Intact DNA Mismatch Repair Research Articles

MHC class III lymphocyte antigens 6 as endogenous immunotoxins: Unlocking immunotherapy in proficient mismatch repair colorectal cancer.

A majority of cancers, including colorectal cancer (CRC) with intact DNA mismatch repair, exhibit a paralyzed antitumor immune response and resistance to immune checkpoint inhibitors. Members of MHC class III lymphocyte antigen 6G (LY6G) encode glycosylphosphatidylinositol (GPI) proteins anchored to the membrane. Snake venom neurotoxins and LY6G proteins share a three-finger (3F) folding domain. LY6 proteins such as LY6G6D are gaining a reputation as excellent tumor-associated antigens that can potently inhibit anti-tumor immunity in cancers with proficient mismatch repair. Thus, we called MHC class III LY6G endogenous immunotoxins. Since the discovery of LY6G6D as a tumor-associated antigen, T-cell engagers (TcEs) have been developed to simultaneously bind LY6G6D on cancer cells and CD3 on T cells, improving the treatment of metastatic solid tumors that are resistant to ICIs. We present a current understanding of how alterations in MHC class III genes inhibit antitumor immunity, and how these understandings can be turned into effective treatments for patients who are refractory to standard immunotherapy. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics Cancer > Molecular and Cellular Physiology.

Abstract 1568: Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma

Abstract The DNA damage repair protein O6-methylguanine methyl transferase (MGMT) reverses O6-alkylguanine lesions via SN2 transfer of the alkyl lesion to an active site cysteine which restores DNA to its native state. MGMT is ubiquitously expressed in healthy tissue but is silenced by promoter hypermethylation in approximately half of glioblastomas and up to 75% of lower grade gliomas. As such, patients with MGMT deficient (MGMT-) tumors benefit from DNA alkylation agents such as temozolomide (TMZ), an imidazotetrazine that deposits methyl lesions at O6-guanine. However, the cytotoxicity of TMZ requires an intact DNA mismatch repair (MMR) pathway and acquired resistance to TMZ via MMR silencing negates the efficacy of the drug. We recently reported the synthesis, discovery, and in vivo evaluation of KL-50, an imidazotetrazine derivative that overcomes this resistance mechanism while maintaining high selectivity for MGMT- cells in vitro and in vivo. Our preliminary mechanistic data suggest that KL-50 induces cell death independent of MMR status via the deposition of 2-fluoroethyl lesions at O6-guanine to give an O6-(2-fluoroethyl)guanine (O6FEtG) lesion that evolves to a deadly DNA interstrand cross-link (ICL). We hypothesize that this occurs via a two-step pathway involving slow cyclization of the initial O6FEtG lesion to an electrophilic N1,O6-ethanoguanine intermediate and subsequent nucleophilic ring-opening by the base-paired cytidine residue to yield a guanine(N1)-cytidine(N3) ethyl ICL. Our data suggest the high therapeutic index of KL-50 derives from the relative rates of MGMT reversal of O6FEtG and its conversion to an ICL, specifically that MGMT proficient healthy cells can repair the O6FEtG before it evolves to the ICL. However, direct MGMT reversal of O6FEtG has not been reported, and the rates of ICL formation are not known. Additionally, the formation of the putative ICL has not been demonstrated. Herein, we demonstrate that MGMT can repair the O6FEtG lesion. Kinetics studies using well-defined, photocaged oligonucleotides containing a single O6FEtG lesion show that ICL formation occurs on the order of hours. Furthermore, addition of MGMT following photo-deprotection prevents the formation of ICLs via rapid reversal of O6FEtG. Finally, we have detected a mass corresponding to the putative ICL in pUC19 DNA treated with KL-50 via LCMS . These findings bolster our mechanistic model and suggest strategies that exploit the relative rates of DNA damage and repair may be extensible to other tumor types harboring specific DNA repair defects. Citation Format: Eric D. Huseman, Anna Lo, Olga Fedorova, Kingson Lin, Susan Gueble, Ranjini Sundaram, Anna M. Pyle, Ranjit S. Bindra, Seth Herzon. Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1568.

