent advances in whole exome sequencing have generated evidence for new CRC-susceptibility genes including POLE, POLD1 and NTHL1¸ but many patients remain unexplained. Whole exome sequencing was performed on DNA from nine patients from five different families with familial clusters of CRC in which traditional genetic testing failed to yield a diagnosis. Variants were filtered by minor allele frequencies, followed by prioritization based on in silico prediction tools, and the presence in cancer susceptibility genes or genes in cancer-associated pathways. Effects of frameshift variants on protein structure were modeled using I-Tasser. One known pathogenic variant in POLD1 was detected (p.S478N), together with variants in 17 candidate genes not previously associated with CRC. Additional in silico analysis using SIFT, PROVEAN and PolyPhen on the 14 missense variants indicated a possible damaging effect in nine of 14 variants. Modeling of the insertions/deletions showed a damaging effect of two variants in NOTCH2 and CYP1B1. One family was explained by a mutation in a known familial CRC gene. In the remaining four families, the most promising candidates found are a frameshift NOTCH2 and a missense RAB25 variant. This study provides potential novel candidate variants in unexplained familial CRC patients, however, functional validation is imperative to confirm the role of these variants in CRC tumorigenesis. Additionally, while whole exome sequencing enables detection of variants throughout the exome, other causes explaining the familial phenotype such as multiple single nucleotide polymorphisms accumulating to a polygenic risk or epigenetic events, might be missed with this approach.

Abstract Background Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL; OMIM# 615381) is a rare autosomal dominant disorder, with only a few reported cases worldwide. Herein, we describe the clinical features and underlying molecular etiology of MDPL syndrome in an 8-year-old Chinese patient. Methods We performed otological, endocrine, ultrasound, and radiological examinations, as well as genetic testing. Additionally, the literature concerning MDPL was reviewed to do a retrospective analysis of the pathogenesis, genotype–phenotype correlation, and clinical management. Results The proband was diagnosed with MDPL, presenting with mandibular hypoplasia, a characteristic facial appearance, lipodystrophy, and sensorineural hearing loss (SNHL). Whole-exome sequencing and bioinformatics analysis revealed a de novo missense variant in the POLD1 gene, NM_002691.4:c.3185A>G (NP_002682.2:p.(Gln1062Arg)). The retrospective analysis showed wide variation in the MDPL phenotype, but the most frequent features included mandibular hypoplasia, characteristic facial appearance, lipodystrophy, and SNHL. Conclusions This study supplements the mutational spectrum of POLD1. The genetic analysis contributes to the diagnosis of syndromic deafness, and it has a vital role in clinical management and future genetic consultation.

DNA polymerase delta 1 catalytic subunit (POLD1) plays a vital role in genomic copy with high fidelity and DNA damage repair processes. However, the prognostic value of POLD1 and its relationship with tumor immunity in clear cell renal cell carcinoma (ccRCC) remains to be further explored. Transcriptional data sets and clinical information were obtained from the TCGA, ICGC, and GEO databases. Differentially expressed genes (DEGs) were derived from the comparison between the low and high POLD1 expression groups in the TCGA–KIRC cohort. KEGG and gene ontology (GO) analyses were performed for those DEGs to explore the potential influence of POLD1 on the biological behaviors of ccRCC. The prognostic clinical value and mutational characteristics of patients were described and analyzed according to the POLD1 expression levels. TIMER and TISIDB databases were utilized to comprehensively investigate the potential relevance between the POLD1 levels and the status of the immune cells, as well as the tumor infiltration of immune cells. In addition, RT-qPCR, Western blot, immunohistochemistry and several functional and animal experiments were performed for clinical, in vitro and in vivo validation. POLD1 was highly expressed in a variety of tumors including ccRCC, and further verified in a validation cohort of 60 ccRCC samples and in vitro cell line experiments. POLD1 expression levels in the ccRCC samples were associated with various clinical characteristics including pathologic tumor stage and histologic grade. ccRCC patients with high POLD1 expression have poor clinical outcomes and exhibit a higher rate of somatic mutations than those with low POLD1 expression. Cox regression analysis also showed that POLD1 could act as a potential independent prognostic biomarker. The DEGs associated with POLD1 were significantly enriched in the immunity-related pathways. Moreover, further immune infiltration analysis indicated that high POLD1 expression was associated with high NK CD56bright cells, Treg cells, and myeloid-derived suppressor cells' (MDSCs) infiltration scores, as well as their marker gene sets of immune cell status. Meanwhile, POLD1 exhibited resistance to various drugs when highly expressed. Finally, the knockdown of POLD1 inhibited the proliferation and migration, and promoted the apoptosis of ccRCC cells in vitro and in vivo, as well as influenced the activation of oncogenic signaling. Our current study demonstrated that POLD1 is a potential prognostic biomarker for ccRCC patients. It might create a tumor immunosuppressive microenvironment and inhibit the susceptibility to ferroptosis leading to a poor prognosis. [ABSTRACT FROM AUTHOR]

Abstract POLE and POLD1 encode the catalytic and proofreading subunits of DNA polymerase ε and polymerase δ, and play important roles in DNA replication and proofreading. POLE/POLD1 exonuclease domain mutations lead to loss of proofreading function, which causes the accumulation of mutant genes in cells. POLE/POLD1 mutations are not only closely related to tumor formation, but are also a potential molecular marker for predicting the efficacy of immunotherapy in pan-carcinomatous species. The association of POLE/POLD1 mutation, ultra-high mutation load, and good prognosis have recently become the focus of clinical research. This article reviews the function of POLE/POLD1, its relationship with deficient mismatch repair/high microsatellite instability, and the role of POLE/POLD1 mutation in the occurrence and development of various tumors.

