TDP43 Interacts with MLH1 and MSH6 Proteins in A DNA Damage-Inducible Manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.

TDP43 interacts with MLH1 and MSH6 proteins in a DNA damage-inducible manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

Gut Microbiome Composition in Lynch Syndrome With and Without History of Colorectal Neoplasia and Non-Lynch Controls.

BACKGROUND: While Lynch syndrome (LS) is a highly penetrant colorectal cancer (CRC) syndrome, there is considerable variation in penetrance; few studies have investigated the association between microbiome and CRC risk in LS. We analyzed the microbiome composition among individuals with LS with and without personal history of colorectal neoplasia (CRN) and non-LS controls. METHODS: We sequenced the V4 region of the 16S rRNA gene from the stool of 46 individuals with LS and 53 individuals without LS. We characterized within community and in between community microbiome variation, compared taxon abundance, and built machine learning models to investigate the differences in microbiome. RESULTS: There was no difference within or between community variations among LS groups, but there was a statistically significant difference in both within and between community variation comparing LS to non-LS. Streptococcus and Actinomyces were differentially enriched in LS-CRC compared to LS-without CRN. There were numerous differences in taxa abundance comparing LS to non-LS; notably, Veillonella was enriched and Faecalibacterium and Romboutsia were depleted in LS. Finally, machine learning models classifying LS from non-LS controls and LS-CRC from LS-without CRN performed moderately well. CONCLUSIONS: Differences in microbiome composition between LS and non-LS may suggest a microbiome pattern unique to LS formed by underlying differences in epithelial biology and immunology. We found specific taxa differences among LS groups, which may be due to underlying anatomy. Larger prospective studies following for CRN diagnosis and microbiome composition changes are needed to determine if microbiome composition contributes to CRN development in patients with LS.

Evaluation of immunotherapy efficacy in gynecologic cancer.

Various immunotherapies have demonstrated remarkable success over the past few decades, and have been approved for the treatment of different cancer types. However, patient responses to immunotherapy are variable, and approximately 50% of cases are refractory to these agents. Tumor biomarker-based stratification of cases may therefore help identify subpopulations that are sensitive/resistant to immunotherapy; it may also improve prediction of response in various cancers including gynecologic cancer. These biomarkers include the tumor mutational burden, microsatellite instability, mismatch repair deficiency, T cell-inflamed gene expression profile, programmed cell death protein 1 ligand 1, tumor-infiltrating lymphocytes, and numerous other genomic alterations. Future directions in the treatment of gynecologic cancer include the utilization of these biomarkers to select ideal candidates. This review focused on recent advances in the predictive ability of molecular biomarkers in patients with gynecologic cancer who undergo immunotherapy. The most recent developments in combined immunotherapy and targeted therapy strategies and novel immune interventions against gynecologic cancers have also been discussed.

"Left in limbo": Exploring how patients with colorectal cancer interpret and respond to a suspected Lynch syndrome diagnosis.

BACKGROUND: A diagnosis of suspected Lynch syndrome (SLS) is given when a tumour displays characteristics consistent with Lynch syndrome (LS), but no germline pathogenic variant is identified. This inconclusive diagnosis results in uncertainty around appropriate cancer risk management. This qualitative study explored how patients with CRC interpret and respond to an SLS diagnosis. METHODS: Semi-structured telephone interviews were conducted with 15 patients with CRC who received an SLS diagnosis, recruited from cancer genetics services across Australia. Interviews were transcribed verbatim and analysed using thematic analysis. Participant responses were compared with appointment summary letters from cancer genetics services. RESULTS: Participants' interpretations of genetic test results were found to vary widely. While this variation often aligned with variation in interpretations by cancer genetics services, participants also had difficulties with the complexity and recall of genetic test results. Participants had a range of psychological responses to the uncertainty that their results presented, from relief to disappointment and doubt. Cancer risk perceptions also varied widely, with participants' interpretations of their genetic test results just one of several influencing factors. Despite this variability, almost all participants adhered to cancer risk management advice, although different participants received different advice. All participants also communicated any cancer risk management advice to first-degree relatives, motivated by protecting them, but information communicated was not always consistent with advice received. CONCLUSIONS: Our study findings highlight the variability in patients' interpretations of their diagnosis, cancer risk management and family communication when a diagnosis of SLS is received, and provide novel insights into how healthcare professionals can better support patients with SLS.

