Exonuclease Domain Research Articles

Exploring somatic mutations in POLE and POLD1: Their role in colorectal cancer pathogenesis and potential therapeutic strategies

Colorectal cancer (CRC) is one of the major causes of morbidity and mortality worldwide, resulting from the accumulation of genetic and epigenetic alterations in several oncogenes and tumor suppressor genes. Recent studies have identified germline and somatic mutations in the exonuclease domain regions of both epsilon polymerase (POLE) and delta polymerase (POLD1) genes in CRCs. We sought to examine the mutation of these genes in a series of sporadic CRCs. To do this, we extracted DNA from 100 primary CRC samples and 40 corresponding normal tissue samples, which had been previously characterized for clinicopathological and molecular features. We employed a combination of quick-multiplex consensus (QMC)-polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR, followed by high-resolution melting (HRM) analysis and Sanger sequencing, to investigate the exonuclease domain regions of POLE and POLD1 genes for somatic mutations that may potentially alter the proofreading activities of these genes. In silico predictions of the functional significance of the identified genetic alterations were performed using the protein variation effect analyzer, sorting intolerant from tolerant, and PON-P2 algorithms. We identified a total of eight new and non-recurrent somatic variants in the endonuclease domains of POLE and two in POLD1. Nine out of ten variants caused amino acid substitutions, whereas one resulted in a stop codon. Although no significant associations or correlations were found between the POLE/POLD1 mutations and the clinicopathological or molecular features of the CRC cases, most of the POLE/POLD1-mutated cases were microsatellite stable (90%) and aneuploid (80%). Furthermore, in silico analyses showed that nine of the ten variants would likely cause some adverse effect on protein function. Ten somatic variants with predicted proofreading activity-altering effects have been identified in the endonuclease domains of POLE and POLD1 in CRC using a combination of QMC-PCR, COLD-PCR, HRM analyses, and Sanger sequencing.

A phase II clinical trial of toripalimab in advanced solid tumors with polymerase epsilon/polymerase delta (POLE/POLD1) mutation

Patients carrying mutations in polymerase epsilon/polymerase delta have shown positive responses to immune checkpoint inhibitors. Yet, prospective trials exploring the efficacy in those with polymerase epsilon/polymerase delta mutations are still lacking. A phase II clinical trial was initiated to evaluate the efficacy of toripalimab, a humanized IgG4K monoclonal antibody to human PD-1, in patients with advanced solid tumors with unselected polymerase epsilon/polymerase delta mutations but without microsatellite instability-high. A total of 15 patients were enrolled, 14 of whom were assessed for treatment efficacy. There was a 21.4% overall response rate, with a disease control rate of 57.1%. The median overall survival and median progression-free survival were 17.9 (95% CI 13.5-not reach) months and 2.5 (95% CI 1.4-not reach) months, respectively. For patients with exonuclease domain mutations, the objective response rate was 66.7% (2/3), with a disease control rate of 66.7% (2/3). For those with non-exonuclease domain mutations, the rates were 9.1% (1/11) and 54.5% (6/11), respectively. Notably, patients with PBRM1 gene mutations exhibited a high response rate to toripalimab at 75.0% (3/4). This study showed that neither the exonuclease domain mutations nor non-exonuclease domain mutations could fully predict the efficacy of immunotherapy, urging the need for more investigations to clarify potential immune sensitization differences within polymerase epsilon/polymerase delta mutation variants.

