Mismatch Repair Components Research Articles

Transcriptomic Meta-Analysis Identifies Upregulated Clotting and Fibrinolysis Pathways in Colorectal Cancer Tumors Containing Hereditary PMS2 Mismatch Repair Deficiency.

Lynch Syndrome is characterized by deficient mismatch repair (dMMR) components. We performed a meta-analysis of multiple RNA-sequencing datasets from patients with different dMMR variants (PMS2, MLH1, and MSH2) to better characterize the unique transcriptional profiles. Our results reveal enriched signaling pathways from tumor samples with germline mutations in the PMS2 gene including upregulation in pathways related to intrinsic and extrinsic prothrombin activation, fibrinolysis, and uPA/uPAR-mediated signaling. These pathways have been associated with tumor growth, invasiveness, and metastasis. This work provides support for further exploration into the role of PMS2 in tumor development, and as a potential therapeutic mechanism.

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 invitro and invivo. 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.

PMON148 ACQUIRED TEMOZOLOMIDE RESISTANCE IN CORTICOTROPE PITUITARY TUMOR CELLS

Abstract Introduction Temozolomide (TMZ) treatment is an effective treatment for aggressive pituitary tumors, leading to tumor regression or stabilization of tumor progression in the majority of cases. In cases of disease recurrence, however, TMZ treatment does not control tumour regrowth. The expression of O6-methyl-guanine-DNA-methyltransferase (MGMT) protein and mismatch repair components (MMR) are implicated in the mechanism of TMZ resistance. In the present study, we aimed to develop an in vitro model of acquired TMZ resistance and to describe its mechanism. Methods The mouse AtT20 corticotrope pituitary tumor cell line was used. Two protocols were developed, one based on high dose of TMZ (high TMZ-challenge) and another one using lower TMZ concentrations (low TMZ-challenge) that are clinically achievable after a standard TMZ schedule. Both protocols consisted in pulse exposures to increasing concentrations of TMZ for 24rhs. TMZ concentrations in the high TMZ-challenge were 100, 200 and 300 µM while in the low TMZ-challenge were 20, 40, 60 and 80 µM. AtT20 cells treated with challenges of the vehicle dimethyl sulfoxide (vehicle-challenged cells) were used as control. Results The TMZ challenges induced a significant increase in EC50 (decreased sensitivity) of the growth inhibitory effect of TMZ in TMZ-challenged cells compared to vehicle-challenged cells in both protocols (high TMZ-challenge, EC50 = 123 µM vs 19 µM, p<0.001; low TMZ-challenge, EC50 = 69 µM vs 21 µM, p<0.001). The TMZ-challenged cells remained resistant compared to vehicle-challenged cells even after 9 weeks following the last TMZ challenge. The apoptotic cell death was higher in vehicle-challenged cells compared to TMZ-challenged cells in both protocols, albeit not statistically significant in the low TMZ-challenge protocol (high TMZ-challenge, p=0.003; low TMZ-challenge, p=0.169). TMZ-induced changes in cell cycle phases were more significant in vehicle-challenged cells compared to TMZ-challenged cells. Differences in cell morphology were observed between vehicle- and TMZ-challenged cells in the high dose protocol. In vehicle-challenged cells, small rounded and fusiform-shaped cells were observed, whereas in the TMZ-challenged cells only rounded cells were detected in culture. mRNA expression of MGMT was significantly higher in TMZ-challenged cells compared to vehicle-challenged cells. MMR components expression was not affected by TMZ challenges. Conclusion The present study describes an acquired TMZ-resistant corticotrope pituitary tumor cell model. TMZ resistance was demonstrated in both protocols by a sustained increase in TMZ EC50, small TMZ-induced cell cycle changes, and lower increase in TMZ-induced apoptosis. Additionally, TMZ challenges affected AtT20 cell morphology. This was evident only in the high TMZ-challenge protocol. Acquired TMZ resistance was associated with a strong increase in MGMT expression. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

Responses of dinoflagellate cells to ultraviolet-C irradiation.

