Academic literature on the topic 'Pol01'

Abstract INTRODUCTION: Acute Graft-versus-host disease (aGVHD) is a major complication of allogeneic stem cell transplantation (alloSCT). Risk factors include patient age, sex matching, CMV status and degree of match. Regulatory T cells are critical for immune tolerance processes such as aGVHD, and express the transcription factor FOXP3, a member of the forkhead/winged-helix family, identified as a key regulatory gene required for the development and activity of these cells. It has been suggested that genetic expression of FOXP3 is inversely correlated with the severity of the GVHD. We studied donor’s DNA looking for 5 polymorphisms on the promoter region of the FOXP3 gene, and we tried to correlate them with presence and degree of aGVHD. PATIENTS AND METHODS: We studied donors of stem cells for allogeneic stem cell transplants. We looked for the presence of the following polymorphisms by PCR: POL01 −5906 T/A rs2869211; POL03 −3279 A/C rs3761548 ; POL04 −2383 C/T rs3761549 ; POL05 −1383 C/T rs2232364 ; POL06 −924 A/G rs2232365. RESULTS: Our sample consisted of 31 donors, all siblings. In them we found only 2 of the 5 FOXP3 polymorphisms, either as homozygous or heterozygous. These polymorphisms were found in 15/31 donors, with 12 being homozygous (38.7%), and 3 heterozygous (9.7%). These genes polymorphisms were POL03 y POL06. The most observed polymorphism was POL-06 with 9 cases, while POL-03 was found in 6 donors. Only sex difference and CMV status had an elevated hazard ratio for developing GVHD (HR=1.18, CI95%: 0.18 to 7.64; p=0.85) and (HR=3.0, CI95%: 0.07 to 126; p=0.46) respectively. We found no statistically significant difference in the incidence of GVHD between patients who had received cells from donor with or without a FOXP3 polymorphism (p=0.87). When we analyzed the risk of presenting GVHD, results suggest that having one of the 2 positive polymorphisms of the FOXP3 gene could have a protective effect for the patient. For POL03 HR=0.87, CI95%:0.18 to 4.14; p=0.86, and for POL06, HR=1, CI95%: 0.37 to 2.64, p=0.67. CONCLUSIONS: Even though our sample is still small to make conclusive remarks, we believe that our results point toward some level of “protection” from acute GVHD in patients receiving cells from donors expressing POL03 and POL06. It is also worthwhile mentioning the relatively high frequency of these polymorphisms, as well as the absence of the other 3.

DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”) in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients’ T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder.

In this paper, the unexpected behavior of object negative quantifiers in some diagnostic tests of sentential negation is accounted for within a Minimalist framework assuming that: (i) negative quantifiers decompose into negation and an existential quantifier; (ii) negative quantifiers are multidominant phrase markers, as Parallel Merge allows the verb to c-select their existential part but not their negative part, thus giving negation remerge flexibility; (iii) tag questions involve or-coordination of TPs, and neither/so clauses involve and-coordination of TPs; (iv) two positions for sentential negation are available in English, one below TP (PolP2), and one above TP (PolP1). Activation of either PolP1 or PolP2 in the absence of other scope-taking operators corresponds to two distinct grammars. If PolP1 is active, the negative part of an object negative quantifier remerges in its Specifier valuing the [upol: ] feature of Pol1 as negative ([upol:neg]) while skipping the TP-domain. As no negative formal feature is present in the TP, a negative question tag is required, as well as so-coordination, too-licensing and Yes, I guess so ‘expression of agreement’. Conversely, if PolP2 is active, the negative part of the object negative quantifier remerges in the TP-domain (in Spec, PolP2), thus requiring a positive question tag, neither-coordination, either-licensing, and No, I guess not.

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3–Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31–Pol3 and Pol31–Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

DNA polymerase B1 (PolB1) is a member of the B-family DNA polymerase family and is a replicative DNA polymerase in Crenarchaea. PolB1 is responsible for the DNA replication of both the leading and lagging strands in the thermophilic crenarchaeon Sulfolobus acidocaldarius. Recently, two subunits, PolB1-binding protein (PBP)1 and PBP2, were identified in Saccharolobus solfataricus. Previous in vitro studies suggested that PBP1 and PBP2 influence the core activity of apoenzyme PolB1 (apo-PolB1). PBP1 contains a C-terminal acidic tail and modulates the strand-displacement synthesis activity of PolB1 during the synthesis of Okazaki fragments. PBP2 modestly enhances the DNA polymerase activity of apo-PolB1. These subunits are present in Sulfolobales, Acidilobales, and Desulfurococcales, which belong to Crenarchaea. However, it has not been determined whether these subunits are essential for the activity of apo-PolB1. In this study, we constructed a pbp1 deletion strain in S. acidocaldarius and characterized its phenotypes. However, a pbp2 deletion strain was not obtained, indicating that PBP2 is essential for replication by holoenzyme PolB1. A pbp1 deletion strain was sensitive to various types of DNA damage and exhibited an increased mutation rate, suggesting that PBP1 contribute to the repair or tolerance of DNA damage by holoenzyme PolB1. The results of our study suggest that PBP1 is important for DNA repair by holoenzyme PolB1 in S. acidocaldarius.