REXO5 promotes genomic integrity through regulating R-loop using its exonuclease activity

Chronic myeloid leukemia (CML), caused by BCR::ABL1 fusion gene, is known to regulate disease progression by altering the expression of genes. However, the molecular mechanisms underlying these changes are largely unknown. In this study, we identified RNA Exonuclease 5 (REXO5/LOC81691) as a novel gene with elevated mRNA expression levels in chronic myeloid leukemia (CML) patients. Additionally, using the REXO5 knockout (KO) K562 cell lines, we revealed a novel role for REXO5 in the DNA damage response (DDR). Compared to wild-type (WT) cells, REXO5 KO cells showed an accumulation of R-loops and increased DNA damage. We demonstrated that REXO5 translocates to sites of DNA damage through its RNA recognition motif (RRM) and selectively binds to R loops. Interestingly, we identified that REXO5 regulates R-loop levels by degrading mRNA within R-loop using its exonuclease domain. REXO5 KO showed ATR-CHK1 activation. Collectively, we demonstrated that REXO5 plays a key role in the physiological control of R-loops using its exonuclease domain. These findings may provide novel insights into how REXO5 expression changes contribute to CML pathogenesis.

A ~40-kb flavi-like virus does not encode a known error-correcting mechanism

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

Glioma oncogenesis in the Constitutional mismatch repair deficiency (CMMRD) syndrome.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition due to biallelic mutations in one of the mismatch repair (MMR) genes associated with early onset of cancers, especially high-grade gliomas. Our aim was to decipher the molecular specificities of these gliomas. Clinical, histopathological, and whole exome sequencing data were analyzed in 12 children with genetically proven CMMRD and a high-grade glioma. PDL1 expression was present in immunohistochemistry in 50% of the samples. In 9 patients, the glioma harbored an ultra-hypermutated phenotype (104-635 coding single nucleotide variants (SNV) per Mb, median 204). Driver mutations in POLE and POLD1 exonuclease domains were described for 8 and 1 patients respectively and were always present in the mutation burst with the highest variant allele frequency (VAF). The mutational signatures were dominated by MMR-related ones and similar in the different mutation bursts of a same patient without subsequent enrichment of the mutation signatures with POL-driven ones. Median number of coding SNV with VAF above one of the driving polymerase mutation per Mb was 57 (17-191). Our findings suggest that somatic polymerase alterations does not entirely explain the ultra-hypermutant phenotype. SETD2, TP53, NF1, EPHB2, PRKDC, and DICER1 genes were frequently mutated with higher VAF than the deleterious somatic polymerase mutation. CMMRD-associated gliomas have a specific oncogenesis that does not involve usual pathways and mutations seen in sporadic pediatric or adult glioblastomas. Frequent alterations in other pathways such as MAPK may suggest the use of other targeted therapies along with PD1 inhibitors.

Human Immunodeficiency Viral Reverse Transcriptases: Analyses of the Active Sites of the Polymerase Domain and Drug-Resistant Mutants of the Reverse Transcriptases

It is well-known that human immunodeficiency viruses (HIVs) cause the chronic, potentially life-threatening condition known as human Acquired Immuno-Deficiency Syndrome (AIDS). As the lifecycle of HIVs heavily depends on the crucial enzyme, the reverse transcriptase (RT), it has been used as a potential therapeutic target to treat and control the spread of AIDS. The active site amino acids of the polymerase domain of the RTs and their anti-HIV drug-binding sites are analyzed. The catalytic region of HIV RTs and the E. coli DNA polymerase I showed very similar active site amino acids, suggesting that HIV RTs would have possibly evolved from the bacterial DNA polymerase. The catalytic proton abstractor is identified as a K and the nucleotide selection amino acid as an N. However, the regular template-binding pair –YG- is slightly modified to a –YXG- in HIV RTs. Three completely conserved Ds in HIV RTs are involved in binding to the catalytic Mg2+. The sensitive and resistant strains of HIV-1 for the HIV antiretroviral drug, azidothymidine (AZT), a nucleoside analogue of thymidine, show only a few non-isofunctional amino acid replacements and are located mainly in the palm and thumb subdomains of the RT polymerase, whereas the rest of the polymerase catalytic core and metal-binding sites are completely conserved in AZT-sensitive and -resistant HIV-1 strains. In the drug-resistant mutants of non-nucleoside RT inhibitors of HIV-2, the crucial mutations are located mainly near the -MDD- motif of the catalytic metal-binding region. Even though a large number of amino acid replacements are seen between the RTs of HIV-1 and HIV-2, the polymerase active sites are completely conserved in both. The HIV-1 and HIV-2 catalytic and metal-binding sites are completely conserved in simian immunodeficiency virus (SIV) as well. The absence of DEDD-superfamily of proofreading exonuclease domain in the HIV RTs, might cause the virus to evolve rapidly in patients. A possible mechanism of action for the HIV RTs is also proposed.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Integrated liver and serum proteomics uncover sexual dimorphism and alteration of several immune response proteins in an aging Werner syndrome mouse model.

Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.

Investigating somatic variants and pathways in mismatch repair-deficient (dMMR) colorectal carcinoma in South Africa

AimsColorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide, and an emerging public health problem in sub-Saharan Africa. Several authors have described an increased frequency of mismatch...

Application of novel algorithm on a retrospective series to implement the molecular classification for endometrial cancer

IntroductionThe study aimed to validate the Betella algorithm, focusing on molecular analyses exclusively for endometrial cancer patients, where molecular classification alters risk assessment based on ESGO/ESTRO/ESP 2020 guidelines. Materials and methodsConducted between March 2021 and March 2023, the retrospective research involved endometrial cancer patients undergoing surgery and comprehensive molecular analyses. These included p53 and mismatch repair proteins immunohistochemistry, as well as DNA sequencing for POLE exonuclease domain. We applied the Betella algorithm to our population and evaluated the proportion of patients in which the molecular analysis changed the risk class attribution. ResultsOut of 102 patients, 97 % obtained complete molecular analyses. The cohort exhibited varying molecular classifications: 10.1 % as POLE ultra-mutated, 30.3 % as mismatch repair deficient, 11.1 % as p53 abnormal, and 48.5 % as non-specified molecular classification. Multiple classifiers were present in 3 % of cases. Integrating molecular classification into risk group calculation led to risk group migration in 11.1 % of patients: 7 moved to lower risk classes due to POLE mutations, while 4 shifted to higher risk due to p53 alterations. Applying the Betella algorithm, we can spare the POLE sequencing in 65 cases (65.7 %) and p53 immunochemistry in 17 cases (17.2 %). ConclusionIn conclusion, we externally validated the Betella algorithm in our population. The application of this new proposed algorithm enables assignment of the proper risk class and, consequently, the appropriate indication for adjuvant treatment, allowing for the rationalization of the resources that can be allocated otherwise, not only for the benefit of settings with low resources, but of all settings in general.

Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells

Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.

Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Abstract 3113: Development of HTS enzymatic assays for WRN helicase and exonuclease functions

Abstract WRN, a double strand break repair enzyme with ATP-dependent DNA-helicase and 3’- &amp;gt;5’ exonuclease activities, is under intense focus as a therapeutic target because of its synthetic lethality in tumors with microsatellite instability. To accelerate discovery of WRN inhibitors, we developed and validated homogenous, fluorescence-based high throughput assays for WRN helicase and exonuclease enzymatic activities using the Transcreener HTS Assay platform, which relies on highly selective antibodies to detect nucleotides in a homogenous (mix-and-read) format with far red fluorescent readouts. We produced the WRN helicase domain (aa 500-946, N-terminal 6xHis) in BaV-infected insect cells and optimized detection of its dsDNA-dependent ATPase activity using the Transcreener ADP2 Assay in FP, TR-FRET, and FI detection modes. Optimal reaction conditions included 50 μM ATP (approximate Km) and saturating concentration (100 nM) of a 3’ flapped duplex DNA oligomer; these conditions yielded a good signal with 250 pM WRN, a kcat of 300 min−1, and a Z’ of 0.85. An IC50 of 35 nM was determined for the probe inhibitor NSC-617145, and a 1,280 compound pilot screen (Tocris 2.0 Micro) identified multiple hits that were confirmed in dose response experiments. We produced the WRN 3’-5’ exonuclease domain (aa 1-133-6xHis) in E. coli and used the Transcreener dAMP Exonuclease Assay to allow direct, FP-based detection of dAMP monomers released from a 52 bp dsDNA oligomer with a 5’ overhang. With saturating DNA present (1 μM), the assay produced a good signal with 1 nM enzyme; the Z’ value was 0.89. There are no published inhibitors for the WRN exonuclease, but suramin inhibited with an IC50 of approximately 100 nM. These assays allow sensitive, robust, quantitative detection of WRN ATPase and exonuclease activities using an extensively validated HTS platform; they should be valuable tools for screening and optimizing small molecules targeting specific WRN functions. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for WRN helicase and exonuclease functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3113.

