MSH2 Research Articles

Jinekolojik Onkoloji Pratiğimizde Risk Azaltıcı Cerrahi Yönetimi Gerektiren Genetik Değişiklikler

Kanser, fonksiyonu hücre büyümesi ve DNA’nın bütünlüğünü koruma olan genlerdeki mutasyonlar sonucu gelişir. Mutasyonlar, germ hücrelerinde(germline)(GLM) ve diğer hücrelerde (somatik)(SM) olabilir. Kanserlerin %5-10’undan germline mutasyonlar sorumludur. Erken dönemde GLM’ların tespit edilmesi; risk altındaki bireylerin belirlenmesi, takibinin yapılması ve risk azaltıcı koruyucu stratejilerin geliştirilmesi açısından önemlidir. Ayrıca son zamanlarda tümör dokusunda SM’ların tespiti, hedefe yönelik ve adjuvan tedaviyi düzenlemede rehber olmaktadır. Over ve meme kanserlerinin yaklaşık %10-30’u ailesel kümelenme göstermekle birlikte vakaların sadece %5-10’u kalıtsaldır. Özellikle, kalıtsal over kanserlerinin yaklaşık %65-85’ini DNA onarım kusurlarına neden olan BRCA1/2 genlerinde GLM’lar sonucu oluşmaktadır. BRCA1/2 genlerinde patojenik mutasyonu olan bireyler için risk azaltıcı mastektomi (RRM) meme kanseri riskini %90-95 oranında azaltırken, salpingo-ooferektomi (RRSO) ile meme ( %50), over ve fallop tüpü kanseri insidansı da ( %70-96) azalmaktadır. Ayrıca RRSO, kansere özgü genel mortaliteyi azaltmakla ve over kanserini önlemede en etkili seçenek olarak kabul edilmektedir. BRCA1/2 dışında homolog rekombinasyon yolu DNA tamirinde rol oynayan RAD51C, RAD51D ve BRIP1 genlerdeki patojenik mutasyonlar over kanserinde rol oynamaktadır. RAD51C, RAD51D ve BRIP1, genlerinin patojenik mutasyon taşıyıcılarında rehberler, RRSO’i önermektedir. Lynch sendromu (LS), kolorektal ve endometriyal kanser gelişme riskinde artış ile karakterize otozomal dominant bir hastalıktır. LS’da endometrium ve over kanseri risk artışı en fazla olan MLH1, MSH2 ve MSH6 genlerinde patojenik mutasyon taşıyıcılarında fertilitesini tamamladıktan sonra risk azaltıcı cerrahi olarak histerektomi ve RRSO önerilmektedir. Bu derlemede amacımız, jinekolojik onkoloji pratiğimizde son zamanlarda kullanımı gittikçe artan risk azaltıcı profilaktik cerrahi uygulamaları gerektiren genetik hastalıkları son rehberler eşliğinde özetlemektir.