Dinoflagellates are important aquatic microbes and major harmful algal bloom (HAB) agents that form invasive species through ship ballast transfer. UV-C installations are recommended for ballast treatments and HAB controls, but there is a lack of knowledge in dinoflagellate responses to UV-C. We report here dose-dependent cell cycle delay and viability loss of dinoflagellate cells irradiated with UV-C, with significant proliferative reduction at 800 Jm-2 doses or higher, but immediate LD50 was in the range of 2400-3200 Jm-2 . At higher dosages, some dinoflagellate cells surprisingly survived after days of recovery incubation, and continued viability loss, with samples exhibiting DNA fragmentations per proliferative resumption. Sequential cell cycle postponements, suggesting DNA damages were repaired over one cell cycle, were revealed with flow cytometric analysis and transcriptomic analysis. Over a sustained level of other DNA damage repair pathways, transcript elevation was observed only for several components of base pair repair and mismatch repair. Cumulatively, our findings demonstrated special DNA damage responses in dinoflagellate cells, which we discussed in relation to their unique chromo-genomic characters, as well as indicating resilience of dinoflagellate cells to UV-C.

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature

Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically. We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes. The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes. MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

Histone demethylase AMX-1 is necessary for proper sensitivity to interstrand crosslink DNA damage.

Histone methylation is dynamically regulated to shape the epigenome and adjust central nuclear processes including transcription, cell cycle control and DNA repair. Lysine-specific histone demethylase 2 (LSD2) has been implicated in multiple types of human cancers. However, its functions remain poorly understood. This study investigated the histone demethylase LSD2 homolog AMX-1 in C. elegans and uncovered a potential link between H3K4me2 modulation and DNA interstrand crosslink (ICL) repair. AMX-1 is a histone demethylase and mainly localizes to embryonic cells, the mitotic gut and sheath cells. Lack of AMX-1 expression resulted in embryonic lethality, a decreased brood size and disorganized premeiotic tip germline nuclei. Expression of AMX-1 and of the histone H3K4 demethylase SPR-5 is reciprocally up-regulated upon lack of each other and the mutants show increased H3K4me2 levels in the germline, indicating that AMX-1 and SPR-5 regulate H3K4me2 demethylation. Loss of AMX-1 function activates the CHK-1 kinase acting downstream of ATR and leads to the accumulation of RAD-51 foci and increased DNA damage-dependent apoptosis in the germline. AMX-1 is required for the proper expression of mismatch repair component MutL/MLH-1 and sensitivity against ICLs. Interestingly, formation of ICLs lead to ubiquitination-dependent subcellular relocalization of AMX-1. Taken together, our data suggest that AMX-1 functions in ICL repair in the germline.

Mismatch Repair: From Preserving Genome Stability to Enabling Mutation Studies in Real-Time Single Cells.

Mismatch Repair (MMR) is an important and conserved keeper of the maintenance of genetic information. Miroslav Radman’s contributions to the field of MMR are multiple and tremendous. One of the most notable was to provide, along with Bob Wagner and Matthew Meselson, the first direct evidence for the existence of the methyl-directed MMR. The purpose of this review is to outline several aspects and biological implications of MMR that his work has helped unveil, including the role of MMR during replication and recombination editing, and the current understanding of its mechanism. The review also summarizes recent discoveries related to the visualization of MMR components and discusses how it has helped shape our understanding of the coupling of mismatch recognition to replication. Finally, the author explains how visualization of MMR components has paved the way to the study of spontaneous mutations in living cells in real time.

Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2-M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo We propose this combinatorial treatment as a potential approach for patients with GBM.

Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus.

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.

Oxidative stress-induced DNA damage and repair in primary human osteoarthritis chondrocytes: focus on IKKα and the DNA Mismatch Repair System