Abstract 3889: Genomic landscape of gynecologic cancers with poor prognosis in Japan, an analysis of the national database of comprehensive genomic profiling tests

Abstract Objective: Comprehensive genomic profiling (CGP) tests were approved in Japan in June 2019 under universal health insurance coverage for cancer patients who have finished (or almost finished) standardized treatment. All the data are collected to the “Center for Cancer Genomics and Advanced Therapeutics (C-CAT)” under written informed consent. Up to date, over 60,000 CGP data have been deposited with a 99.7% consent rate. This study aims to analyze mutational landscape of gynecologic cancers, especially with poor prognosis, by using this database. Methods: We analyzed CGP datasets from 3,006 gynecologic cancer patients using FoundationOne® CDx (F1CDx), the most commonly tested in Japan. This study was ethically approved by our institutional ethics committee (#2021341G) and the Information Utilization Review Board of C-CAT (#CDU2022-026N). Results: Endometrial cancers (n=561) showed high rates of tumor mutational burden-high (TMB-H) (13.9%) and MSI-high (MSI-H) (10.8%), especially in endometrioid carcinomas. POLE exonuclease mutations were less frequent (1.4%) compared to TCGA data (7%), while TP53 mutations were more common (54.4% vs. 29% in TCGA), suggesting the impact of prognosis of the molecular subtypes. Other frequently mutated genes included PIK3CA (41%), PTEN (35%), ARID1A (31%) and KRAS (26%). In 839 cervical cancers, TMB-H prevalence was 14.2%, with low MSI-H at 1.5%. TMB-H was significantly higher in squamous cell carcinomas (20.6%) than adenocarcinomas (8.3%). Frequent mutations included PIK3CA (32%), STK11 (20%), TP53 (20%), KRAS (14%), and CDKN2A (11%). TP53 mutations were significantly higher than TCGA data (&amp;lt;5%), particularly in mucinous carcinomas (60%), indicating a prevalence of HPV-independent tumors with poorer prognosis. Actionable mutations were generally more frequent in adenocarcinomas than in squamous cell carcinomas, including KRAS (32.2%), TP53 (29.4%), CDKN2A (18.3%), and ERBB2 (16.7%). In 1,606 ovarian cancers, the ratio of TMB-H was 5.0%, and MSI-H was 1.2%. TMB-H rates varied between 3.3-6.5% across histological types, while the ratio of MSI-H was significantly lower in serous carcinomas (0.3%) than non-serous carcinomas. Pathogenic mutations in the POLE exonuclease domain were rare in cervical (0.0%) and ovarian (0.2%) cancers. Conclusion: The C-CAT database offers insights into the mutational landscape of various cancers and histological subtypes, especially those with poor prognosis, highlighting the unmet needs for drug development in these gynecologic cancers. Citation Format: Qian Xi, Hidenori Kage, Miho Ogawa, Asami Matsunaga, Akira Nishijima, Kenbun Sone, Kei Kawana, Katsutoshi Oda. Genomic landscape of gynecologic cancers with poor prognosis in Japan, an analysis of the national database of comprehensive genomic profiling tests [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3889.