Abstract 5219: Frequency of germline mutations in Mexican children with cancer

Abstract Childhood cancer is a leading cause of death by disease in children from 5 -14 ages, for whom there are no prevention strategies. Due to early-age of diagnosis and short-time environmental factors exposition, increasing evidence suggests childhood cancer could have stronger association with germline alterations in predisposition cancer genes. It has been estimated that up to 30% all childhood cancer diagnosis may be caused by germline variants, but their frequency and distribution remain unclear. Elucidating the prevalence and role of germline mutations in the development of childhood cancer is crucial for understanding its bases, causes, cancer-risk reduction plan, surveillance, and treatment. Here, we present a pilot study on cancer children’s patients and their relatives. A total of 40 Mexican-mestizo children with any cancer diagnosis were recruited from several Institutions of Health in Mexico and a sample of saliva or blood was obtained. Whole Exome Sequencing was performed and bioinformatic analysis was carried out following GATK best practices for germline genetic variants. Additionally, we evaluated a risk selection tool for recognition of genetic predisposition in pediatric cancer patients. Pathogenic or likely-pathogenic genetic variants (mutations) were identified from the application of a filtering chain based on ACMG guidelines, and they were subsequently validated by Sanger sequencing. Germline mutations in MSH6, NF1, MUTYH, CDKN2A, CHEK2, DICER1, FANCA and SBDS genes were identified in 5/40 (12.5%) patients with cancer, only 3 of 5 index cases (60%) reported family history of cancer and all patients meet at least one criterion of the risk selection tool. Two patients were carriers of one mutation, while in three patients were identified 2 pathogenic variants. Additionally, we identified four nonsense candidate genetic variants on EWSR1, FANCL, EPCAM and HRAS genes and two pathogenic variants in non-cancer predisposition gene, ABCA4. Our results show for the first time a higher frequency (12.5%) of germline mutations in cancer predisposition genes in Mexican-mestizo children with cancer. The lack of family history in two carriers showed the need to establish standardized suspicion criteria for identification of children with higher risk of cancer who can benefitfrom genetic testing. Further characterization of additional genetic variants identified without clinical classification will be important to evaluate their real contribution, as well as the influence of environmental factors on de novo mutation promotion. Citation Format: Oscar Alonso Luna, Jorge Meléndez Zajgla, Marta Zapata Tarres, Luis Enrique Juárez Villegas, Elvia Cristina Mendoza Caamal, Elianeth Rey Helo, Socorro Aida Borges Yanez, Gabriela Elisa Mercado Celis, Rodrigo Barquera Lozano. Frequency of germline mutations in Mexican children with cancer [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 5219.

Abstract 2781: De novo molecular mechanisms of resistance to mobocertinib

Abstract Mobocertinib is an oral tyrosine kinase inhibitor recently approved by the FDA for the treatment of patients (pts) with locally advanced or metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutations who have progressed on or after platinum-based chemotherapy. To understand the impact of co-occurring genetic alterations on the therapeutic response to mobocertinib, tumors from a subset of 72 platinum-pretreated pts with EGFR exon 20 insertions from the phase 1/2 study (NCT02716116) were collected before treatment and analyzed by next-generation sequencing. More than 200 co-occurring hotspot genetic alterations were detected in 43 pts, with some alterations detected with greater than 20% frequency, such as PTCH1 (67%), HNF1A (58%), TP53 (58%), and GNAQ (35%). In addition to EGFR exon 20 insertion mutations, co-occurring EGFR substitutions were also observed in the exon 20 loop following the C-helix (amino acid positions 770, 771, 772) in 4 pts and L858Q in 1 pt. The pt with the L858Q mutation had a confirmed partial response as assessed by an independent review committee, while the 4 pts with exon 20 substitutions were non-responders (2 had stable disease and 2 had progressive disease as best response). An abnormally high level of mutational burden was seen in 4 pts with EGFR exon 20 insertions, along with alterations in the mismatch repair gene MSH6 and/or the elongation factor MLLT3. Association analysis of mutations reported increased odds of association between the presence of PIK3CA, TP53, and EGFR alterations and a lack of mobocertinib response. Gene amplifications were observed in 12 pts (16%) affecting EGFR (6 pts), MYC (4 pts), CDK4 (3 pts), and CCND1 (2 pts). When assessed for association with lack of response (P values from the Fisher exact test), odds ratios were 2.47 (P=0.66) and 6.34 (P=0.085) for EGFR amplification and any-gene amplification, respectively. When combined, the presence of either PIK3CA mutations or any-gene amplification was associated with a lack of response to mobocertinib treatment with an odds ratio of 12.93. Following adjustment for selected clinical covariates in a multivariate model, the presence of either PIK3CA mutation or gene amplification was an independent predictor of lack of response to mobocertinib (P value of 0.047). Given the retrospective nature of these analyses and the insufficient control of statistical multiplicity, this finding should be considered exploratory. These data suggest that intrinsic genetic alterations, present at baseline in patients with NSCLC harboring an EGFR exon 20 insertion mutation, might impact the response to mobocertinib treatment. The presence of PIK3CA mutations, EGFR gene amplification, or cell cycle regulator MYC-CDK4-CCND1 amplification is associated with lack of response to mobocertinib, which might guide rational combinations for future clinical investigation. Citation Format: Sylvie Vincent, Zhenqiang Su, Adarsh Joshi, Kalyani Penta, Pingkuan Zhang. De novo molecular mechanisms of resistance to mobocertinib [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 2781.