The mutational landscape of upper gastrointestinal adenocarcinomas- A study of similarities and differences

PurposeThe gastrointestinal tract is home to a wide variety of neoplasms. Gastrointestinal adenocarcinomas display distinct prognostic patterns. With the advent of next generation sequencing, attempts are being made to delineate distinct molecular characteristics of these adenocarcinomas from adjoining anatomical sites. MethodsThirty-seven cases of upper gastrointestinal adenocarcinomas including those of the esophagus, gastroesophageal junction, stomach, small intestine and gallbladder were retrieved. Next generation sequencing data consisting of base substitutions, copy number variations, indels and rearrangements, in 324 genes, were analyzed for recurrent genetic abnormalities. Statistical analysis was performed using IBM SPSS25 and SAS software. ResultsGenetic alterations were found in 181 genes. APC mutations were found in 50% of the esophageal adenocarcinomas, 5% of the gastric adenocarcinomas and 33.3% of the small intestinal adenocarcinomas (p = 0.04). PIK3 gene family mutations were found in 10% of the gastric adenocarcinomas, 66% of the gall bladder adenocarcinomas and 66% of the small intestinal adenocarcinomas (p = 0.002). The mutations were found exclusively in the PIK3 class 1 family. TP53 mutations were more common in tumors with intact DNA mismatch repair protein expression as assessed by immunohistochemistry (p = 0.042). ConclusionIn this study, APC gene mutations were found to be more frequent in esophageal and small intestinal adenocarcinomas than previously reported. PIK3 class 1 gene family mutations were found to be more frequent in gallbladder and small intestinal adenocarcinomas. An inverse relationship was found between TP53 mutations and loss of DNA mismatch repair protein expression.

Clinicopathologic features of endometrial carcinomas from patients with Lynch syndrome, Lynch-like syndrome and intact DNA mismatch repair: A systematic review and meta-analysis

Clinicopathologic features of endometrial carcinomas from patients with Lynch syndrome, Lynch-like syndrome and intact DNA mismatch repair: A systematic review and meta-analysis

Abstract 1540: CCCTC-binding factor (CTCF) mutation in endometrioid endometrial carcinoma (EEC): Master epimutator contributing to biologic aggressiveness

Abstract Endometrial cancer is the most common gynecologic malignancy in the US. At present, therapies for recurrent or advanced stage disease are largely ineffective. Understanding the genetic and epigenetic factors that contribute to biologic aggressiveness holds promise for new treatment strategies. CTCF, a master regulator of gene expression, was identified as a significantly mutated gene in endometrial cancers by TCGA. Our group identified a hot spot mutation in CTCF in microsatellite instability positive (MSI) endometrial cancers not reported by TCGA. It is noteworthy that among cancer types analyzed to date, EEC is the only malignancy in which CTCF mutation is a frequent defect. CTCF has thousands of binding sites throughout the human genome and is capable of direct activation of genes, enhancer binding and far-reaching chromatin modification, and controls expression of numerous critical regulators of cellular growth/survival (e.g. c-myc, p53, p19/ARF, p16/INK4A, BRCA1, p27, E2F1). However, the role CTCF plays in EEC is largely unknown. Our analysis of a large cohort of EEC offers further insights into CTCF's role in EEC. Targeted ultra-deep sequencing of 540 primary ECC samples (966X mean coverage) identified 97 different variants (excluding SNPs, low quality variant calls, and synonymous variants) in 136 samples (25.2%). Mutations were more common in tumors with MSI than tumors with intact DNA mismatch repair (40% and 14% respectively p<.0001). Sixty-five of the 97 variants identified (67%) are novel and sequencing of matched tumor/normal DNA confirmed variants are somatic mutations. Most mutated samples (79%) harbored stop, frameshift or splice site variants. As we have shown that CTCF frameshift and stop mutations are subject to nonsense-mediated decay, these data strongly implicate CTCF as a haploinsufficient tumor suppressor. Mutation was not associated with age, stage, grade and disease-free or overall survival. However, among patients who recurred (n=67) the recurrence free interval (RFI) was shorter for mutation carriers (n=15) than for patients whose tumors had no mutation (8.5 months and 17.9 months respectively, p=0.02). Because we saw frequent loss of function mutations we undertook studies to determine if CTCF deletion is also a feature of EECs. Q-PCR for 149 samples revealed 8 cases with heterozygous CTCF deletion (5%), and in one tumor a 1741bp homozygous deletion (chr16: 67643579-67645330). Two patients with CTCF deletion recurred and both had short RFIs (<wild-type mean). We are currently validating CTCF deletion in 33 of the 540 samples analyzed by deep sequencing (6.1%) that had low normalized CTCF read depth (>1 SD below the mean). Investigation of the effects of CTCF mutation on gene expression in primary tumors is ongoing. Taken together, our analyses serve to contextualize the effects of CTCF mutations in EEC and implicate loss of CTCF in biologic aggressiveness. Citation Format: Christopher J. Walker, Mario A. Miranda, David G. Mutch, Paul Goodfellow. CCCTC-binding factor (CTCF) mutation in endometrioid endometrial carcinoma (EEC): Master epimutator contributing to biologic aggressiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2014-1540