Abstract Background DNA polymerase delta 1 catalytic subunit (POLD1) plays a key role in DNA replication and damage repair. A defective DNA proofreading function caused by POLD1 mutation contributes to carcinogenesis, while POLD1 overexpression predicts poor prognosis in cancers. However, the effect of POLD1 in hepatocellular carcinoma (HCC) is not well-understood. Methods Expression patterns of POLD1 were evaluated in TCGA and the HPA databases. Kaplan-Meier curves and Cox regression were used to examine the prognostic value of POLD1. The prognostic and predictive value of POLD1 was further validated by another independent cohort from ICGC database. The influences of DNA copy number variation, methylation and miRNA on POLD1 mRNA expression were examined. The correlation between infiltrating immune cells and POLD1 expression was analyzed. GO and KEGG enrichment analyses were performed to detect biological pathways associated with POLD1 expression in HCC. Results POLD1 was overexpressed in HCC (n = 369) compared with adjacent normal liver (n = 50). POLD1 upregulation was significantly correlated with positive serum AFP and advanced TNM stage. Kaplan–Meier and multivariate analyses suggested that POLD1 overexpression predicts poor prognosis in HCC. DNA copy gain, low POLD1 methylation, and miR‑139-3p downregulation were associated with POLD1 overexpression. Besides, POLD1 expression was associated with the infiltration levels of dendritic cell, macrophage, B cell, and CD4 + T cell in HCC. Functional enrichment analysis suggested “DNA replication”, “mismatch repair” and “cell cycle” pathways might be involved in the effect of POLD1 on HCC pathogenesis. Additionally, POLD1 mRNA expression was significantly associated with tumor mutation burden, microsatellite instability, and prognosis in various tumors. Conclusions POLD1 may be a potential prognostic marker and promising therapeutic target in HCC.

Serrated polyposis syndrome (SPS) is one of the most frequent polyposis syndromes characterized by an increased risk for developing colorectal cancer (CRC). Although SPS etiology has been mainly associated with environmental factors, germline predisposition to SPS could also be relevant for cases with familial aggregation or a family history of SPS/CRC. After whole-exome sequencing of 39 SPS patients from 16 families, we identified a heterozygous germline frameshift variant in the POLD1 gene (c.1941delG, p.(Lys648fs*46)) in a patient with SPS and CRC. Tumor presented an ultra-hypermutated phenotype and microsatellite instability. The POLD1 germline variant segregated in three additional SPS-affected family members. We attempted to create yeast and cellular models for this variant but were no viable. Alternatively, we generated patient-derived organoids (PDOs) from healthy rectal tissue of the index case, as well as from a control donor. Then, we challenged PDOs with a DNA-damaging agent to induce replication stress. No significant differences were observed in the DNA damage response between control and POLD1-Lys648fs PDOs, nor specific mutational signatures were observed. Our results do not support the pathogenicity of the analyzed POLD1 frameshift variant. One possible explanation is that haplosufficiency of the wild-type allele may be compensating for the absence of expression of the frameshift allele. Overall, future work is required to elucidate if functional consequences could be derived from POLD1 alterations different from missense variants in their proofreading domain. To our knowledge, our study presents the first organoid model for germline POLD1 variants and establishes the basis for its use as a model for disease in SPS, CRC and other malignancies.

Objective: POLE and POLD1 mutations cause loss of DNA polymerase function, which may affect genome stability and lead to increased mutations and tumor formation. This article combines online databases and real world samples to further analyze the clinical significance of POLE and/or POLD1 mutations in lung adenocarcinoma (LUAD) patients. Methods: During Jan 2021 to Aug2021,115 cases of postoperative LUAD tissue specimens were enrolled from the Affiliated Hospital of Xuzhou Medical University.Next-generation sequencing (NGS) was used to detect gene mutations. LUAD data sets were download from The Cancer Genome Atlas (TCGA) database, tumor mutation distribution maps were obtained through the Cbioportal online database, and the immune-related cell infiltration status of the samples were calculated through the Cibersort method. Results: The proportion of POLE/POLD1 mutations in real world samples was 7.83% (9/115). TCGA data shows that overall survival (OS) of LUAD patients with POLE/POLD1 mutations tend to have a worse prognosis (P=0.0359). However, patients with this mutation showed a higher mutation count, especially positively correlated with TP53 mutation; at the same time, POLE/POLD1 mutation was positively correlated with LUAD tissue infiltrating immune killer cells, and negatively correlated with immunosuppressive cells, suggesting these patients are sensitive to immune checkpoint inhibitor (ICI). Conclusion: POLE/POLD1 mutations in LUAD patients indicate higher tumor mutation burden and immune microenvironment changes, which can be used as potential biomarkers for the prediction of ICI efficacy and deserve more clinical attention. [ABSTRACT FROM AUTHOR]