Potential Influence of Age and Diabetes Mellitus Type 1 on MSH2 (MutS homolog 2) Expression in a Rat Kidney Tissue

Background: Homeostasis of proliferating tissues is strongly dependent on intact DNA. Both neoplastic and non-neoplastic diseases have been associated with MSH2 (MutS homolog 2, a mismatch repair protein) deficiency. In this study, we examined how age and diabetes mellitus influence the expression of MSH2 in the kidney. Methods: To study the effect of age, three groups of healthy rats were formed: 2 months, 8 months, and 14 months old. Two groups of diabetic rats were formed: 8 months old and 14 months old. Expression of MSH2 in the kidney was studied by quantifying immunofluorescent staining. Results: Age was identified as the main factor that influences MSH2 expression in kidneys. The effect of age followed parabolic dynamics, with peak expression at 8 months of age and similar levels at 2 and 14 months. Diabetes had an age-dependent effect, which manifested as the increase of MSH2 expression in 14-month-old diabetic rats in comparison to healthy animals. Conclusions: Age influences MSH2 expression in the kidney more than diabetes mellitus. Since ageing is a risk factor for kidney neoplasia, downregulation of MSH2 in older rats might represent one of the pro-oncogenic mechanisms of ageing at a molecular level.

Complex mutation profiles in mismatch repair and ribonucleotide reductase mutants reveal novel repair substrate specificity of MutS homolog (MSH) complexes.

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.

HGG-51. Uncovering therapeutic vulnerabilities in mismatch repair-deficient gliomas

INTRODUCTION: We have observed that approximately 26% of recurrent gliomas acquire hypermutation following treatment with temozolomide (TMZ). Intriguingly, 91% of these tumors harbor mutations in mismatch repair (MMR) genes. Since MMR deficiency confers resistance to TMZ, strategies to target MMR-deficient gliomas stand to impact many patients. METHODS: We ablated the MMR genes MSH2, MSH6, MLH1, and PMS2 using an all-in-one sgRNA-CRISPR/Cas9 expression vector to generate isogenic MMR knockouts in patient-derived glioma cell lines. We characterized the gene expression profiles of these MMR-deficient glioma models and leveraged high-throughput drug screens and genome-scale CRISPR/Cas9 dropout screens to identify therapeutic vulnerabilities induced by loss of MMR. RESULTS: We show that loss of each major MMR gene confers resistance to TMZ. Gene set enrichment analysis of our MMR-deficient knockouts shows enrichment of several hallmark gene sets including DNA repair and G2M checkpoint signatures, and our genome-wide CRISPR dropout screen reveals that MMR-deficient cells are preferentially dependent on a number of genes involved in DNA repair and cell cycle, along with several other pathways. Lastly, the high-throughput drug repurposing (REPO) screen shows that loss of MMR confers differential dependencies to small molecule inhibitors. CONCLUSIONS: Using CRISPR/Cas9 to knock out individual MMR pathway members allows us to systematically study the response of MMR-deficient cells to alkylating agents in an isogenic context. Importantly, these isogenic models reveal that MMR-deficient glioma cells possess novel genetic dependencies and sensitivities to small molecules, which may inform future therapies for MMR-deficient tumors.

Novel targets in rectal cancer by considering lncRNA–miRNA–mRNA network in response to Lactobacillus acidophilus consumption: a randomized clinical trial

