RESUMEN
Lynch syndrome (LS) is a prevalent genetic condition associated with colorectal cancer (CRC). Accurate identification of LS patients is challenging, and a universal tumor screening approach has been recommended. We present the methodology and results of universal LS screening in our hospital's Pathology Department. This retrospective study analyzed CRC tumors from a 5-year period (2017-2021). Immunohistochemistry was used to assess MMR protein expression, followed by BRAF V600E analysis and MLH1 promoter methylation. Statistical analysis examined associations between clinicopathologic variables MMR status and LS-suspected tumors. The study analyzed 939 colorectal carcinomas, with 8.7% exhibiting mismatch repair (MMR) deficiency, significantly lower than previous research. After applying the algorithm, 24 LS-suspected cases were identified, accounting for 2.6% of tested patients and 29.3% of MMR-deficient tumors. Our study establishes the feasibility of universal testing for all new cases of CRC in detecting individuals at risk for LS, even in the absence of clinical information. To gain a comprehensive understanding of the MMR status in our population, further investigations are warranted.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis , Neoplasias Colorrectales , Reparación de la Incompatibilidad de ADN , Homólogo 1 de la Proteína MutL , Humanos , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Femenino , Masculino , Persona de Mediana Edad , Portugal/epidemiología , Estudios Retrospectivos , Anciano , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Neoplasias Colorrectales/diagnóstico , Adulto , Proteínas Proto-Oncogénicas B-raf/genética , Metilación de ADN , Detección Precoz del Cáncer , InmunohistoquímicaRESUMEN
OBJECTIVE: Although early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor of prognosis and response to immune checkpoint inhibitor(s) in several cancer types, but its value in cervical cancer remains unclear. This study aimed to define the prevalence of MMR-D in cervical cancer and assess the prognostic value of MMR protein expression. METHODS: Expression of the MMR proteins MLH-1, PMS-2, MSH-2, and MSH-6 was investigated by immunohistochemical staining in a prospectively collected cervical cancer cohort (n=508) with corresponding clinicopathological and follow-up data. Sections were scored as either loss or intact expression to define MMR-D, and by a staining index, based on staining intensity and area, evaluating the prognostic potential. RNA and whole exome sequencing data were available for 72 and 75 of the patients and were used for gene set enrichment and mutational analyses, respectively. RESULTS: Five (1%) tumors were MMR-deficient, three of which were of neuroendocrine histology. MMR status did not predict survival (HR 1.93, p=0.17). MSH-2 low (n=48) was associated with poor survival (HR 1.94, p=0.02), also when adjusting for tumor stage, tumor type, and patient age (HR 2.06, p=0.013). MSH-2 low tumors had higher tumor mutational burden (p=0.003) and higher frequency of (frameshift) mutations in the double-strand break repair gene RAD50 (p<0.01). CONCLUSION: MMR-D is rare in cervical cancer, yet low MSH-2 expression is an independent predictor of poor survival.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN , Proteína 2 Homóloga a MutS , Neoplasias del Cuello Uterino , Humanos , Femenino , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/mortalidad , Pronóstico , Persona de Mediana Edad , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 2 Homóloga a MutS/biosíntesis , Proteína 2 Homóloga a MutS/genética , Adulto , Anciano , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/metabolismo , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/genética , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/biosíntesisRESUMEN
The incidence of colorectal cancer (CRC) is closely linked to metabolic diseases. Accumulating evidence suggests the regulatory role of AMP-activated protein kinase (AMPK) in cancer metabolic reprogramming. In this study, wild-type and AMPK knockout mice were subjected to azoxymethane-induced and dextran sulfate sodium (AOM/DSS)-promoted colitis-associated CRC induction. A stable AMPK-deficient Caco-2 cell line was also established for the mechanistic studies. The data showed that AMPK deficiency accelerated CRC development, characterized by increased tumor number, tumor size, and hyperplasia in AOM/DSS-treated mice. The aggravated colorectal tumorigenesis resulting from AMPK ablation was associated with reduced α-ketoglutarate production and ten-eleven translocation hydroxylase 2 (TET2) transcription, correlated with the reduced mismatch repair protein mutL homolog 1 (MLH1) protein. Furthermore, in AMPK-deficient Caco-2 cells, the mRNA expression of mismatch repair and tumor suppressor genes, intracellular α-ketoglutarate, and the protein level of TET2 were also downregulated. AMPK deficiency also increased hypermethylation in the CpG islands of Mlh1 in both colonic tissues and Caco-2 cells. In conclusion, AMPK deficiency leads to reduced α-ketoglutarate concentration and elevates the suppressive epigenetic modifications of tumor suppressor genes in gut epithelial cells, thereby increasing the risk of colorectal tumorigenesis. Given the modifiable nature of AMPK activity, it holds promise as a prospective molecular target for the prevention and treatment of CRC.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Azoximetano , Carcinogénesis , Neoplasias Colorrectales , Metilación de ADN , Dioxigenasas , Animales , Humanos , Ratones , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Azoximetano/toxicidad , Azoximetano/efectos adversos , Células CACO-2 , Carcinogénesis/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/etiología , Sulfato de Dextran/toxicidad , Dioxigenasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Ácidos Cetoglutáricos/metabolismo , Ratones Noqueados , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