Long-Term Survival and Clinicopathological Implications of DNA Mismatch Repair Status in Endometrioid Endometrial Cancers in Hong Kong Chinese Women.

To investigate the role of DNA mismatch repair status (MMR) in survival of endometrioid endometrial cancer in Hong Kong Chinese women and its correlation to clinical prognostic factors, 238 patients with endometrioid endometrial cancer were included. Tumor MMR status was evaluated by immunohistochemistry. Clinical characteristics and survival were determined. Association of MMR with survival and clinicopathological parameters were assessed. MMR deficiency (dMMR) was found in 43 cases (16.5%). dMMR was associated with poor prognostic factors including older age, higher stage, higher grade, larger tumor size and more radiotherapy usage. Long-term survival was worse in dMMR compared to the MMR proficient group. The dMMR group had more deaths, shorter disease-specific survival (DSS), shorter disease-free survival (DFS), less 10-year DSS, less 10-year DFS, and more recurrence. The 5-year DSS and 5-year DFS in the dMMR group only showed a trend of worse survival but did not reach statistical significance. In conclusion, dMMR is present in a significant number of endometrioid endometrial cancers patients and is associated with poorer clinicopathological factors and survival parameters in the long run. dMMR should be considered in the risk stratification of endometrial cancer to guide adjuvant therapy and individualisation for longer follow up plan.

The contribution of Lynch syndrome to early onset malignancy in Ireland.

BACKGROUND: Lynch syndrome (LS) is an autosomal dominant hereditary cancer syndrome responsible for 2-4% of hereditary colorectal cancers (CRC). Mismatch repair protein deficiency (dMMR) is a characteristic feature of LS. It has been associated with a poor response to standard chemotherapy in metastatic colorectal cancer (mCRC). There is currently no LS database to monitor trends of disease in Ireland. We aim to centralise LS data in Ireland to assess the burden of LS in Ireland and guide improvements in prevention and treatment of LS-associated cancer. METHODS: A retrospective review was carried out including all medical records for LS patients from two of the three cancer genetics clinics in Ireland between 2000 and 2018 was carried out. Clinicopathological data of probands (n = 57) and affected family members including demographics, mutation status, cancer diagnosis and outcome was recorded. Statistical analysis was carried out using SPSS software. RESULTS: Fifty-seven families including three-hundred and forty-five individuals affected by cancer were identified. The most common cancers recorded were colorectal (53%), breast (12%) and endometrial (10%). One-hundred and thirty-eight confirmed carriers were identified: 65 path_MLH1 (47%), 43 path_MSH2 (31%), 11 path_MSH6 (8%), 17 path_PMS2 (12%) and two path_EPCAM (1%). Cancer type varied significantly by gene. Median age of first diagnosis was 44.5 years (range 23-81). Half of all deceased patients (n = 11) in this group died within 2.5 years of first diagnosis. These deaths were directly related to cancer in 59% of cases. CONCLUSIONS: Under diagnosis of LS misses a powerful preventive and therapeutic opportunity. LS causes early onset dMMR cancer diagnoses with substantial societal impact. Implementation of ICBs into treatment policy for this small cohort of dMMR mCRC is an achievable therapeutic goal that may significantly improve survival. A prospective database for LS in Ireland is necessary to maximise prevention in this population.

The association of four SNPs in DNA mismatch repair genes with idiopathic male infertility in northwest China.

DNA mismatch repair (MMR) plays a critical role in the maintenance of genetic integrity. The failure of MMR in sperm DNA was found in male infertility. However, its aetiology in idiopathic male infertility (IMI) remains unknown. The present study was to investigate whether the four SNPs (rs26279 in MSH3, rs1800734 and rs4647269 in MLH1 and rs175080 in MLH3) in MMR genes were associated with IMI or not. The interactions of the SNPs were also performed to clarify its genetic aetiology. In the present study, 209 clinically diagnosed IMI men and 201 fertile men were recruited. Four SNPs were genotyped by DNA sequencing. It was the first time to investigate the association between rs26279 in MSH3 and IMI. The genotype frequency distribution of rs26279 (A>G) in MSH3 was found to be significantly different between IMI and control (p < 0.05), as well as azoospermia. The rs1800734 and rs4647269 in MLH1 were found to be significantly different between severe oligozoospermia and control groups (p < 0.05). However, rs175080 in MLH3 was not significantly different between IMI and control (p > 0.05). Multifactor dimensionality reduction (MDR) for detecting interactions showed that there were no interactions among the four SNPs on IMI.