We aimed to explore the lncRNA–miR–mRNA network in response to Lactobacillusacidophilus (L. acidophilus) consumption in rectal cancer patients. The candidate miRs were first taken from the GEO and TCGA databases. We constructed the lncRNA–miR–mRNA network using the high-throughput sequencing data. At last, we created a heatmap based on the experimental data to show the possible correlation of the selected targets. The expression levels of selected targets were measured in the samples of 107 rectal cancer patients undergoing placebo and probiotic consumption and 10 noncancerous subjects using Real-Time PCR. Our analysis revealed a group of differentially expressed 12 miRs and 11 lncRNAs, and 12 genes in rectal cancer patients. A significant expression increase of the selected tumor suppressor miRs, lncRNAs, and genes and a substantial expression decrease of the selected oncomiRs, onco-lncRNAs, and oncogenes were obtained after the probiotic consumption compared to the placebo group. There is a strong correlation between some network components, including miR-133b and IGF1 gene, miR-548ac and MSH2 gene, and miR-21 and SMAD4 gene. In rectal cancer patients, L.acidophilus consumption was associated with improved expression of the lncRNA–miR–mRNA network, which may provide novel monitoring and therapeutic approaches.

15 years of experience of an oncogenetics service in the northeast of Brazil.

e22522 Background: The Oncogenetics service at the Federal University of Bahia was implemented in 2007 and since then has been offering pre- and post-test genetic counseling for cancer susceptibility genes, mainly tumors associated with King's syndrome in the population of the state of Bahia. This is a mixed population of about 80% of African ancestry. Methods: Between the years 2007 to 2021, 462 probands and their families have already been evaluated by NGS analysis, Sanger sequencing. Large rearrangements were detected by multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 and BRCA2 genes. Results: The mean age at diagnosis was 41 years. Pathogenic/probably pathogenic mutations were founded in 118 patients: 67 had breast cancer, 09 with ovarian cancer, 26 with more than one tumor, 15 without cancer, and 01 with another tumor type. Most of the pathogenic variants were located in the BRCA1 gene (27 variants), the two most frequent variants being: c.211G > A (13 probands) and c.3331_3334delCAAG (18 probands), both of Iberian origin. Of patients with mutation, 9.8% had large rearrangements in BRCA1 and 11.5% in BRCA2. Bilateral breast cancer appears predominantly in patients with BRCA1 (4) and BRCA2 (1) variants. In this study, only two mutations in BRCA described as of African origin were found in two patients. Other pathogenic mutations appear in ATM, BARD1, BRIP1, CDH1, CHEK2, FAM175A, FANCM, MSH6, NBN, NF2, PALB2, RAD51, SDHB, SLX4, and TP53 genes. Variants of uncertain significance were identified in 71 patients. Conclusions: The population of the state of Bahia has a peculiar mutational profile, containing mutations that are not very frequent in other regions of the country. Despite being a population with high African ancestry, most mutations are of European origin.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

Andrias davidianus Ranavirus (ADRV) Genome Replicate Efficiently by Engaging Cellular Mismatch Repair Protein MSH2.

As nucleocytoplasmic large DNA viruses, replication of ranaviruses (genus Ranavirus, family Iridoviridae) involves a series of viral and host proteins. We have described that the replication and transcription machinery of Andrias davidianus ranavirus (ADRV) which was isolated from the Chinese giant salamander contained host factors. Here, a new host factor, the MutS homolog 2 (MSH2), was proved as an important protein that participated in ADRV infection. Expression of MSH2 was stable during ADRV infection in cultured cells and it localized at the cytoplasmic viral factories and colocalized with virus nascent DNA, indicating its possible role in virus genome replication. Investigation of the viral proteins that interacted with MSH2 by co-immunoprecipitation showed that A. davidianus MSH2 can interact with ADRV-35L (possible components associated with virus transcription), ADRV-47L (virus DNA polymerase), and ADRV-98R. Further knockdown MSH2 expression by RNAi significantly reduced the late gene expression of ADRV. Additionally, MSH2 knockout by CRISPR/Cas9 significantly reduced viral titers, genome replication, and late gene transcription of ADRV. Thus, the current study proved that ADRV can engage cellular MSH2 for its efficient genome replication and late gene transcription, which provided new information for understanding the roles of host factors in ranavirus replication and transcription.