Both hMutSα and hMutSß DNA mismatch repair complexes participate in 5-fluorouracil cytotoxicity.

BackgroundPatients with advanced microsatellite unstable colorectal cancers do not show a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. We and others have shown that the DNA mismatch repair (MMR) complex hMutSα binds 5-FU incorporated into DNA. Although hMutSß is known to interact with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen, it has not been demonstrated to interact with 5-FU incorporated into DNA. Our aim was to examine if hMutSß plays a role in 5-FU recognition.MethodsWe compared the normalized growth of 5-FU treated cells containing either or both mismatch repair complexes using MTT and clonogenic assays. We utilized oligonucleotides containing 5-FU and purified baculovirus-synthesized hMutSα and hMutSß in electromobility shift assays (EMSA) and further analyzed binding using surface plasmon resonance.ResultsMTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutSα and hMutSß, intermediate cytotoxicity in cells with hMutSα alone, and the least cytotoxicity in cells with hMutSß alone, hMutSß binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutSα.ConclusionCytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutSα and/or hMutSß 5-FU binding ability (hMutSα>hMutSß). The MMR complexes provide a hierarchical chemosensitivity for 5-FU cell death, and may have implications for treatment of patients with certain MMR-deficient tumors.

Abstract 904: Obesity is an independent prognostic variable in colon cancer survivors

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the prognosis of colon cancer survivors and its relationship to gender remains unknown. Experimental Design: BMI (kg/m2) was categorized in patients with TNM stage II and III colon carcinomas (n= 4,381) enrolled in 7 randomized trials of 5-fluorouracil-based adjuvant chemotherapy. Cox proportional hazards models were used to determine the association of BMI with disease-free (DFS) and overall survival (OS). Results: Among colon cancer patients, 868 (20%) were obese (BMI >30 kg/m2) of which 606 were class 1 (BMI 30-34 kg/m2) and 262 were class 2,3 (BMI >35 kg/m2). Obese versus normal-weight patients were more likely to be younger (p=0.038), have distal tumors (0.058), show intact DNA mismatch repair (MMR) (p=0.035), and have more lymph node metastases (p=0.017). In a multivariate analysis, BMI was significantly associated with both DFS (p=0.030) and OS (p=0.0017). Men with class 2,3 obesity showed reduced OS compared to normal weight men [HR 1.35 (1.02, 1.79); p=0.039]. Women with class I obesity had reduced OS [HR 1.24 (1.01, 1.53), p=0.045] compared to normal weight women. Overweight status was associated with improved OS in men (p= 0.006) and underweight women had significantly worse OS (p= 0.019). BMI was not predictive of therapeutic benefit. Conclusions: Obesity is an independent prognostic variable in colon cancer survivors and shows gender-related differences. These data suggest that obesity-related biological factors can influence clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 904.

Obesity Is an Independent Prognostic Variable in Colon Cancer Survivors

Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) on the prognosis of colon cancer survivors and its relationship to gender remains unknown. BMI (kg/m(2)) was categorized in patients with tumor-node-metastasis stage II and III colon carcinomas (n = 4,381) enrolled in seven randomized trials of 5-fluorouracil-based adjuvant chemotherapy. Cox proportional hazards models were used to determine the association of BMI with disease-free survival (DFS) and overall survival (OS). Among colon cancer patients, 868 (20%) were obese (BMI, >or=30 kg/m(2)), of which 606 were class 1 (BMI, 30-34 kg/m(2)) and 262 were class 2,3 (BMI, >or=35 kg/m(2)). Obese versus normal-weight patients were more likely to be younger, have distal tumors, show intact DNA mismatch repair, and have more lymph node metastases (P < 0.017). In a multivariate analysis, BMI was significantly associated with both DFS (P = 0.030) and OS (P = 0.0017). Men with class 2,3 obesity showed reduced OS compared with normal-weight men [hazard ratio, 1.35; 95% confidence interval, 1.02-1.79; P = 0.039]. Women with class I obesity had reduced OS [hazard ratio, 1.24; 95% confidence interval, 1.01-1.53; P = 0.045] compared with normal-weight women. Overweight status was associated with improved OS in men (P = 0.006), and underweight women had significantly worse OS (P = 0.019). BMI was not predictive of therapeutic benefit. Obesity is an independent prognostic variable in colon cancer survivors and shows gender-related differences. These data suggest that obesity-related biological factors can influence clinical outcome.