During osteoarthritis development, chondrocytes are subjected to a functional derangement. This increases their susceptibility to stressful conditions such as oxidative stress, a characteristic of the aging tissue, which can further provoke extrinsic senescence by DNA damage responses. It was previously observed that IκB kinase α knockdown increases the replicative potential of primary human OA chondrocytes cultured in monolayer and the survival of the same cells undergoing hypertrophic-like differentiation in 3-D. In this paper we investigated whether IKKα knockdown could modulate oxidative stress-induced senescence of OA chondrocytes undergoing a DDR and particularly the involvement in this process of the DNA mismatch repair system, the principal mechanism for repair of replicative and recombinational errors, devoted to genomic stability maintenance in actively replicating cells. This repair system is also implicated in oxidative stress-mediated DNA damage repair. We analyzed microsatellite instability and expression of the mismatch repair components in human osteoarthritis chondrocytes after IKKα knockdown and H2O2 exposure. Only low MSI levels and incidence were detected and exclusively in IKKα proficient cells. Moreover, we found that IKKα proficient and deficient chondrocytes differently regulated MMR proteins after oxidative stress, both at mRNA and protein level, suggesting a reduced susceptibility of IKKα deficient cells. Our data suggest an involvement of the MMR system in the response to oxidative stress that tends to be more efficient in IKKαKD cells. This argues for a partial contribution of the MMR system to the better ability to recover DNA damage already observed in these cells.

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.

Irinotecan or Oxaliplatin: Which is the First Move for the Mate?

The aim of the present review is to discuss the potential link between RAS, BRAF and microsatellite instability (MSI) mutational patterns and chemotherapeutic agent efficacy [Irinotecan (IRI) vs. Oxaliplatin (OXA)], and how this can potentially influence the choice of the chemotherapy backbone. Following a review of the research literature, all pertinent articles published in the core journals were selected for the study. The inclusion criteria regarded relevant clinical and pre-clinical studies on the topic of interest (Relationship of OXA and IRI to KRAS/BRAF mutations and MSI). Excision repair cross complementation group 1 (ERCC1) expression is inhibited by KRAS mutation, making tumor cells more sensitive to OXA. Results from OPUS, COIN and PRIME trials support that no conclusive data are available for BRAF mutant population because of the small number of patients. Enhanced IRI cytotoxicity to MSI cell lines is due to the participation of some of the mismatch repair (MMR) components in various DNA repair processes and their role in the maintenance of the pro-apoptotic effect of IRI and G2/M cell arrest. OXA and IRI are indispensable drugs for mCRC treatment and their selection must be as careful as that of targeted agents. We suggest taking into consideration the interaction between known genomic alterations and OXA and IRI activity to personalize chemotherapy in mCRC patients.

Yeast mismatch repair components are required for stable inheritance of gene silencing.

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.

Trypanosoma brucei and Trypanosoma cruzi DNA Mismatch Repair Proteins Act Differently in the Response to DNA Damage Caused by Oxidative Stress.

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites' MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei.

Alterations in DNA damage repair (DDR) genes and outcomes to systemic therapy in 225 immune-oncology (IO) versus tyrosine kinase inhibitor (TKI) treated metastatic clear cell renal cell carcinoma (mccRCC) patients (pts).

551 Background: Loss of function alterations in DDR genes including core components of mismatch repair and homologous recombination deficiency pathways are associated with tumorigenesis and may determine benefit from IO therapies as shown in colon cancer. The significance for standard IO and TKI treatments in mccRCC is unknown. Methods: Genomic data and treatment outcomes were retrospectively collected for two large cohorts of mccRCC: pts treated with pure IO therapy (cohort 1); and pts receiving first-line TKI (cohort 2). Tumor and germline DNA was subject to targeted panel testing across >400 genes of interest, including 34 DDR machinery genes. Presence of truncating mutations, deletions and functionally validated missense mutations identified individual patient as ‘DDR altered’ (DDRa). Tumor mutational burden (TMB) was inferred for all pts. Non-parametric tests were applied to determine association between DDR status, TMB and treatment outcomes. Results: 225 pts were included (cohort 1=107, cohort 2=118), 37 (16%) were DDRa. Most commonly altered genes were ATM (n=8, 4%) and CHEK2 (n=8, 4%). DDR germline alterations were seen in 12 pts (5%). Median TMB was 4.1 per megabase (range: 0-21.7), and higher TMB (≥ median) was associated with being DDRa (Fisher’s exact, p=0.03). DDRa status correlated significantly with longer overall survival (OS) in the IO cohort (HR 0.29, logrank p=0.04) but not in the TKI cohort (HR 0.74, logrank p=0.44). We found no interaction between objective response and DDR status in either cohort. Conclusions: Loss of function in DDR genes was associated with superior OS in IO-treated but not in TKI-treated RCC pts. Possible underlying mechanisms beyond increase in TMB observed here deserve further study. [Table: see text]