A Review of MutSα and its Absence in Mismatch Repair Related Ovarian Carcinomas

This review investigates the roles of microsatellite instability (MSI) and the DNA mismatch repair (MMR) pathway in ovarian carcinomas. MMR promotes genome stability post‐replication by repairing DNA mismatches; defective MMR (dMMR) leaves genomic errors that can cause MSI. To repair these mismatches, the MMR system employs two sets of proteins that form heterodimers: MutL Homolog 1 (MLH1) in complex with post meiotic segregation increased 2 (PMS2) and MutS Homolog 2 (MSH2) in complex with MutS Homolog 6 (MSH6). The MSH2/MSH6 heterodimer (MutSα) recognizes the mismatch, while the MLH1/PMS2 heterodimer (MutLα) repairs the mismatch. The MSH2/MSH6 proteins are each composed of 5 asymmetrical structural domains, the first being responsible for mismatch binding, followed by the connector, lever, clamp, and ATPase domains, or domains I‐V, respectively. The MSH2 mismatch binding domain associates directly with DNA and extends a helix to block the polymerase, while MSH6 associates with DNA via its Phe‐X‐Glu motif. The MutSα complex has two states: an ADP‐bound state in which it searches for mismatches, and an ATP‐bound state in which it signals for repair. Lack of effective repair can contribute to carcinogenesis including ovarian carcinogenesis. Mismatches in microsatellites are phenotypic evidence that the MMR pathway is non‐functional in these tumors, and differences in MMR status are observed across ovarian carcinoma subtypes. For example MSH6 deficiency is associated with three subtypes of ovarian cancer: clear cell, mucinous, and endometrioid carcinomas, and there is a higher survival rate for patients with these MSH6 deficient tumors. Tumor MMR status also influences treatment efficacy and age of disease onset. Interestingly, only in familial carcinomas was MSI demonstrated as a supportive diagnostic tool. Tumors with high MSI can be treated with immunotherapy and immune checkpoint inhibitors; therefore investigating the MMR pathway functionality and MSI status of ovarian carcinomas can shape the course of treatment and patient outcomes.

Potential genetic biomarker of Saudi Arabian patients with colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths globally. We implemented a comprehensive literature review regarding CRC genetics studies to offer a perception into the genes associated with CRC recognized in Saudi patients. Definite genetic variants in ABCB1, ADIPOQ, CTNNB1, SFRP3, LRP6, CYP19A1, PARP-1, TDG genes exhibited significant protection against CRC development in Saudi population. Whereas, other gene mutations in ABCB1, ABCC1, CASR, IL-17F, NOTCH1, NOTCH4, PRNCR1, TDG, TLR2, TLR4, TLR-9, TSLP, TSLPR and TNF-α genes showed irrelevant correlation with CRC risk in Saudi Arabia. On the other hand, specific mutations in ABCC1, ADIPOQ, CYP1A1, KIR, IL-17A, MMP2, NOTCH3, PRNCR1, RETN, TDG, TLR2, BRAF, PARP-1, TLR4, TLR-9, TNF-α, TSLP and XRCC1 genes demonstrated a substantial augmented CRC risk development in Saudi patients. Furthermore, ATR, ATM, BMI1, CCAT1, Chk1, Chk2, COX-2, FoxM1, FSCN1, Ki67, MALAT1, miR-29, miR-34a, miR-92, miR-182-5, PANDAR, PIK3CA, TIGAR over-expression revealed a robust association with CRC in Saudi Arabia (KSA). Moreover, gene alterations in APC, EGFR, FBXW7, TP53, PTEN, K-ras genes were concomitant in CRC. As well as, lower expression of MLH1, MSH2, MSH6, PMS2, EPCAM and MUTYH genes were recognized in LS patients and future CRC Saudi patients. These gene mutations may be used as diagnostic and/or prognostic genetic markers in CRC Saudi patients and could offer a potential therapeutic target for CRC management.