Background: Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL; OMIM# 615381) is a rare autosomal dominant disorder, with only a few reported cases worldwide. Herein, we describe the clinical features and underlying molecular etiology of MDPL syndrome in an 8-year-old Chinese patient. Methods: We performed otological, endocrine, ultrasound, and radiological examinations, as well as genetic testing. Additionally, the literature concerning MDPL was reviewed to do a retrospective analysis of the pathogenesis, genotype–phenotype correlation, and clinical management. Results: The proband was diagnosed with MDPL, presenting with mandibular hypoplasia, a characteristic facial appearance, lipodystrophy, and sensorineural hearing loss (SNHL). Whole-exome sequencing and bioinformatics analysis revealed a de novo missense variant in the POLD1 gene, NM_002691.4:c.3185A>G (NP_002682.2:p.(Gln1062Arg)). The retrospective analysis showed wide variation in the MDPL phenotype, but the most frequent features included mandibular hypoplasia, characteristic facial appearance, lipodystrophy, and SNHL. Conclusions: This study supplements the mutational spectrum of POLD1. The genetic analysis contributes to the diagnosis of syndromic deafness, and it has a vital role in clinical management and future genetic consultation. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) in adolescents and young adults (AYA) is rare. Genetic causes include autosomal recessive and dominant monogenic disorders due to pathogenic variants (PVs) in genes involved in DNA repair. However, the genetic etiology of the majority of AYA-CRC remains unidentified. In two teenage siblings with CRC, we show to our knowledge for the first time that AYA-CRC cases can be caused by digenic inheritance of each a heterozygous pathogenic variant (PV) in the mismatch-repair (MMR) gene PMS2 and the proofreading polymerase (PP) Pol δ gene POLD1. With the aim to elucidate how the constitutional polymerase proofreading defect and the high propensity to MMR deficiency (MMRd) interact, we performed a comprehensive tumor analysis of the two siblings' tumors. Results indicate that tumorigenesis is initiated by MMRd and the inherited POLD1 PV contributes to fast tumor progression reflected by an ultra-high tumor mutational burden (TMB) and specific mutational signatures. Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings' CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient's CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd. [ABSTRACT FROM AUTHOR]

POLE and POLD1 encode the catalytic and proofreading subunits of DNA polymerase ε and polymerase δ, and play important roles in DNA replication and proofreading. POLE/POLD1 exonuclease domain mutations lead to loss of proofreading function, which causes the accumulation of mutant genes in cells. POLE/POLD1 mutations are not only closely related to tumor formation, but are also a potential molecular marker for predicting the efficacy of immunotherapy in pan-carcinomatous species. The association of POLE/POLD1 mutation, ultra-high mutation load, and good prognosis have recently become the focus of clinical research. This article reviews the function of POLE/POLD1, its relationship with deficient mismatch repair/high microsatellite instability, and the role of POLE/POLD1 mutation in the occurrence and development of various tumors. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of resveratrol (RSV) on epithelial-mesenchymal transition of MDA-MB-231 cells by down-regulating POLD1 expression. Methods CCK-8 was used to detect the effect of RSV on the activity of MDA-MB-231 cells. POLD1-OE and POLD1-NC cell lines were constructed by transfecting MDA-MB-231 cells with recombinant lentivirus. Western blot was used to detect the expression of POLD1, E-cadherin, N-cadherin and Vimentin after RSV treatment. Transwell invasion experiment and the scratch test were used to detect the cells invasion and migration abilities of each experimental group. Results RSV could significantly inhibit the survival of MDA-MB-231 cells, reduce the expression of POLD1, N-cadherin and Vimentin, increase the expression of E-cadherin, and inhibit the abilities of cell invasion and migration. Increasing the POLD1 expression could reduce the above-mentioned biological effects of RSV on MDA-MB-231 cells. Conclusion RSV could significantly inhibit the viability and EMT of MDA-MB-231 cells by down-regulating the expression of POLD1.

Formation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations of embryo size, pre-gastrulation mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle-related genes frequently lead to cell-cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase δ. Impaired cell proliferation in the tyrn mutant leaves anterior–posterior patterning unperturbed during gastrulation but results in reduced embryo size and severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth.

BackgroundLines of evidence implicate CENPF and FOXM1 may have novel co-operative roles in driving hepatocellular carcinoma (HCC).ObjectiveWe investigated the clinicopathological correlation, functional characterization, molecular mechanism and translational significance of CENPF and FOXM1.MethodsWe carried out integrative studies investigating functional synergism of CENPF and FOXM1 in HCC and its metastasis. Human HCC samples, HCC cell lines and mouse model were used in the studies. Stable knockdown, q-PCR, Western blotting, whole-transcriptomic sequencing (RNA-seq), as well as cell and mouse assays were performed.ResultsUpon clinicopathological correlation, we found that co-overexpression of CENPF and FOXM1 in human HCCs was associated with more aggressive tumor behavior including presence of venous invasion, tumor microsatellite formation, and absence of tumor encapsulation. Moreover, co-silencing FOXM1 and CENPF using shRNA approach in HCC cell lines resulted in significantly reduced cell proliferation. Furthermore, our RNA-seq and differential gene expression analysis delineated that CENPF and FOXM1 co-regulated a specific set of target genes in various metabolic processes and oncogenic signaling pathways. Among them, POLD1, which encodes the catalytic subunit of DNA polymerase δ, was ranked as the top downstream target co-regulated by CENPF and FOXM1. POLD1 expression was positively correlated with that of FOXM1 and CENPF in HCCs. In addition, POLD1 expression was significantly upregulated in HCC tumors. Functionally, in vivo orthotopic injection model showed that stable knockdown of POLD1 in HCC cells suppressed tumor incidence and tumorigenicity and had a trend of diminished lung metastasis.ConclusionTaken together, our data suggest that CENPF and FOXM1 could synergistically support hepatocarcinogenesis via the regulation of POLD1. CENPF and FOXM1 may represent new vulnerabilities to novel drug-based therapy in HCC.