e14031 Background: POLE and POLD1 gene variations have been suggested as potential markers for immunotherapy due to their significant association with the tumor mutation burden (TMB), an effective indicator for response prediction in immunotherapy. Methods: In this study, we systematically studied the spectrum and characteristics of POLE and POLD1 gene variations from 1,392 Chinese cancer patients, and their correlation with existing immunotherapeutic markers and cancer associated genes. Results: We found that the frequency of POLE variations was not statistically different from that in COSMIC database, while the frequency of POLD1 variations in Chinese lung cancer patients was significantly higher than that in COSMIC database. Variations frequency analysis showed that c.857C > G and c.2091dupC were potential high frequency variations in Chinese cancer patients. Patients carrying POLE damaging variations were significantly younger, and patients carrying POLD1 damaging variations exhibited significantly higher frequency of MSI than POLE/POLD1 WT patients. Further studies found that patients carrying POLE exonuclease domain damaging variations, POLD1 exonuclease and non-exonuclease domain damaging variations exhibited significantly higher TMB than POLE/POLD1 WT patients, while no such difference was found in patients carrying POLE and POLD1 neutral variations. Moreover, patients with POLE exonuclease domain damaging variations showed significantly higher frequency of MMR gene and driver gene variations than POLE/POLD1 WT patients, including genes associated with RTK/RAS/RAF pathway. Patients with damaging POLD1 variations also exhibited higher frequency of MMR gene variations than POLE/POLD1 WT patients. The high frequency variant genes in patients with POLE exonuclease domain damaging variations or POLD1 damaging variations were associated with MMR, RTK/RAS/RAF, TGFβ, WNT, PI3K-Akt and TP53 pathways. Conclusions: This study identified key characteristics and domains of POLE and POLD1 gene variations that related to TMB, MSI, MMR gene variations and key driver gene variations, and provided theoretical and practical basis for patient selection based on POLE or POLD1 gene status in immunotherapy.

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.

199 Background: The progression of neoplasia in colorectal cancer (CRC) has been well characterized, with the evolution typically involving mutations in APC, KRAS, and p53, among others. POLE/POLD1 genes encode for proteins essential in enzymatic DNA polymerase function, yet POLE/POLD1 mutations in tumors are poorly characterized. Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H) independent of deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H). Studies have shown associations between this hypermutated phenotype and susceptibility to immune checkpoint inhibition, which may be attributable to neoantigen creation. As such, these tumors are a clinically relevant phenotype requiring further investigation. Here, we characterized POLE/POLD1 alterations in a large, real-world cohort of patients with CRC. Methods: We retrospectively analyzed de-identified records of 9,136 primary CRC patients profiled with the Tempus xT assay. The assay includes DNA-seq of 595-648 genes at 500x to evaluate single-nucleotide variants, insertions/deletions, and copy number changes. Immunological markers analyzed included tumor mutational burden (TMB), MSI, and dMMR. MSI-H was determined by the assay through assessment of 239 loci. dMMR was determined by immunohistochemistry. Results: A total of 217 patient tumors harbored somatic POLE/POLD1 mutations (n = 203 POLE, n = 13 POLD1, and n = 1 POLE and POLD1; none germline), with no differences noted in baseline demographics including gender and age. Among the POLE/POLD1-mutant cohort, POLE copy number losses accounted for most mutations (n = 127, 59%), with short variants and copy number amplifications accounting for 35% (n = 76). The remainder of tumors exhibited POLD1 copy number changes (n = 14), including one with both POLE and POLD1 copy number loss. POLE/POLD1-mutated tumors presented with a lower frequency of MSI-H compared to wild-type (1.8% vs 6.1%, P= 0.018). Conversely, there was a higher frequency of TMB-high cases ( > 10 mut/Mb) for POLE/POLD1-mutated compared to wild-type tumors (22% vs 9.9%, P< 0.001). Differences between POLE/POLD1-mutated and wild-type tumors were also observed among many co-mutated genes, including APC (80% vs 65%, P< 0.001), ALK (31% vs 11%, P< 0.001), ATM (12% vs 3.6%, P< 0.001), BRCA2 (12% vs. 3.2%), and RET (24% vs 8.9%, P< 0.001). Conclusions: Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers and molecular co-alterations. POLE/POLD1 mutations in CRC have been previously described to present with a hypermutated phenotype; however, 78% of tumors exhibited low TMB despite POLE/POLD1 mutations. Thus, these results have identified POLE/POLD1-mutated tumors as a unique genomic subpopulation, and further studies are needed to better characterize POLE/POLD1 mutations in CRC.