Function of ATM and MSH2 during nonhomologous end joining in DNA repair

Abstract Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also play a role in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, ATM−/− mice were bred to mice deficient for the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/−MSH2−/− adult mice were not observed. To obtain ATM- and MSH2-deficient B cells, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression (CD23-cre) to generate a deleted ATM allele (ATMD). As compared to the ATMD/D and MSH2−/− mice and B cells, the ATMD/DMSH2−/− mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from ATMD/DMSH2−/− B cells that were induced to switch to IgG1 in vitro revealed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, ATMD/D, or MSH2−/− B cells. This data suggests that the absence of both ATM and MSH2 blocks NHEJ and leads to inefficient end joining and the reduced CSR. We identify complementary roles for ATM and MSH2 in NHEJ and A-EJ during CSR and propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID. This work was supported by The National Institute on Minority Health and Health Disparities (5G12MD007603), The National Cancer Institute (2U54CA132378), and The National Institute of General Medical Sciences (1SC1GM132035-01).

Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.

As a biomarker that affects treatment decisions of immune checkpoint inhibitors, the accuracy, reliability, and comparability of tumor mutational burden (TMB) estimation is of paramount importance. To improve the consistency and reliability of these tests, qualified reference materials providing ground-truth data are crucial. In this study, we developed a set of formalin-fixed and paraffin-embedded (FFPE) samples with different TMB values as the novel reference materials for TMB estimation. By introducing several clinically relevant variants in MutS Homolog 2 (MSH2) gene and DNA polymerase epsilon (POLE) gene into human cell lines using CRISPR/Cas9 technology, we first constructed four typical cell lines which verified with hypermutator or ultramutator phenotype. Followed by cell mixing and paraffin embedding, the novel FFPE samples were prepared. It was confirmed that our novel FFPE samples have sufficient quantity of cells, high reproducibility, and they can provide matched wild type sample as the genetic background. The double-platform whole exome sequencing validation showed that our FFPE samples were also highly flexible as they containing different TMB values spanning a clinically relevant range (2.0–106.1 mut/Mb). Without limitations on production and TMB values, our novel FFPE samples based on CRISPR/Cas9 editing are suitable as candidate reference materials. From a practical point of view, these samples can be used for the validation, verification, internal quality control, and proficiency testing of TMB assessment.

GSTM3 deficiency impedes DNA mismatch repair to promote gastric tumorigenesis via CAND1/NRF2-KEAP1 signaling

Gastric cancer (GC) is one of the most severe gastric diseases worldwide. However, the molecular basis that drives tumorigenesis and progression is not completely understood, which hinders the efficacy and development of therapeutic options. Glutathione-S-transferases (GSTs) are a group of phase II detoxification enzymes that maintain redox homeostasis; however, their roles in cancers are not well defined. Here, we revealed that the expression of GST family members is significantly impaired in GC tissues. Glutathione-S-transferase mu 3 (GSTM3), a member of GST family, is dramatically downregulated in cancerous tissues and has been identified as an independent prognostic factor in GC associated with tumor differentiation, inhibiting GC cell proliferation and migration in vitro and in vivo. Mechanistically, GSTM3 is transcriptionally activated by NRF2/KEAP1 signaling. As a feedback loop, GSTM3 binds to Cullin-associated and neddylation-dissociated 1 protein (CAND1), an exchange factor for integrating Kelch-like ECH-associated protein 1 (KEAP1) into Cul3-RING ubiquitin ligases (CRL3), to disrupt nuclear factor-erythroid factor 2-related factor 2 (NRF2)/KEAP1 binding and prevent NRF2 ubiquitination and degradation, leading to its activation. A deficiency in glutathione S-Transferase Mu 3 (GSTM3) reduces DNA mismatch repair (MMR) gene expression and increases mutagenesis via CAND1/NRF2 binding. Importantly, GSTM3/NRF2 and KEAP1 were negatively and positively associated with the genomic signature for microsatellite instability, respectively. Clinically, GSTM3, NRF2, and MutS homolog 6 (MSH6) were positively correlated in the GC specimens. This study uncovered a reciprocal regulation between GSTM3 and NRF2 and established a functional and clinical link between GSTM3-NRF2/KEAP1 and MMR during GC cell proliferation and progression, thus providing potential therapeutic targets for GC.

Nuclear respiratory factor 1 transcriptomic signatures as prognostic indicators of recurring aggressive mesenchymal glioblastoma and resistance to therapy in White American females.