Abstract B111: Obesity is associated with worse outcome in colon cancer survivors

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the outcome of colon cancer survivors and its relationship to gender remains unknown. Experimental Design: BMI (kg/m2) was calculated and categorized in patients with TNM stage II and III colon carcinomas (n= 4,381) enrolled in 7 randomized trials of 5-fluorouracil-based adjuvant chemotherapy. Cox proportional hazards modeling was used to determine the association of BMI with disease-free and overall survival. Results: Among colon cancer patients, 868 (20%) were obese (BMI ≥30 kg/m2) of which 606 were class 1 (BMI 30–34 kg/m2) and 262 were class 2,3 (BMI ≥35 kg/m2). Obese versus normal-weight patients were more likely to be younger, have distal tumors, show intact DNA mismatch repair (MMR), and have increased lymph node metastases (p &amp;lt;0.017). The association of BMI and MMR status was dependent upon patient age (p= 0.031). In a multivariate analysis, BMI was significantly associated with both disease-free (p=0.030) and overall survival (p=0.0017). Men with class 2,3 obesity showed reduced overall survival compared to normal weight men [HR 1.35 (1.02, 1.79); p=0.039]. Women with class I obesity had reduced overall survival [HR 1.24 (1.01, 1.53), p=0.045] compared to normal weight women. BMI was not predictive of therapeutic benefit. Conclusions: Obesity is associated with intact DNA mismatch repair status and was an adverse, independent prognostic variable in colon cancer survivors. Interventions to reduce obesity have the potential to improve patient outcomes in colon cancer survivors. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B111.

Improved Survival With an Intact DNA Mismatch Repair System in Endometrial Cancer

To correlate survival and surgical-pathologic factors with DNA mismatch repair status in patients with endometrial cancer. Specimens from 336 patients with endometrial cancer were used to create a tissue microarray. Immunohistochemistry with antibodies against the mismatch repair genes MLH1, MSH2, MSH6, and PMS2 were used to stain the tissue microarray. Clinical, pathologic, and survival data were collected and correlated with the immunohistochemistry results. Mismatch repair deficiency was seen in 29% (84 of 294) of cases. Correlation was noted between lack of expression of MLH1 and an increased risk for lymphvascular space involvement (32% versus 21%, P=.05) and cervical involvement (26% versus 14%, P=.02). Lack of expression of either MLH1 or MSH2 was associated with thinner patients (85% had a body mass index less than 40 versus 73% of patients with normal expression, P=.02), as well as with the absence of a history of previous primary malignancy (0 verus 13 cases [4%], P=.023). The estimated disease-free survival is 88%; despite a small number of recurrences, there was a nonsignificant improvement in disease-free survival in tumors with an intact mismatch repair system (P=.1). Significantly improved disease-free survival was seen in patients with normal MLH1 and MSH2 expression compared with those with abnormal expression (92% versus 81%, P=.035). Defects in DNA mismatch repair in endometrial cancer is correlated with negative prognostic factors and worse progression-free survival (without a difference in overall survival) compared with tumors with an intact mismatch repair system. II-3.

Chemotherapeutic implications in microsatellite unstable colorectal cancer.