Muts Homolog Sliding Clamps Shield the DNA from Binding Proteins

Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch. Specific or nonspecific binding of other proteins to the DNA between the mismatch and the distant excision-initiation site could conceivably obstruct the free diffusion of these MMR sliding clamps, inhibiting their ability to initiate repair. Here, we employed bulk biochemical analysis, single-molecule fluorescence imaging, and mathematical modeling to determine how sliding clamps might overcome such hindrances along the DNA. Using both bacterial and human MSH proteins, we found that increasing the number of MSH sliding clamps on a DNA decreased the association of the Escherichia coli transcriptional repressor LacI to its cognate promoter LacO. Our results suggest a simple mechanism whereby thermal diffusion of MSH sliding clamps along the DNA alters the association kinetics of other DNA-binding proteins over extended distances. These observations appear generally applicable to any stable sliding clamp that forms on DNA.

MutS homolog sliding clamps shield the DNA from binding proteins

Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch. Specific or nonspecific binding of other proteins to the DNA between the mismatch and the distant excision-initiation site could conceivably obstruct the free diffusion of these MMR sliding clamps, inhibiting their ability to initiate repair. Here, we employed bulk biochemical analysis, single-molecule fluorescence imaging, and mathematical modeling to determine how sliding clamps might overcome such hindrances along the DNA. Using both bacterial and human MSH proteins, we found that increasing the number of MSH sliding clamps on a DNA decreased the association of the Escherichia coli transcriptional repressor LacI to its cognate promoter LacO. Our results suggest a simple mechanism whereby thermal diffusion of MSH sliding clamps along the DNA alters the association kinetics of other DNA-binding proteins over extended distances. These observations appear generally applicable to any stable sliding clamp that forms on DNA.

Abstract 917: Defects in multiple DNA damage repair pathways render endocrine treatment resistance in ER+ breast cancer patients

Abstract Endocrine therapy resistance results in the death of majority of women diagnosed with breast cancer in the USA every year. Although a few markers can predict endocrine treatment failure, comprehensive understanding of the underlying cause and early detection of resistant population remains biggest challenge in the field. Recently, deficient expression or mutation in MutL mismatch repair components was found to cause 20% of intrinsic resistance in primary ER+ HER2- breast cancers. The role of other DNA damage repair (DDR) defects in inducing endocrine resistance therefore requires exploration. To address this question, a systematic study of associations between DDR dysregulation at DNA and RNA level and response to endocrine therapy was conducted using aggregated data from patient tumors from several neoadjuvant aromatase inhibitor (NeoAI) clinical trials. METABRIC, TCGA and Loi et al. datasets were used as validation datasets. Statistically significant correlations between endocrine therapy resistance and mutations in two single strand break repair (SSBR) pathways, i.e. nucleotide excision repair (NER; p=0.04) and base excision repair (BER; p=0.03), and one double strand break repair (DSBR) pathway, i.e. non-homologous end joining (NHEJ; p=0.004) were identified. A second set of analyses identified loss of expression of three genes: CETN2 and ERCC1 from the NER pathway and NEIL2 from BER as specifically associating with poor outcome despite endocrine treatment, emphasizing potential connections between NER and BER defects and endocrine treatment response. The functional role of CETN2, NEIL2 and ERCC1 loss in inducing intrinsic endocrine therapy resistance was experimentally validated in two breast cancer cell lines, and in ER+ PDX models. Finally, a DDR signature score was devised from baseline expression levels of candidate genes and was able to predict endocrine treatment failure in >30% of patients.In conclusion, this study describes a screening strategy for identifying novel predictive markers of endocrine therapy resistance in primary patient tumors. Three DDR pathways, NER, BER and NHEJ, were identified in clinical data as associating with endocrine resistance and validated using experimental model systems. Results reveal an understudied but impactful role for DDR pathways that may explain up to half of all endocrine therapy failure and open new avenues for early screening, predictive diagnostics and personalized medicine. Citation Format: Meenakshi Anurag, Nindo Punturi, Jeremy Hoog, Matthew N. Bainbridge, Matthew J. Ellis, Svasti Haricharan. Defects in multiple DNA damage repair pathways render endocrine treatment resistance in ER+ breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 917.