The mechanisms contributing to recurrence of glioblastoma (GBM), an aggressive neuroepithelial brain tumor, remain unknown. We have recently shown that nuclear respiratory factor 1 (NRF1) is an oncogenic transcription factor and its transcriptional activity is associated with the progression and prognosis of GBM. Herein, we extend our efforts to (1) identify influential NRF1-driven gene and microRNA (miRNA) expression for the aggressiveness of mesenchymal GBM; and (2) understand the molecular basis for its poor response to therapy. Clinical data and RNA-Seq from four independent GBM cohorts were analyzed by Bayesian Network Inference with Java Objects (BANJO) and Markov chain Monte Carlo (MCMC)-based gene order to identify molecular drivers of mesenchymal GBM as well as prognostic indicators of poor response to radiation and chemotherapy. We are the first to report sex-specific NRF1 motif enriched gene signatures showing increased susceptibility to GBM. Risk estimates for GBM were increased by greater than 100-fold with the joint effect of NRF1-driven gene signatures-CDK4, DUSP6, MSH2, NRF1, and PARK7 in female GBM patients and CDK4, CASP2, H6PD, and NRF1 in male GBM patients. NRF1-driven causal Bayesian network genes were predictive of poor survival and resistance to chemoradiation in IDH1 wild-type mesenchymal GBM patients. NRF1-regulatable miRNAs were also associated with poor response to chemoradiation therapy in female IDH1 wild-type mesenchymal GBM. Stable overexpression of NRF1 reprogramed human astrocytes into neural stem cell-like cells expressing SOX2 and nestin. These cells differentiated into neurons and form tumorospheroids. In summary, our novel discovery shows that NRF1-driven causal genes and miRNAs involved in cancer cell stemness and mesenchymal features contribute to cancer aggressiveness and recurrence of aggressive therapy-resistant glioblastoma.

The CtIP-CtBP1/2-HDAC1-AP1 transcriptional complex is required for the transrepression of DNA damage modulators in the pathogenesis of osteosarcoma

Most tumors, including osteosarcomas, have deficiencies in DNA damage repair. However, the regulatory mechanisms underlying dysregulation of DNA damage repair genes are still being investigated. In this study, we reveal that C-terminal binding protein (CtBP) interacting protein (CtIP) couples with three transcriptional regulators, CtBP1/2 heterodimer, histone deacetylase 1 (HDAC1), and two subunits of the activating protein 1 (AP1) transcription factor to assemble a transcriptional complex. This complex specifically controls the expression of four genes involved in DNA damage and repair processes: MutL homolog 1 (MLH1), MutS Homolog 3 (MSH3), breast cancer type 1 (BRCA1), and cyclin dependent kinase inhibitor 1A (CDKN1A). Chromatin immunoprecipitation (ChIP) assay results revealed that the CtIP-CtBP1/2-HDAC1-AP1 complex regulated these four genes by binding to their promoters through the TGAT/CTCA consensus sequence. The depletion of CtIP, CtBP1/2, and HDAC1 increased the expression levels of MLH1, MSH3, BRCA1, and CDKN1A and inhibited in vitro and in vivo osteosarcoma cell growth. Overexpression of MLH1, MSH3, BRCA1, or CDKN1A in osteosarcoma cells can reduce cell viability, colony formation, cell migration, and tumor growth. Our findings suggest that the CtIP-CtBP1/2-HDAC1-AP1 complex is required for mediation of DNA damage processes for the pathogenesis of osteosarcoma.

Combination of HLA-DQ2/-DQ8 Haplotypes and a Single MSH5 Gene Variant in a Polish Population of Patients with Type 1 Diabetes as a First Line Screening for Celiac Disease?