Background/Aim: DNA polymerase delta 1 catalytic subunit (POLD1 or POLD1/p125) plays a crucial role in DNA synthesis and proofreading during the semiconservative genome replication. Mutations of POLD1 are associated with abnormal cell division in various human tumors. However, the significance of altered POLD1 expression in malignant diseases and its usefulness as a prognostic factor is not fully understood. This study aimed to determine POLD1 immunoexpression levels in paired sections of tumor and normal kidney derived from 56 patients with clear cell renal cell carcinoma (ccRCC) and evaluate the significance of POLD1 protein as a potential prognostic factor in ccRCC. Materials and Methods: Tissue samples were collected from 56 patients (27 females and 29 males, mean age 62.6, range=27-83 years) who underwent nephrectomy due to ccRCC. Paired tissue samples were obtained from the tumor and unchanged part of the kidney. The expression of POLD1 protein was assessed by immunohistochemistry. Clinical and pathological data of patients were also collected. Patients were followed-up and the median time of observation period was 39.3 months. Results: The study revealed a significantly higher POLD1 nuclear expression in ccRCC tumor tissue samples and this was correlated with longer survival rates (better prognosis) of ccRCC patients. Conclusion: POLD1 immunoreactivity in ccRCC postoperative material could be helpful as a prognostic marker in the ccRCC patient group. [ABSTRACT FROM AUTHOR]

Pathogenic germline exonuclease domain (ED) variants of POLE and POLD1 cause the Mendelian dominant condition polymerase proof-reading associated polyposis (PPAP). We aimed to describe the clinical features of all PPAP patients with probably pathogenic variants. We identified patients with a variants mapping to the EDs of POLE or POLD1 from cancer genetics clinics, a colorectal cancer (CRC) clinical trial, and systematic review of the literature. We used multiple evidence sources to separate ED variants into those with strong evidence of pathogenicity and those of uncertain importance. We performed quantitative analysis of the risk of CRC, colorectal adenomas, endometrial cancer or any cancer in the former group. 132 individuals carried a probably pathogenic ED variant (105 POLE, 27 POLD1). The earliest malignancy was colorectal cancer at 14. The most common tumour types were colorectal, followed by endometrial in POLD1 heterozygotes and duodenal in POLE heterozygotes. POLD1-mutant cases were at a significantly higher risk of endometrial cancer than POLE heterozygotes. Five individuals with a POLE pathogenic variant, but none with a POLD1 pathogenic variant, developed ovarian cancer. Nine patients with POLE pathogenic variants and one with a POLD1 pathogenic variant developed brain tumours. Our data provide important evidence for PPAP management. Colonoscopic surveillance is recommended from age 14 and upper-gastrointestinal surveillance from age 25. The management of other tumour risks remains uncertain, but surveillance should be considered. In the absence of strong genotype–phenotype associations, these recommendations should apply to all PPAP patients. [ABSTRACT FROM AUTHOR]

Thyroid cancer is the most frequent endocrine cancer with an increasing incidence rate worldwide and is the second most common malignancy among females in Saudi Arabia. Papillary thyroid cancer (PTC) is the most common subtype. Germline pathogenic variants in the proofreading domain of the POLE and POLD1 genes predispose to several types of cancers. However, the role of pathogenic variants of these two genes in PTC remains unknown. Capture sequencing, Sanger sequencing and immunohistochemistry were performed on 300 PTC cases from the Middle Eastern region. One germline pathogenic variant each of POLE (1/300, 0.33%) and POLD1 (1/300, 0.33%) genes was identified. Low expression of POLD1 was detected in 46.5% (133/286) of cases an d was significantly associated with the follicular variant of PTC (P = 0.0006), distant metastasis (P = 0.0033) and stage IV tumours (P = 0.0081). However, no somatic pathogenic variant was detected in POLE gene. Furthermore, low expression of POLE was noted in 61.7% (175/284) of cases with no significant clinicopathological associations. Our study shows that pathogenic variant in the POLE and POLD1 proofreading domain is a cause of PTC and low expression of POLD1 is associated with poor prognostic markers in the Middle Eastern population. Further studies from different geographic populations are needed to determine the frequency and spectrum of proofreading domain pathogenic variants in POLE and POLD1 genes and in PTC from different ethnicities.

Abstract A patient with a history of Mandibular hypoplasia, Deafness, Progeroid Features Associated Lipodystrophy Syndrome (MDPL), familial lipodystrophy presented with hypertriglyceridemia induced pancreatitis with triglycerides in the 3000s. This lipodystrophy occurs due to a mutation in the POLD1 gene (DNA polymerase delta 1). MDPL, hypertriglyceridemia, pancreatitis, POLD1.