Abstract Background: Mutations in genes encoding DNA polymerase epsilon (POLE) and delta 1 (POLD1) can lead to defects in the DNA replication, resulting in a hypermutated tumor phenotype, which might help identify potential responders to immunotherapy. This study aims to comprehensively investigate the different variants of POLE/POLD1 mutants and how it related to MSI/MMR status and TMB levels within a large Chinese population. Methods:Among 15640 Chinese patients who underwent NGS testing, the prevalence of POLE/POLD1 mutants was analyzed and the high frequencies of POLE/POLD1 variants as well as their relations with MMR/MSI and TMB were explored. Results: POLE and POLD1 mutations were highly distributed in endometrial cancer (11.1% and 9.3%), colorectal cancer (5.9% and 4.5%), and gastric cancer (4.6% and 3.3%). In our study, the mutation frequency of POLE was 3.4%, including p.Ala252Val (9.2%), p.Pro286Arg (2.7%), p.Val411Leu (1.3%), p.Asp601Glu (6.0%), p.Lys425Gln (2.9%) and p.Phe1849Val (1.8%). Variants of p.Ala252Val, p.Pro286Arg and p.Val411Leu displayed high TMB levels (median 71.4 muts/Mb, 236.7 muts/Mb and 220 muts/Mb, respectively) while variants of p.Asp601Glu, p.Lys425Gln, p.Phe1849Val and p.Asn2098His had a rather low TMB, ranging from 5.2-8.1 muts/Mb. It is worth mentioning that 37.5% of p. Pro286Arg and 87.5% of Val411Leu variants had concurrent loss-of-function mutations of MMR (MMRLOF), while MMRLOF rarely occurred in the other variants.For POLD1, the mutation frequency in Chinese patients was 2.8%, including p.Arg119His (18.8%), p.Glu57del (16.5%) and p.Asp987Thrfs (3.3%). p.Arg119His variants showed a high TMB level of 65.6 muts/Mb and most occurred in lung cancer. Variants of p.Asp987Thrfs, p.Pro116Hisfs and p.Arg19His had similar TMB levels (p>0.05), while much lower TMB levels was found in p.Glu57del (3.3 muts/Mb, p<0.001) compared to p.Arg119His.Compared with POLE/POLD1 wild-type (POLDE/POLD1-WT), the percentage of MSI-H samples in POLE/POLD1 mutants was much higher (14% vs 0.7, p<0.001). About 10% of POLE/POLD1 mutations carried MMRLOF mutations while it was barely observed in POLE/POLD1-WT (0.5%). The TMB levels of POLE/POLD1 mutations were significantly higher than that of the POLE/POLD1-WT (17.7 vs 2.9 muts/Mb, p<0.001). Furthermore, POLE/POLD1 mutations was found to be a main cause leading to high levels of TMB. Among samples with TMB >10 muts/Mb, 22.6% of them were POLE/POLD1 mutations. And in samples with TMB >100 muts/Mb, as high as 82% of samples were found carrying POLE/POLD1 mutations. Conclusion: POLE/POLD1 mutations were found in Chinese patients in multiple tumors. Different POLE/POLD1 variants were associated with different MMR status and TMB levels. In addition to MMR, POLE/POLD1 mutations might be another distinct means of inducing high TMB. Citation Format: Yongning Jia, Yaqun Xin, Hongling Yuan, Fei Pang, Fei Shan, Shuangxi Li, Honglin Zhu, Ziyu Li. Comprehensive analysis of POLE/POLD1 variants, MMR deficient/MSI, and tumor mutational burden in Chinese population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5751.

184 Background: POLE and POLD1 alterations (DNA polymerase ε and DNA polymerase δ) are key biomarkers of immune response for patients with MSS colorectal cancers (CRC). The clinical and molecular characteristics of POLE/POLD1 alterations are not yet well defined across CRCs. In this study, we investigated the clinical/molecular features of POLE and POLD1 alterations and its association with MSI-H CRC. Methods: All the patients and mutation data were selected from the cBioPortal database (https://www.cbioportal.org). All nonsynonymous mutations including missense, frameshift, nonsense, nonstop, splice site, and translation start site changes of POLE/POLD1 were considered. 21 studies were used in the analysis. 7179 data samples were obtained from 7179 patients. Tumors included were from all stages of CRCs. POLE/POLD1 mutations were from mutation tables, only protein-changing mutations were kept. MSI-H status was based on mutation data (presence of MLH1/PMS2/MSH2/MSH6 mutations). Results: Of 7179 patients with all stage CRCs, 6.1% (439) were found to harbor mutated POLE/POLD1 genes. Among those, 3446 patients had known MSI status and 14.9% (514/3446) had MSI-H disease. Notably, 3.3% patients with POLE/POLD1 mutations were found to have concurrent MSI-H disease (co-existence) and only 1.1% of patients with POLE/POLD1 mutations had MSS CRC. MSI status was unknown for 1.7% of patients with POLE/POLD mutant CRC. The mean age for patients with POLE/POLD1 + with or without MSI-H disease was 67. POLE/POLD mutations were exceedingly more common in colon than rectum regardless of MSI-H status (85% vs 15% overall and 88% vs 12% in MSI-H subgroup). No difference was seen among genders for the distribution of POLE/POLD1 mutations. Conclusions: POLE/POLD1 mutations frequently co-exist with MSI-H disease in CRC. Patients with MSS-CRC harboring POLE/POLD1 mutations represent a smaller subgroup of CRC (~1%). The incidence of POLE/POLD1 somatic mutations was more common among patients with colon cancer than those with rectal cancer.