Patients with type 1 diabetes (T1D) are at increased risk for developing celiac disease (CD). The aim of the study was to assess the usefulness of celiac-specific human leukocyte antigen (HLA) haplotype and the rs3130484 variant of MSH5 gene, a previously described non-HLA variant associated with CD in the Polish population as a first-line screening for CD in T1D pediatric patients. Serological CD screening performed in the T1D group (n = 248) and healthy controls (n = 551) allowed for CD recognition in 20 patients (8.1%) with T1D (T1D + CD group). HLA-DQ2, HLA-DQ8 and the rs3130484 variant were genotyped with TaqMan SNP Genotyping Assays. The T1D + CD group presented a higher, but not statistically significant, frequency of HLA-DQ2 in comparison with T1D subjects. Combining the rs3130484 with HLA-DQ2/HLA-DQ8 typing significantly increased the sensitivity of HLA testing from 32.7% to 68.7%, and the accuracy of estimating CD prediction from 51.7% to 86.4% but decreased the specificity from 100% to 78.2%. The receiver operating characteristic curve analysis confirmed the best discrimination for the combination of both genetic tests with an area under curve reaching 0.735 (95% CI: 0.700–0.7690) in comparison with 0.664 (95% CI: 0.632–0.696) for HLA typing alone. Results show the low utility of HLA-DQ2/HLA-DQ8 typing for CD screening in T1D pediatric patients. Combination of the rs3130484 variant of the MSH5 gene and HLA testing increases both the sensitivity and the predictive value of the test accuracy, but still, the obtained values are not satisfactory for recommending such testing as the first-line screening for CD in T1D patients.

Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT-methylated glioblastoma

The methylation status of the O6-methylguanine methyltransferase (MGMT) gene promoter has been widely accepted as a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ). In the absence of promoter methylation, the MGMT enzyme removes O6-methylguanine (O6-meG) lesions. In the setting of MGMT-promoter methylation (MGMT-), the O6-meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Our group reported that loss of MGMT expression via MGMT promoter silencing modulates activation of ataxia telangiectasia and RAD3 related protein (ATR) in response to TMZ treatment, which is associated with synergistic tumor-cell killing. Whether or not MMR proteins are involved in ATR activation in MGMT-cells upon alkylation damage remains poorly understood. To investigate the function of MMR in ATR activation, we created isogenic cell lines with knockdowns of the individual human MMR proteins MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutS homolog 3 (MSH3), MutL homolog 1 (MLH1), and PMS1 homolog 2 (PMS2). Here, we demonstrate that MSH2, MSH6, MLH1 and PMS2, specifically, are involved in the activation of the ATR axis after TMZ exposure, whereas MSH3 is likely not. This study elucidates a potential mechanistic understanding of how the MMR system is involved in ATR activation by TMZ in glioblastoma cells, which is important for targeting MMR-mutated cancers.

Correlation of mismatch repair deficiency with clinicopathological features and programmed death-ligand 1 expression in thyroid carcinoma.

Mutations in DNA mismatch repair (MMR) genes associated with thyroid carcinoma (TC) have rarely been reported, especially in East Asian populations. We examined tumor tissue from a cohort of 241 patients diagnosed with TC between 2008 and 2020. MMR proteins were detected using tissue microarray-based immunohistochemistry in order to identify MMR-protein-deficient (MMR-D) and MMR-protein-intact (MMR-I) tumors. We retrospectively summarized the clinicopathologic characteristics of patients with MMR-D TC, measured the expression of PD-L1, and recorded overall survival (OS) and other clinical outcomes. In our cohort, there were 18 (7.5%) MMR-D (MLH1, MSH2, MSH6, and PMS2) patients, including 12 with papillary TC (PTC) (6.7%), 2 with poorly differentiated TC (PDTC) (4.7%), and 4 with anaplastic TC (ATC) (22.2%). Half of them (9/18) showed a specific deletion in MSH6, and 6 of them also carried variants in the MSH6 and PMS2 gene. Survival was significantly better in patients with MMR-D ATC than in those with MMR-I tumors (p = 0.033). Four of the 18 MMR-D patients (22%) were found to be PD-L1 positive. Their OS was much shorter than that of PD-L1-negative patients. MMR-D and PD-L1 positivity appear to be associated with clinicopathological characteristics and prognosis in TC. The results indicate that MMR status may have important prognostic significance in TC. Therefore, immune checkpoint inhibitors that target the PD-1/PD-L1 pathway may be a treatment option for TCs.