A patient with a history of Mandibular hypoplasia, Deafness, Progeroid Features Associated Lipodystrophy Syndrome (MDPL), familial lipodystrophy presented with hypertriglyceridemia induced pancreatitis with triglycerides in the 3000s. This lipodystrophy occurs due to a mutation in the POLD1 gene (DNA polymerase delta 1). MDPL, hypertriglyceridemia, pancreatitis, POLD1. [ABSTRACT FROM AUTHOR]

Background: DNA polymerase delta 1 catalytic subunit (POLD1) plays a key role in DNA replication and damage repair. A defective DNA proofreading function caused by POLD1 mutation contributes to carcinogenesis, while POLD1 overexpression predicts poor prognosis in cancers. However, the effect of POLD1 in hepatocellular carcinoma (HCC) is not well-understood.Methods: Expression patterns of POLD1 were evaluated in TCGA and the HPA databases. Kaplan-Meier curves and Cox regression were used to examine the prognostic value of POLD1. The prognostic and predictive value of POLD1 was further validated by another independent cohort from ICGC database. The influences of DNA copy number variation, methylation and miRNA on POLD1 mRNA expression were examined. The correlation between infiltrating immune cells and POLD1 expression was analyzed. GO and KEGG enrichment analyses were performed to detect biological pathways associated with POLD1 expression in HCC.Results: POLD1 was overexpressed in HCC (n = 369) compared with adjacent normal liver (n = 50). POLD1 upregulation was significantly correlated with positive serum AFP and advanced TNM stage. Kaplan-Meier and multivariate analyses suggested that POLD1 overexpression predicts poor prognosis in HCC. DNA copy gain, low POLD1 methylation, and miR‑139-3p downregulation were associated with POLD1 overexpression. Besides, POLD1 expression was associated with the infiltration levels of dendritic cell, macrophage, B cell, and CD4 + T cell in HCC. Functional enrichment analysis suggested "DNA replication", "mismatch repair" and "cell cycle" pathways might be involved in the effect of POLD1 on HCC pathogenesis. Additionally, POLD1 mRNA expression was significantly associated with tumor mutation burden, microsatellite instability, and prognosis in various tumors.Conclusions: POLD1 may be a potential prognostic marker and promising therapeutic target in HCC. [ABSTRACT FROM AUTHOR]

Background and ObjectiveHepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the DNA polymerase delta (POLD) family is significantly related to cancer prognosis. This study aimed to explore the significance of the POLD family in HCC via the DNA damage repair (DDR) pathway.MethodsData mining was conducted using bioinformatics methods. RNA sequencing and clinicopathological data were collected from The Cancer Genome Atlas, GTEx database and the Gumz Renal cohort. Statistical analyses were also performed in cancer samples (n>12,000) and the Affiliated Hospital of Youjiang Medical University for Nationalities (AHYMUN, n=107) cohort.ResultsThe POLD family (POLD1–4) was identified as the most important functional component of the DDR pathway. Based on the analysis of independent cohorts, we found significantly elevated POLD expression in HCC compared with normal tissues. Second, we investigated the prognostic implication of elevated POLD1 expression in HCC and pan-cancers, revealing that increased POLD1 levels were correlated to worse prognoses for HCC patients. Additionally, we identified 11 hub proteins interacting closely with POLD proteins in base excision repair, protein-DNA complex and mismatch repair signaling pathways. Moreover, POLD1 mutation functioned as an independent biomarker to predict the benefit of targeted treatment. Importantly, POLD1 expression was associated with immune checkpoint molecules, including CD274, CD80, CD86, CTLA4, PDCD1 and TCGIT, and facilitated an immune-excluded tumor microenvironment. Additionally, we confirmed that elevated POLD1 expression was closely correlated with the aggressive progression and poor prognosis of HCC in the real-world AHYMUN cohort.ConclusionWe identified a significant association between elevated POLD1 expression and poor patient survival and immune-excluded tumor microenvironment of HCC. Together, these findings indicate that POLD1 provides a valuable biomarker to guide the molecular diagnosis and development of novel targeted therapeutic strategies for HCC patients.

Background and Objective: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the DNA polymerase delta (POLD) family is significantly related to cancer prognosis. This study aimed to explore the significance of the POLD family in HCC via the DNA damage repair (DDR) pathway. Methods: Data mining was conducted using bioinformatics methods. RNA sequencing and clinicopathological data were collected from The Cancer Genome Atlas, GTEx database and the Gumz Renal cohort. Statistical analyses were also performed in cancer samples (n>12,000) and the Affiliated Hospital of Youjiang Medical University for Nationalities (AHYMUN, n=107) cohort. Results: The POLD family (POLD1–4) was identified as the most important functional component of the DDR pathway. Based on the analysis of independent cohorts, we found significantly elevated POLD expression in HCC compared with normal tissues. Second, we investigated the prognostic implication of elevated POLD1 expression in HCC and pan-cancers, revealing that increased POLD1 levels were correlated to worse prognoses for HCC patients. Additionally, we identified 11 hub proteins interacting closely with POLD proteins in base excision repair, protein-DNA complex and mismatch repair signaling pathways. Moreover, POLD1 mutation functioned as an independent biomarker to predict the benefit of targeted treatment. Importantly, POLD1 expression was associated with immune checkpoint molecules, including CD274, CD80, CD86, CTLA4, PDCD1 and TCGIT, and facilitated an immune-excluded tumor microenvironment. Additionally, we confirmed that elevated POLD1 expression was closely correlated with the aggressive progression and poor prognosis of HCC in the real-world AHYMUN cohort. Conclusion: We identified a significant association between elevated POLD1 expression and poor patient survival and immune-excluded tumor microenvironment of HCC. Together, these findings indicate that POLD1 provides a valuable biomarker to guide the molecular diagnosis and development of novel targeted therapeutic strategies for HCC patients. [ABSTRACT FROM AUTHOR]