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Dietary restriction fails to extend lifespan of Drosophila model of Werner syndrome.

Werner syndrome (WS) is a rare genetic disease in humans, caused by mutations in the WRN gene that encodes a protein containing helicase and exonuclease domains. WS is characterized by symptoms of accelerated aging in multiple tissues and organs, involving increased risk of cancer, heart failure, and metabolic dysfunction. These conditions ultimately lead to the premature mortality of patients with WS. In this study, using the null mutant flies (WRNexoΔ) for the gene WRNexo (CG7670), homologous to the exonuclease domain of WRN in humans, we examined how diets affect the lifespan, stress resistance, and sleep/wake patterns of a Drosophila model of WS. We observed that dietary restriction (DR), one of the most robust nongenetic interventions to extend lifespan in animal models, failed to extend the lifespan of WRNexoΔ mutant flies and even had a detrimental effect in females. Interestingly, the mean lifespan of WRNexoΔ mutant flies was not reduced on a protein-rich diet compared to that of wild-type (WT) flies. Compared to WT control flies, the mutant flies also exhibited altered responses to DR in their resistance to starvation and oxidative stress, as well as changes in sleep/wake patterns. These findings show that the WRN protein is necessary for mediating the effects of DR and suggest that the exonuclease domain of WRN plays an important role in metabolism in addition to its primary role in DNA-repair and genome stability.

Abstract IA021: Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles

Abstract Alterations in the exonuclease domain of POLE drive the development of ultramutated endometrial tumors with better prognosis. A hallmark of these tumors is the accumulation of G&amp;gt;T mutations in AGA sequences; however, understanding of their genomic features beyond the trinucleotide motifs is limited. We applied a novel computational framework to the whole-exome sequencing data of 524 endometrial tumors reported by The Cancer Genome Atlas network to analyze the extended DNA sequence context of ultramutation. We found that the presence of POLE driver alleles is associated with an increased frequency of G&amp;gt;T mutations in polypurine tracts. Sequences containing three consecutive purines, including the classic AGA context, are only moderately mutable, but the mutability increased abruptly as the tract length reached six or more purines. Moreover, mutations showed a strong preference for certain positions within the tracts, creating a characteristic quantifiable pattern. Using this signature, we developed a machine learning classifier to identify tumors with hitherto unknown POLE drivers and validated two new drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike previously known pathogenic variants, the E978G substitution affects the DNA polymerase rather than exonuclease domain of POLE. We further show that the extended genomic signature of POLE ultramutation is shared by tumors with POLD1 drivers. These findings uncover new mechanisms of ultramutation and highlight the limitations of the current focus on POLE exonuclease domain variants when stratifying patients. Citation Format: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova. Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA021.