Chemotherapy for colorectal cancer is currently offered to patients based on the stage of their cancer, and there is evidence to show an overall survival benefit with 5-fluorouracil-based (5-FU) therapy for patients with lymph node metastasis who receive it. The pathogenesis of colorectal cancer involves genomic instability, with about 15% of tumors demonstrating a form of genomic instability called high-frequency microsatellite instability (MSI-H) and due to loss of DNA mismatch repair function, and the remainder of colorectal tumors lacking MSI-H with retained DNA mismatch repair function and called microsatellite stable (MSS), with a large proportion of these tumors demonstrating another form of genomic instability called chromosomal instability. There is now evidence to show that the form of genomic instability that is present in a patient's colorectal cancer may predict a survival benefit from 5-FU. In particular, patients whose colorectal tumors have MSI-H do not gain a survival benefit with 5-FU as compared to patients with MSS tumors. In vitro evidence supports these findings, as MSI-H colon cancer cell lines are more resistant to 5-FU compared to MSS cell lines. More specifically, components of the DNA mismatch repair system have been shown to recognize and bind to 5-FU that becomes incorporated into DNA and which could be a trigger to induce cell death. The binding and subsequent cell death events would be absent in colorectal tumors with MSI-H, which have lost intact DNA mismatch repair function. These findings suggest that: (a) tumor cytotoxicity of 5-FU is mediated by DNA mechanisms in addition to well-known RNA mechanisms, and (b) patients whose tumors demonstrate MSI-H may not benefit from 5-FU therapy. Future studies should include a better understanding of the cellular mechanisms of the DNA recognition of 5-FU, multi-centered prospective trials investigating the survival benefit of 5-FU based on genomic instability, and the investigation of alternative chemotherapeutic regimens for patients with MSI-H tumors to improve survival.

Evaluation of expression of DNA repair molecules as a prognostic factor for long-term survivors with glioblastoma multiforme (GBM) treated on RTOG protocols with irradiation and nitrosoureas

1509 Background: Adding BCNU to irradiation appears to modestly increase one and two year survival in GBM patients. BCNU attaches a chloroethyl adduct to the O6 position of guanine followed by formation of a DNA interstrand cross-link. These lesions may be repaired by alkyl-guanine transferase (AGT) and nucleotide excision repair (NER)/homologous recombinational repair (HRR) pathways, respectively. BCNU-initiated apoptosis signaling depends on intact DNA mismatch repair (MMR). Defects in MMR and intact NER/HRR and p53 expression may be linked to BCNU insensitivity. We hypothesized that these may also be prognostic factors in GBM. Methods: We compared the expression of AGT, ERCC1 (involved in the DNA nicking step of NER/HRR), the MMR proteins hMLH1 and hMSH2, and p53 between tumor samples, selected from the RTOG tissue bank, from 28 long term (>18 months) and 35 short term (< 6 months) GBM survivors receiving irradiation and nitrosoureas on RTOG protocols. Tumor cell nuclear expression was determined by immunohistochemistry using the Chromovision automated cell imaging system. Results: The two groups vary in age (p=0.003) and KPS (p=0.02) but not extent of resection (p=0.12). Conclusions: Within this patient population the levels of the DNA repair molecules examined do not predict for long term survivorship. No interaction between MMR and AGT was observed. Larger future studies should examine the expression of these and additional factors involved in the repair of nitrosourea induced DNA adducts. No significant financial relationships to disclose.

Mismatch repair proficiency and in vitro response to 5-fluorouracil

Background & Aims: The DNA mismatch repair (MMR) system recognizes certain DNA adducts caused by alkylation damage in addition to its role in recognizing and directing repair of interstrand nucleotide mismatches and slippage mistakes at microsatellite sequences. Because defects in the MMR system can confer tolerance to acquired DNA damage and, by inference, the toxic effects of certain chemotherapeutic agents, we investigated the effect of 5-fluorouracil (5-FU) on colon cancer cell lines. Methods: We determined growth selection by cell enrichment assay and cloning efficiency after treatment with 5 μmol/L 5-FU, assayed nucleic 3H–5-FU incorporation, and analyzed the cell cycle by flow cytometry. Results: 5-FU treatment provided a growth advantage for MMR-deficient cell lines, indicating a relative degree of tolerance to 5-FU by the MMR-deficient cell lines. Enhanced survival was statistically significant after 5 days of growth, and a 28-fold reduction in survival was noted in the MMR-proficient cells by clonagenic assays after 10 days of growth. Differences in nucleotide uptake of 5-FU did not account for the observed growth differences, and specific cell cycle checkpoint arrest was not detected. Conclusions: Intact DNA MMR seems to recognize 5-FU incorporated into DNA but may do so in a different manner than other types of alkylation damage. Defective DNA MMR might be one mechanism for tumor resistance to 5-FU. GASTROENTEROLOGY 1999;117:123-131