Abstract Background Endometrial carcinoma (EC) accounts for 5.8% of all cancers in Saudi females. Although most ECs are sporadic, 2–5% tend to be familial, being associated with Lynch syndrome and Cowden syndrome. In this study, we attempted to uncover the frequency, spectrum and phenotype of germline mutations in the proofreading domain of POLE and POLD1 genes in a large cohort of ECs from Middle Eastern region. Methods We performed Capture sequencing and Sanger sequencing to screen for proofreading domains of POLE and POLD1 genes in 432 EC cases, followed by evaluation of protein expression using immunohistochemistry. Variant interpretation was performed using PolyPhen-2, MutationAssessor, SIFT, CADD and Mutation Taster. Results In our cohort, four mutations (0.93%) were identified in 432 EC cases, two each in POLE and POLD1 proofreading domains. Furthermore, low expression of POLE and POLD1 was noted in 41.1% (170/1414) and 59.9% (251/419) of cases, respectively. Both the cases harboring POLE mutation showed high nuclear expression of POLE protein, whereas, of the two POLD1 mutant cases, one case showed high expression and another case showed low expression of POLD1 protein. Conclusions Our study shows that germline mutations in POLE and POLD1 proofreading region are a rare cause of EC in Middle Eastern population. However, it is still feasible to screen multiple cancer related genes in EC patients from Middle Eastern region using multigene panels including POLE and POLD1.

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POLD1, the catalytic subunit of DNA polymerase δ, plays a critical role in DNA synthesis and DNA repair processes. Moreover, POLD1 is downregulated in replicative senescence to mediate aging. In any case, the components of age-related downregulation of POLD1 expression have not been fully explained. In this article, we elucidate the mechanism of the regulation of POLD1 at the transcription level and found that the transcription factor CCCTC-binding factor (CTCF) was bound to the POLD1 promoter area in two sites. The binding level of CTCF for the POLD1 promoter appeared to be related to aging and was confirmed to be positively controlled by the CTCF level. Additionally, cell senescence characteristics were detected within the cells transfected with short hairpin RNA (shRNA)-CTCF, pLenti-CMV-CTCF, shRNA-POLD1, and pLenti-CMV-POLD1, and the results showed that the CTCF may contribute to the altered expression of POLD1 in aging. In conclusion, the binding level of CTCF for the POLD1 promoter intervened by an age-related decrease in CTCF and downregulated the POLD1 expression in aging. Moreover, the decrease in CTCF-mediated POLD1 transcription accelerates the progression of cell aging.

Resveratrol (RSV) is known to possess anticancer properties in many types of cancers like breast cancer, in which POLD1 may serve as a potential target. However, the anticancer mechanism of RSV on triple negative breast cancer (TNBC) remains unclear. In the present study, the antitumor effects and mechanism of RSV on TNBC cells were analyzed by RNA sequencing (RNA-seq), which was then verified via cell counting kit-8 (CCK8), immunofluorescence, immunohistochemistry, Western Blot (WB), flow cytometry, and hematoxylin-eosin (HE) staining. According to the corresponding findings, the survival rate of MDA-MB-231 cells gradually decreased as RSV treatment concentration increased. The RNA-seq analysis results demonstrated that genes affected by RSV treatment were mainly involved in apoptosis and the p53 signaling pathway. Moreover, apoptosis of MDA-MB-231 cells induced by RSV was observed to be mainly mediated by POLD1. When treated with RSV, the expression levels of full length PARP1, PCNA, and BCL-2 were found to be significantly reduced, and the expression level of Cleaved-PARP1 as well as Cleaved-Caspase3 increased significantly. Additionally, the mRNA expression of POLD1 was significantly reduced after treatment with RSV, and the protein expression level was also inhibited by RSV in a concentration-dependent manner. The prediction of domain interaction suggested that RSV may bind to at least five functional domains of the POLD1 protein (6s1m, 6s1n, 6s1o, 6tny and 6tnz). Furthermore, after RSV treatment, the anti-apoptotic index (PCNA, BCL-2) of MDA-MB-231 cells was found to decrease while the apoptosis index (caspase3) increased. Moreover, the overexpression of POLD1 reduced the extent of apoptosis observed in MDA-MB-231 cells following RSV treatment. Moreover, animal experimental results showed that RSV had a significant inhibitory effect on the growth of live tumors, while POLD1 overexpression was shown to antagonize this inhibitory effect. Accordingly, this study’s findings reveal that RSV may promote the apoptosis of TNBC cells by reducing the expression of POLD1 to activate the apoptotic pathway, which may serve as a potential therapy for the treatment of TNBC.

Abstract Background Colorectal cancer (CRC) is a major contributor to morbidity and mortality related to cancer. Only ~5% of all CRCs occur as a result of pathogenic variants in well‐defined CRC predisposing genes. The frequency and effect of exonuclease domain pathogenic variants of POLE and POLD1 genes in Middle Eastern CRCs is still unknown. Methods Targeted capture sequencing and Sanger sequencing technologies were employed to investigate the germline exonuclease domain pathogenic variants of POLE and POLD1 in Middle Eastern CRCs. Immunohistochemical analysis of POLE and POLD1 was performed to look for associations between protein expression and clinico‐pathological characteristics. Results Five damaging or possibly damaging variants (0.44%) were detected in 1,135 CRC cases, four in POLE gene (0.35%, 4/1,135) and one (0.1%, 1/1,135) in POLD1 gene. Furthermore, low POLE protein expression was identified in 38.9% (417/1071) cases and a significant association with lymph node involvement (p = .0184) and grade 3 tumors (p = .0139) was observed. Whereas, low POLD1 expression was observed in 51.9% (555/1069) of cases and was significantly associated with adenocarcinoma histology (p = .0164), larger tumor size (T3 and T4 tumors; p = .0012), and stage III tumors (p = .0341). Conclusion POLE and POLD1 exonuclease domain pathogenic variants frequency in CRC cases was very low and these exonuclease domain pathogenic variants might be rare causative events of CRC in the Middle East. POLE and POLD1 can be included in multi‐gene panels to screen CRC patients.