Abstract B014: Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer

Abstract Introduction: The lack of reliable pre-clinical models has significantly impeded the progress of drug discovery for specific molecular subtypes of endometrial cancer. This study aimed to develop patient-derived organoid models for use in preclinical screening of novel and repurposed therapies in endometrial cancer molecular subtypes. Method: Endometrial cancer tissue (n=27) and ascites fluid (n=1) were collected for this study (ethics approval SESLHD HREC 2021_ETH11975 and 19/001) from patients of various subtypes (17 endometrioid, 3 high grade serous, 2 carcinosarcoma, 2 clear cell, 3 mixed) undergoing treatment at the Royal Hospital for Women and Prince of Wales Private Hospital in Sydney, Australia. Tumor cells were isolated, embedded in 70% Matrigel and cultured in defined culture medium to form organoids. Molecular subtyping of organoids and matched parental tissue was undertaken by mismatch repair (MMR) and p53 immunohistochemistry (IHC), and sequencing of POLE exonuclease domain mutations (EDMs). A unique matched two-dimensional (2D) cell line and three-dimensional (3D) organoid culture derived from ascites isolated from a high grade serous endometrial cancer patient was established. These molecularly stratified pre-clinical models were then used to test drug activity. As a proof of principle, response to paclitaxel and carboplatin was undertaken. Result: 20 organoids representing all major molecular subtypes were established (71% success rate), including one derived from ascites fluid. Organoids are presented with either a cystic or solid structure that highly resembles the morphology of the original tissue. Organoid culture can faithfully retain key features including MMR, p53 status as well as POLE EDMs of the parental tissue. The drug screening results identified contrasting responses of 2D and 3D models obtained from the same patient tissue, highlighting the importance of selecting appropriate pre-clinical models for drug development and testing. Conclusion: This study demonstrated that patient-derived organoids can successfully recapitulate the molecular characterization of endometrial cancer. Molecularly defined organoids represent a promising preclinical resource for drug screening in endometrial cancer. In addition, the model structure should be taken into account while conducting drug screening. Citation Format: Dongli Liu, Elyse Powell, King Man Wan, Ramanand Athavale, Christine Loo, Caroline E. Ford. Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B014.

Polymerase and Proofreading Exonuclease Domains in the DNA Polymerase γ and Nuclear-encoded RNA Polymerase of the Human Mitochondria and Identification of Mitochondrial Disease Mutations in the Polymerase Active Site Region of DNA Polymerase γ

Recent reports have made it clear that mutations in the mitochondrial DNA (mtDNA) polymerase γ (POLG1) are a major cause of many human diseases. Mutations in the mtDNA polymerase leads to defective oxidative phosphorylation and ATP production, resulting in many mitochondrial diseases. For their mitochondrial genome replication, humans and animals use POLG1, whose catalytic site is essentially similar to the E. coli DNA polymerase I (DNA pol I). Multiple sequence alignment (MSA) analysis have shown that the POLG1 and E. coli DNA pol I use identical amino acids in their polymerase catalytic sites, viz. –943R-4EHAKI1FNYGRI955Y8G- (human DNA pol γ) as –R-4RSAKA1INFGLIY8G- (E. coli DNA pol I). However, the human POLG1 shows only 31.25% identity with the E. coli DNA pol I, suggesting a highly divergent evolution. Mutation(s) in the POLG1 gene is one of the most common causes of many inherited mitochondrial diseases in children and adults. Depending on their location within the enzyme, mutations either lead to mtDNA depletion or accumulation of multiple mtDNA deletions leading to various mitochondrial diseases. The most common POLG1 dominant mutation, viz. Y955→H/C, which lead to a severe, early-onset of multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure is located in the template-binding pair of the polymerase catalytic site region by MSA. Another dominant mutation, R943→H/C is observed in patients with Progressive External Ophthalmoplegia (adPEO, an autosomal, dominant, heritable mitochondrial disorder) is located in the nucleoside triphosphate (NTP) selection amino acid in the polymerase catalytic site region. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the mitochondrial genes. The human mitochondrial NEP showed 39.30%, 40.12% and 26.98% identities to the NEPs of the mitochondria and chloroplasts of Arabidopsis thaliana, and T7 RNA polymerase, respectively, suggesting that the human and plant mitochondrial NEPs are distinctly different. Interestingly, the human NEP’s catalytic core is almost completely conserved in the plant mitochondrial and chloroplast NEPs, viz. –R-4KVVKQ1TVMTVVY8G- (Human) and –R-4KLVKQ1TVMTSVY8G- (A. Thaliana). Furthermore, the mitochondrial NEP’s catalytic core from human and different animal sources is remarkably conserved and is in close agreement with other NEPs of plant sources. Both the human DNA pol γ and NEP possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from the catalytic K. The PR exonucleases of POLG1 and NEP belong to the DEDD-superfamily of exonucleases and uses a Y or H as the proton acceptor, respectively.

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity.

Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes.