Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo.

The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

MSH3: a confirmed predisposing gene for adenomatous polyposis.

BACKGROUND: The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS: We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS: All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION: This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.

MSH3-related adenomatous polyposis in a patient with the negative family history of colorectal polyps.

Recently, biallelic MSH3 germline pathogenic/likely pathogenic variants have been recognized as a rare cause of adenomatous polyposis. We present a 49-year-old woman who was admitted to our high-risk colorectal cancer clinic after incidental detection of a biallelic MSH3 (likely) pathogenic variant when tested for the germline (likely) pathogenic variants in hereditary breast and ovarian cancer related genes. The focus of this case report is to describe the genotype and phenotype of our patient with MSH3-related adenomatous polyposis. More than half of the polyps (13/19) were located in the right colon. In addition, benign and malignant extraintestinal lesions may be common as our patient had simple liver and kidney cysts and two basal cell skin carcinomas.

Coordinated roles of SLX4 and MutSß in DNA repair and the maintenance of genome stability.

SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSß, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSß promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSß with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSß and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSß and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.

Prevalence of comprehensive DNA damage repair gene germline mutations in Chinese prostate cancer patients.

Germline DNA damage repair (DDR) deficiency has been associated with increased cancer risk, poor prognosis and therapeutic opportunity for prostate cancer (PCa) patients. However, the landscape of germline mutations in PCa covering comprehensive DDR genes has not been reported. We performed whole-exome sequencing in 246 patients who meet the National Cancer Center Network guidelines for genetic testing and analyzed variants in 276 DDR genes, which was from the Cancer Genome Atlas. A total of 79 deleterious germline alterations in 60 DDR genes were identified in 31% (76/246) patients. Mutations were found in nine DDR pathways, including 11.8% men in homologous recombination repair (HR) pathways, 2.4% men in mismatch repair (MMR) pathway and 16.7% (41/246) patients in non-HR/MMR pathways. In HRR and MMR pathways, mutations were mostly identified in BRCA2 (5.3%), HFM1 (0.8%), ZSWIM7 (0.8%), MSH2 (0.8%) and MSH3 (0.8%). When compared with the cancer-free cohort, POLN and POLG conferred high risk to PCa with odds ratio 6.9 and 20.5, respectively. We provided a comprehensive view of germline DDR gene mutations in PCa patients. We also identified two potential PCa predisposition genes: POLN and POLG, which have not been reported in the Western population, confirming the necessity of customizing a multigene panel for Chinese PCa patients.

EMAST status as a beneficial predictor of fluorouracil-based adjuvant chemotherapy for Stage II/III colorectal cancer.

BACKGROUND: Elevated microsatellite alteration at selected tetranucleotide repeats (EMAST) is a type of microsatellite instability that occurs in ∼60% of colorectal cancers (CRCs) and associated with MSH3 dysfunction. A 5-fluorouracil (5-FU)-related cytotoxicity is attenuated in MSH3-deficient colon cancer cells. Reported here is the predictive value of EMAST in CRCs with Stage II or III disease treated with 5-FU-based chemotherapy. METHODS: EMAST status was analyzed in 157 patients with CRC with Stage II or III disease and MSH3 expression was analyzed using immunohistochemistry. The patients treated with 5-FU-based chemotherapy were studied in terms of the links of EMAST status with MSH3 expression, clinicopathological features, and overall survival (OS). RESULTS: A total of 63 patients (40.1%) had EMAST positive (EMAST+ ) CRC and 77 patients (49.0%) had low MSH3 expression. EMAST+ tumors were associated with advanced TNM stage and poor and moderately differentiated tumor. EMAST CRC was more frequently observed in tumors with low expression of MSH3 in the nucleus (n = 53; 84.1%, p < .001). On multivariate analysis, patients with EMAST+ status had a worse OS (hazard ratio: 2.489, 95% confidence interval [1.149-5.394], and p = .021). Worse OS in EMAST+ patients who received 5-FU-based chemotherapy was significantly more common compared with EMAST- CRCs. CONCLUSION: There is a link between EMAST and reduced nuclear expression of MSH3. There is worse survival in patients with EMAST+ CRC after 5-FU-based chemotherapy. According to our findings, adjuvant 5-FU-based chemotherapy might not be advantageous in EMAST+ CRCs with Stage II or III disease.

Recurrent secondary genomic alterations in desmoplastic small round cell tumors.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare, highly aggressive, translocation-associated soft-tissue sarcoma that primarily affects children, adolescents, and young adults, with a striking male predominance. It is characterized by t(11;22) generating a novel EWSR1-WT1 fusion gene. Secondary genomic alterations are rarely described. METHODS: Tumor tissue from 83 DSRCT patients was assayed by hybrid-capture based comprehensive genomic profiling, FoundationOne® Heme next generation sequencing analysis of 406 genes and RNA sequencing of 265 genes. Tumor mutation burden was calculated from a minimum of 1.4 Mb sequenced DNA. Microsatellite instability status was determined by a novel algorithm analyzing 114 specific loci. RESULTS: Comprehensive genomic profiling identified several genomically-defined DSRCT subgroups. Recurrent genomic alterations were most frequently detected in FGFR4, ARID1A, TP53, MSH3, and MLL3 genes. With the exception of FGFR4, where the genomic alterations predicted activation, most of the alterations in the remaining genes predicted gene inactivation. No DSRCT were TMB or MSI high. CONCLUSIONS: In summary, recurrent secondary somatic alterations in FGFR4, ARID1A, TP53, MSH3, and MLL3 were detected in 82% of DSRCT, which is significantly greater than previously reported. These alterations may have both prognostic and therapeutic implications.

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Contribution to colonic polyposis of recently proposed predisposing genes and assessment of the prevalence of NTHL1- and MSH3-associated polyposes.

Technological advances have allowed the identification of new adenomatous and serrated polyposis genes, and of several candidate genes that require additional supporting evidence of causality. Through an exhaustive literature review and mutational screening of 177 unrelated polyposis patients, we assessed the involvement of MCM9, FOCAD, POLQ, and RNF43 in the predisposition to (nonserrated) colonic polyposis, as well as the prevalence of NTHL1 and MSH3 mutations among genetically unexplained polyposis patients. Our results, together with previously reported data and mutation frequency in controls, indicate that: MCM9 and POLQ mutations are not associated with polyposis; germline RNF43 mutations, with a prevalence of 1.5-2.5% among serrated polyposis patients, do not cause nonserrated polyposis; MSH3 biallelic mutations are highly infrequent among European polyposis patients, and the prevalence of NTHL1 biallelic mutations among unexplained polyposes is ~2%. Although nonsignificant, FOCAD predicted deleterious variants are overrepresented in polyposis patients compared to controls, warranting larger studies to provide definite evidence in favor or against their causal association with polyposis predisposition.

Environmentally relevant exposure to dibutyl phthalate disrupts DNA damage repair gene expression in the mouse ovary†.

Phthalates have a history of reproductive toxicity in animal models and associations with adverse reproductive outcomes in women. Human exposure to dibutyl phthalate (DBP) occurs via consumer products (7-10 µg/kg/day) and medications (1-233 µg/kg/day). Most DBP toxicity studies have focused on high supraphysiological exposure levels; thus, very little is known about exposures occurring at environmentally relevant levels. CD-1 female mice (80 days old) were treated with tocopherol-stripped corn oil (vehicle control) or DBP dissolved in oil at environmentally relevant (10 and 100 µg/kg/day) or higher (1000 µg/kg/day) levels for 30 days to evaluate effects on DNA damage response (DDR) pathway genes and folliculogenesis. DBP exposure caused dose-dependent effects on folliculogenesis and gene expression. Specifically, animals exposed to the high dose of DBP had more atretic follicles in their ovaries, while in those treated with environmentally relevant doses, follicle numbers were no different from vehicle-treated controls. DBP exposure significantly reduced the expression of DDR genes including those involved in homologous recombination (Atm, Brca1, Mre11a, Rad50), mismatch repair (Msh3, Msh6), and nucleotide excision repair (Xpc, Pcna) in a dose-specific manner. Interestingly, staining for the DNA damage marker, γH2AX, was similar between treatments. DBP exposure did not result in differential DNA methylation in the Brca1 promoter but significantly reduced transcript levels for the maintenance DNA methyltransferase, Dnmt1, in the ovary. Collectively, these findings show that oral exposure to environmentally relevant levels of DBP for 30 days does not significantly impact folliculogenesis in adult mice but leads to aberrant ovarian expression of DDR genes.

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.

MutSß abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions.

CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSß (Msh2-Msh3 heterodimer). To evaluate properties of MutSß that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSß, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSß. Msh3-/- cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3-/- cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSß on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSß complex, indicating that MutSß abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3-/- cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSß and rely heavily on Msh3 ATPase function.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

HEREDITARY, SPORADIC AND METASTATIC COLORECTAL CANCER ARE COMMONLY DRIVEN BY SPECIFIC SPECTRUMS OF DEFECTIVE DNA MISMATCH REPAIR COMPONENTS.

DNA mismatch repair (MMR) is one of several human cell mechanisms utilized to repair mutable mistakes within DNA, particularly after DNA is replicated. MMR function is dependent upon heterodimerization of specific MMR proteins that can recognize base-base mispairs as well as frameshifts at microsatellite sequences, followed by the triggering of other complementary proteins that execute excision and repair or initiate cell demise if repair is futile. MMR function is compromised in specific disease states, all of which can be biochemically recognized by faulty repair of microsatellite sequences, causing microsatellite instability. Germline mutation of an MMR gene causes Lynch syndrome, the most common inherited form of colorectal cancer (CRC), and biallelic germline mutations cause the rare constitutional mismatch repair deficiency syndrome. Somatic inactivation of MMR through epigenetic mechanisms is observed in 15% of sporadic CRC, and a smaller portion of CRCs possess biallelic somatic mutations. A novel inflammation-driven nuclear-to-cytoplasmic shift of the specific MMR protein hMSH3 is seen in up to 60% of sporadic CRCs that associates with metastasis and poor patient prognosis, unlike improved outcome when MMR is genetically inactivated. The mechanism for MMR inactication as well as the component affected dictates the clinical spectrum and clinical response for patients.

Homopolymer switches mediate adaptive mutability in mismatch repair-deficient colorectal cancer.

Mismatch repair (MMR)-deficient cancer evolves through the stepwise erosion of coding homopolymers in target genes. Curiously, the MMR genes MutS homolog 6 (MSH6) and MutS homolog 3 (MSH3) also contain coding homopolymers, and these are frequent mutational targets in MMR-deficient cancers. The impact of incremental MMR mutations on MMR-deficient cancer evolution is unknown. Here we show that microsatellite instability modulates DNA repair by toggling hypermutable mononucleotide homopolymer runs in MSH6 and MSH3 through stochastic frameshift switching. Spontaneous mutation and reversion modulate subclonal mutation rate, mutation bias and HLA and neoantigen diversity. Patient-derived organoids corroborate these observations and show that MMR homopolymer sequences drift back into reading frame in the absence of immune selection, suggesting a fitness cost of elevated mutation rates. Combined experimental and simulation studies demonstrate that subclonal immune selection favors incremental MMR mutations. Overall, our data demonstrate that MMR-deficient colorectal cancers fuel intratumor heterogeneity by adapting subclonal mutation rate and diversity to immune selection.

Mono- and Biallelic Replication-Coupled Gene Editing Discriminates Dominant-Negative and Loss-of-Function Variants of DNA Mismatch Repair Genes.

Replication-coupled gene editing using locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.

Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms.

BACKGROUND: Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. METHODS: From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. FINDINGS: In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. INTERPRETATION: This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. FUNDING: This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).