Mandibular hypoplasia, deafness, progeroid feature, and lipodystrophy syndrome (MDPL, MIM# 615381) is an extremely rare and recently recognized early adult onset of progeroid syndrome, with features of generalized lipodystrophy, dysmorphic features, telangiectasia, early onset hearing loss, insulin resistance, and dyslipidemia. Here, we present a 31‐year‐old Chinese woman with MDPL, harboring the recurrent pathogenic variant p.(Ser605del) in POLD1, illustrating the evolving manifestations of this premature aging disorder from infancy to adulthood. [ABSTRACT FROM AUTHOR]

Resveratrol (RSV) is known to possess anticancer properties in many types of cancers like breast cancer, in which POLD1 may serve as a potential target. However, the anticancer mechanism of RSV on triple negative breast cancer (TNBC) remains unclear. In the present study, the antitumor effects and mechanism of RSV on TNBC cells were analyzed by RNA sequencing (RNA-seq), which was then verified via cell counting kit-8 (CCK8), immunofluorescence, immunohistochemistry, Western Blot (WB), flow cytometry, and hematoxylin-eosin (HE) staining. According to the corresponding findings, the survival rate of MDA-MB-231 cells gradually decreased as RSV treatment concentration increased. The RNA-seq analysis results demonstrated that genes affected by RSV treatment were mainly involved in apoptosis and the p53 signaling pathway. Moreover, apoptosis of MDA-MB-231 cells induced by RSV was observed to be mainly mediated by POLD1. When treated with RSV, the expression levels of full length PARP1, PCNA, and BCL-2 were found to be significantly reduced, and the expression level of Cleaved-PARP1 as well as Cleaved-Caspase3 increased significantly. Additionally, the mRNA expression of POLD1 was significantly reduced after treatment with RSV, and the protein expression level was also inhibited by RSV in a concentration-dependent manner. The prediction of domain interaction suggested that RSV may bind to at least five functional domains of the POLD1 protein (6s1m, 6s1n, 6s1o, 6tny and 6tnz). Furthermore, after RSV treatment, the anti-apoptotic index (PCNA, BCL-2) of MDA-MB-231 cells was found to decrease while the apoptosis index (caspase3) increased. Moreover, the overexpression of POLD1 reduced the extent of apoptosis observed in MDA-MB-231 cells following RSV treatment. Moreover, animal experimental results showed that RSV had a significant inhibitory effect on the growth of live tumors, while POLD1 overexpression was shown to antagonize this inhibitory effect. Accordingly, this study's findings reveal that RSV may promote the apoptosis of TNBC cells by reducing the expression of POLD1 to activate the apoptotic pathway, which may serve as a potential therapy for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is a major contributor to morbidity and mortality related to cancer. Only ~5% of all CRCs occur as a result of pathogenic variants in well‐defined CRC predisposing genes. The frequency and effect of exonuclease domain pathogenic variants of POLE and POLD1 genes in Middle Eastern CRCs is still unknown. Methods: Targeted capture sequencing and Sanger sequencing technologies were employed to investigate the germline exonuclease domain pathogenic variants of POLE and POLD1 in Middle Eastern CRCs. Immunohistochemical analysis of POLE and POLD1 was performed to look for associations between protein expression and clinico‐pathological characteristics. Results: Five damaging or possibly damaging variants (0.44%) were detected in 1,135 CRC cases, four in POLE gene (0.35%, 4/1,135) and one (0.1%, 1/1,135) in POLD1 gene. Furthermore, low POLE protein expression was identified in 38.9% (417/1071) cases and a significant association with lymph node involvement (p =.0184) and grade 3 tumors (p =.0139) was observed. Whereas, low POLD1 expression was observed in 51.9% (555/1069) of cases and was significantly associated with adenocarcinoma histology (p =.0164), larger tumor size (T3 and T4 tumors; p =.0012), and stage III tumors (p =.0341). Conclusion: POLE and POLD1 exonuclease domain pathogenic variants frequency in CRC cases was very low and these exonuclease domain pathogenic variants might be rare causative events of CRC in the Middle East. POLE and POLD1 can be included in multi‐gene panels to screen CRC patients. [ABSTRACT FROM AUTHOR]

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS‐SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30–40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS‐SNHL. [ABSTRACT FROM AUTHOR]