Implementation of a Systematic Tumor Screening Program for Lynch Syndrome in an Integrated Health Care Setting.

A subset of colorectal cancer (CRC) cases are attributable to Lynch syndrome (LS), a hereditary form of CRC. Effective evaluation for LS can be done on CRC tumors to guide diagnostic testing. Increased diagnosis of LS allows for surveillance and risk reduction, which can mitigate CRC-related burden and prevent cancer-related deaths. We evaluated participation in LS screening among newly diagnosed adult CRC patients. Some cases were referred for genetics evaluation prior to study recruitment (selective screening). Those not referred directly were randomized to the intervention or control (usual care) arms. Control cases were observed for one year, then given information about LS screening. Patients who declined participation were followed through the medical record. Of 601 cases of CRC, 194 (32%) enrolled in our study and were offered LS screening, 43 (7%) were followed as a control group, 148 (25%) declined participation and 216 (36%) were ineligible [63 (10%) of which received prior selective screening]. Six and nine cases of LS were identified through the intervention and selective screening groups, respectively. Overall, a higher proportion of PMS2 variants were identified in the intervention (3/6, 50%) versus selective screening groups (2/9, 22%) (not statistically significant). Eighty-eight percent and 23% of intervention and control patients, respectively, received LS screening. No control patients were found to have LS. Systems-based approaches are needed to ensure we fully identify LS cases. The proportion of LS cases from this program was 4% of newly diagnosed cases of CRC, similar to other programs.

Prognostic Value of Mismatch Repair Genes for Patients With Colorectal Cancer: Meta-Analysis.

DNA mismatch repair was proposed to play a pivotal role in the development and prognosis of colorectal cancer. However, the prognostic value of mismatch repair on colorectal cancer is still unknown. The PubMed, EMBASE, and Cochrane Central Register of Controlled Trials databases were searched. The articles about mismatch repair (including hMLH1, hMSH2, hMSH3, hMSH6, hPMSH1, and hPMSH2) deficiency for the prognosis of patients with colorectal cancer were included in the study. The hazard ratio and its 95% confidence interval were used to measure the impact of mismatch repair deficiency on survival time. Twenty-one articles were included. The combined hazard ratio for mismatch repair deficiency on overall survival was 0.59 (95% confidence interval: 0.50-0.69) and that on disease-free survival was 0.57 (95% confidence interval: 0.43-0.75). In subgroup analysis, there were a significant association between overall survival and mismatch repair deficiency in Asian studies (hazard ratio: 0.67; 95% confidence interval: 0.50-0.91) and Western studies (hazard ratio: 0.56; 95% confidence interval: 0.46-0.67). For disease-free survival, the hazard ratios in Asian studies and Western studies were 0.55 (95% confidence interval: 0.38-0.81) and 0.62 (95% confidence interval: 0.50-0.78), respectively. Our meta-analysis indicated that mismatch repair could be used to evaluate the prognosis of patients with colorectal cancer.

Fusobacterium nucleatum Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations.

It has been recently reported that the population of Fusobacterium, particularly Fusobacterium nucleatum (Fn), is overrepresented in colorectal cancers and adenomas. The promoting effects of Fn infection on adenoma and/or carcinoma formation have been shown in ApcMin/+mice. Characteristics of Fn-associated CRC were identified through studies using human CRC cohorts, and include right-sided colon location, CpG island methylation phenotype-high (CIMP-H), high level of microsatellite instability (MSI-H), and poor patient prognosis. A subset of Fn-associated CRC exhibits a low level of microsatellite instability (MSI-L) and elevated microsatellite alterations in selected tetra-nucleotide repeats (EMAST) induced by translocation of MSH3 from the nucleus to the cytoplasm in response to oxidative DNA damage or inflammatory signals. The association between CIMP/MSI-H and Fn-infection can be explained by the role of the mismatch repair (MMR) protein complex formed between MSH2 and MSH6 (MutSα) to repair aberrant bases generated by ROS to form 7,8-dihydro-8-oxo-guanine (8-oxoG). Clustered 8-oxoGs formed at CpG-rich regions including promoters by ROS is refractory to base excision repair (BER). Under these conditions, MutSα initiates repair in cooperation with DNA methyltransferases (DNMTs) and the polycomb repressive complex 4 (PRC4). DNMTs at damaged sites methylate CpG islands to repress transcription of target genes and promote repair reactions. Thus, continuous generation of ROS through chronic Fn infection may initiate 1) CIMP-positive adenoma and carcinoma in an MSH2/MSH6-dependent manner, and/or 2) MSI-L/EMAST CRC in an MSH3-dependent manner. The poor prognosis of Fn-associated CRC can be explained by Fn-induced immune-evasion and/or chemo-resistance.

Comprehensive clinical genetics care for patients with inherited colorectal cancer associated with Lynch syndrome: Western and Asian perspectives.

Lynch syndrome (LS) arises from germline mutations that lead to defective functioning of the DNA mismatch repair (MMR) system. It is the most common cause of inherited colorectal cancer (CRC), and predisposes individuals to significantly elevated risks for extracolonic cancers. A sensitive and accurate diagnostic approach will enable proactive management of the proband and at-risk relatives in order to minimize their cancer burden. Comprehensive clinical genetics care should include personalized and tailored multidisciplinary oncologic care, with consideration for the extent of surgical resection, the choice of systemic agents, and the use of radiation. Preventive strategies including lifelong multi-organ surveillance, testing of family members, prophylactic-intent surgery and chemoprevention should be considered. In this review, we aim to provide an update on the diagnostic approach to LS, and to summarize key components of comprehensive clinical genetics care, through an examination of existing data and guidelines from Asian and from Western perspectives.

Programmed Death-Ligand 1 Expression, Microsatellite Instability, Epstein-Barr Virus, and Human Papillomavirus in Nasopharyngeal Carcinomas of Patients from the Philippines.

Most nasopharyngeal carcinomas (NPCs) in a high-incidence population are driven by Epstein-Barr virus (EBV) infection. EBV-associated malignancies have increased expression of the programmed death-ligand 1 (PD-L1). Immunotherapy agents targeting the PD-1/PD-L1 pathway have achieved durable treatment effects in patients with various cancer types including EBV-associated malignancies. In this study, we sought to investigate PD-L1 expression in a cohort of patients with NPCs from the Philippines. Fifty-six NPCs were studied for PD-L1, p16, and DNA mismatch repair (MMR) deficiency by immunohistochemistry. One case with MMR deficiency was also assessed for microsatellite instability (MSI) by polymerase chain reaction. EBV and human papillomavirus (HPV) status were tested by in situ hybridization. All NPCs were p16 negative. Three of the 56 NPCs (5%) were EBV negative (EBV-) and HPV negative, while one NPC (1/56, 2%) was EBV positive and showed MSI (EBV+/MSI). Positive PD-L1 expression (PD-L1+), defined as membranous staining in ≥1% tumor cells, was seen in 64% (36/56) of NPCs. All three EBV- NPCs were PD-L1+ as was the EBV+/MSI NPC. PD-L1+ was seen significantly more often in NPCs from non-smokers than those from smokers (23/28, 82% vs 9/18, 50%; P = 0.047). PD-L1+ was not associated with pT, pN, distant metastasis, or clinical stage (P > 0.05). PD-L1+ was not associated with overall survival (P = 0.473). In summary, our results show frequent PD-L1 expression in NPCs regardless of EBV status and a preferential PD-L1 expression in non-smokers. MSI and HPV positivity are exceedingly rare in NPCs.

MutS Homologues hMSH4 and hMSH5: Genetic Variations, Functions, and Implications in Human Diseases.

The prominence of the human mismatch repair (MMR) pathway is clearly reflected by the causal link between MMR gene mutations and the occurrence of Lynch syndrome (or HNPCC). The MMR family of proteins also carries out a plethora of diverse cellular functions beyond its primary role in MMR and homologous recombination. In fact, members of the MMR family of proteins are being increasingly recognized as critical mediators between DNA damage repair and cell survival. Thus, a better functional understanding of MMR proteins will undoubtedly aid the development of strategies to effectively enhance apoptotic signaling in response to DNA damage induced by anti-cancer therapeutics. Among the five known human MutS homologs, hMSH4 and hMSH5 form a unique heterocomplex. However, the expression profiles of the two genes are not correlated in a number of cell types, suggesting that they may function independently as well. Consistent with this, these two proteins are promiscuous and thought to play distinct roles through interacting with different binding partners. Here, we describe the gene and protein structures of eukaryotic MSH4 and MSH5 with a particular emphasis on their human homologues, and we discuss recent findings of the roles of these two genes in DNA damage response and repair. Finally, we delineate the potential links of single nucleotide polymorphism (SNP) loci of these two genes with several human diseases.

Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLß (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.