Microsatellite instability (MSI) and POLD1 mutations are usually described in colorectal tumours in patients with polyposis syndrome but rarely found in breast tumours. This case describes a metastatic luminal B breast tumour in a young patient with an important family history of cancer. Mutational studies found a Variant of Uncertain Significance (VUS)-type alteration in POLD1 that motivated the study for MSI, which was found positive. Recent data point towards the use of pembrolizumab as a treatment option for tumour presenting with MSI instead of chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Endometrial carcinoma (EC) accounts for 5.8% of all cancers in Saudi females. Although most ECs are sporadic, 2–5% tend to be familial, being associated with Lynch syndrome and Cowden syndrome. In this study, we attempted to uncover the frequency, spectrum and phenotype of germline mutations in the proofreading domain of POLE and POLD1 genes in a large cohort of ECs from Middle Eastern region. Methods: We performed Capture sequencing and Sanger sequencing to screen for proofreading domains of POLE and POLD1 genes in 432 EC cases, followed by evaluation of protein expression using immunohistochemistry. Variant interpretation was performed using PolyPhen-2, MutationAssessor, SIFT, CADD and Mutation Taster. Results: In our cohort, four mutations (0.93%) were identified in 432 EC cases, two each in POLE and POLD1 proofreading domains. Furthermore, low expression of POLE and POLD1 was noted in 41.1% (170/1414) and 59.9% (251/419) of cases, respectively. Both the cases harboring POLE mutation showed high nuclear expression of POLE protein, whereas, of the two POLD1 mutant cases, one case showed high expression and another case showed low expression of POLD1 protein. Conclusions: Our study shows that germline mutations in POLE and POLD1 proofreading region are a rare cause of EC in Middle Eastern population. However, it is still feasible to screen multiple cancer related genes in EC patients from Middle Eastern region using multigene panels including POLE and POLD1. [ABSTRACT FROM AUTHOR]

POLD1, the catalytic subunit of DNA Pol δ, plays an important role in DNA synthesis and DNA damage repair, and POLD1 is downregulated in replicative senescence and mediates cell aging. However, the mechanisms of age-related downregulation of POLD1 expression have not been elucidated. In this study, four potential CpG islands in the POLD1 promoter were found, and the methylation levels of the POLD1 promoter were increased in aging 2BS cells, WI-38 cells and peripheral blood lymphocytes, especially at a single site, CpG 36, in CpG island 3. Then, the transcription factor E2F1 was observed to bind to these sites. The binding affinity of E2F1 for the POLD1 promoter was found to show age-related attenuation and was confirmed to be positively regulated by the E2F1 level and negatively regulated by POLD1 promoter methylation. Moreover, cell senescence characteristics were observed in the cells transfected with shRNA-E2F1 and could contribute to the downregulation of POLD1 induced by the E2F1 decline. Collectively, these results indicated that the attenuation of the binding affinity of E2F1 for the POLD1 promoter, mediated by an age-related decline in E2F1 and increased methylation of CpG island 3, downregulates POLD1 expression in aging. [ABSTRACT FROM AUTHOR]

Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE -mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L , by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome. [Display omitted] • POLE variants promote genome-wide mutations at defined positions of polypurine tracts • Polypurine-motif-based classifier identifies tumors with POLE and POLD1 drivers • Two POLE drivers, S461L and E978G, are validated experimentally • E978G driver mutation affects the DNA polymerase domain of Pol ε Using a computational approach, Ostroverkhova et al. show that POLE - and POLD1 -mutant tumors accumulate G>T mutations at defined positions of polypurine tracts, creating a characteristic quantifiable pattern. A classifier based on this signature facilitates the discovery of POLE driver alleles, including a driver affecting the DNA polymerase domain of the protein. [ABSTRACT FROM AUTHOR]

Background/aim: The prognosis of patients with malignant pleural mesothelioma (MPM) remains poor due to lack of effective therapeutic targets. DNA damage caused by long-time exposure to asbestos fibers has been associated with the development of MPM, with mutations at genes encoding DNA damage repair (DDR)-related molecules frequently expressed in patients with MPM. The present study was designed to identify novel therapeutic targets in MPM using large public databases, such as The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) focused on DDR pathways., Materials and Methods: The correlations between mRNA expression levels of DDR-related genes and overall survival (OS) were analyzed in mesothelioma patients in TCGA mesothelioma (TCGA-MESO) datasets. The anti-tumor effects of small interfering RNAs (siRNA) against DDR-related genes associated with OS were subsequently tested in MPM cell lines., Results: High levels of mRNA encoding DNA polymerase delta 1, catalytic subunit (POLD1) were significantly associated with reduced OS in patients with MPM (p<0.001, Log-rank test). In addition, siRNA targeting POLD1 (siPOLD1) caused cell cycle arrest at the G 1 /S checkpoint and induced apoptosis involving accumulation of DNA damage in MPM cell lines., Conclusion: POLD1 plays essential roles in overcoming DNA damage and cell cycle progression at the G 1 /S checkpoint in MPM cells. These findings suggest that POLD1 may be a novel therapeutic target in MPM., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Abstract The DNA polymerase δ catalytic subunit (PolD1) is a highly conserved protein with established functions in both the nucleus and the cytoplasm: whereas PolD1 participates in the replication and repair of nuclear DNA, it plays a role in the control of cytoplasmic microtubule growth by directly acting on microtubule-nucleator γ-tubulin ring complexes. Here, we show that PolD1 shuttles between the nucleus and the cytoplasm. PolD1 harbors two nuclear localization signals that mediate the active transport of PolD1 to the nucleus; conversely, PolD1 is exported from the nucleus by the exportin CRM1-dependent mechanism, a major nuclear-export pathway that mediates the export of various cargos. These findings suggest that the nucleocytoplasmic distribution of PolD1 is influenced by both the nuclear import and export activities of the protein. [ABSTRACT FROM AUTHOR]