MLH1 plays a critical role in DNA mismatch repair and genome maintenance. MLH1 deficiency promotes cancer development and progression, but the mechanism underlying MLH1 regulation remains enigmatic. In this study, we demonstrated that MLH1 protein is degraded by the ubiquitin-proteasome system and have identified vital cis-elements and trans-factors involved in MLH1 turnover. We found that the region encompassing the amino acids 516 to 650 is crucial for MLH1 degradation. The mismatch repair protein PMS2 may shield MLH1 from degradation as it binds to the MLH1 segment key to its turnover. Furthermore, we have identified the E3 ubiquitin ligase UBR4 and the deubiquitylase USP5, which oppositely modulate MLH1 stability. In consistence, UBR4 or USP5 deficiency affects the cellular response to nucleotide analog 6-TG, supporting their roles in regulating mismatch repair. Our study has revealed important insights into the regulatory mechanisms underlying MLH1 proteolysis, critical to DNA mismatch repair related diseases.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Homólogo 1 de la Proteína MutL , Proteolisis , Ubiquitina-Proteína Ligasas , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Estabilidad Proteica , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/metabolismo , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/genética , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Células HEK293RESUMEN
Objective: To investigate the clinicopathological characteristics and prognostic factors of sporadic mismatch repair deficient (dMMR) colorectal cancer. Methods: A total of 120 cases of sporadic dMMR colorectal cancer from July 2015 to April 2021 were retrospectively collected in Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Patients with Lynch syndrome; synchronous multiple colorectal cancers; preoperative anti-tumor treatments such as chemotherapy and radiotherapy; and those with incomplete follow-up information were excluded based on family history and next-generation sequencing (NGS) test results. Immunohistochemical stains were used to detect the expression of mismatch repair proteins, methylation-specific PCR for methylation testing, and fluorescent PCR for BRAF V600E gene mutation detection. The clinical and pathological data, and gene mutation status were analyzed. Follow-up was done to assess survival and prognosis including progression-free survival and overall survival rate. Results: Sporadic dMMR colorectal cancer occurred more frequently in the right side of the colon, in females, and in the elderly. Morphologically, it was mostly moderately-differentiated, and most patients had low-grade tumor budding. In terms of immunohistochemical expression, MLH1 and PMS2 loss were dominant, and there were age and location-specificities in protein expression. MLH1 methylation was commonly detected in elderly female patients and rare in young male patients; while MLH1 and PMS2 deficiency, and BRAF V600E mutation occurred more often on the right side (P<0.05). The 3-year and 5-year progression-free survival rates were 90.7% and 88.7% respectively, and the 3-year and 5-year overall survival rates were 92.8% and 90.7% respectively. Tumor budding status was an independent risk factor affecting patient recurrence (hazard ratio=3.375, 95% confidence interval: 1.060-10.741, P=0.039), patients with low-grade tumor budding had better prognosis, and those with medium or high-grade tumor budding had poor prognosis. Conclusion: For dMMR colorectal cancer patients, tumor budding status is an independent risk factor for recurrence.
Asunto(s)
Neoplasias Colorrectales , Reparación de la Incompatibilidad de ADN , Proteínas Proto-Oncogénicas B-raf , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Masculino , Femenino , Pronóstico , Estudios Retrospectivos , Proteínas Proto-Oncogénicas B-raf/genética , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Mutación , Tasa de Supervivencia , Persona de Mediana Edad , Anciano , Metilación de ADN , Adulto , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismoRESUMEN
BACKGROUND: Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients until immunotherapy was introduced. Studies indicate a noteworthy prevalence (0.2% to 18.6%) of mismatch repair protein (MMRP) deficiency in TNBC, with recent research highlighting the potential of immunotherapy for MMRP-deficient metastatic breast cancer. This study aims to identify MMRP deficiency in TNBC patients using immunohistochemistry. METHODS: A retrospective cohort study design was used and included TNBC patients treated between 2015 and 2021 at King Hussein Cancer Center. Immunohistochemistry was conducted to assess MMRP expression. RESULTS: Among 152 patients, 14 (9.2%) exhibited deficient MMR (dMMR). Loss of PMS2 expression was observed in 13 patients, 5 of whom showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient. The median follow-up duration was 44 (3-102) months. Despite the higher survival rate (80.8%, 5 years) of dMMR patients than of proficient MMR patients (62.3%), overall survival did not significantly differ between the two groups. CONCLUSION: Approximately 9% of TNBC patients exhibit dMMR. dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Persona de Mediana Edad , Adulto , Estudios Retrospectivos , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/metabolismo , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/genética , Anciano , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Proteína 2 Homóloga a MutS/metabolismo , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Tasa de Supervivencia , Inmunohistoquímica , Anciano de 80 o más Años , PronósticoRESUMEN
Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.
Asunto(s)
Butadienos , Neoplasias Colorrectales , Fluorouracilo , Interleucina-8 , Nitrilos , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Interleucina-8/metabolismo , Interleucina-8/genética , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Butadienos/farmacología , Nitrilos/farmacología , Línea Celular Tumoral , Compuestos Organoplatinos/farmacología , Compuestos Organoplatinos/uso terapéutico , Leucovorina/uso terapéutico , Leucovorina/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Femenino , Masculino , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HT29 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Persona de Mediana Edad , Anciano , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fosforilación/efectos de los fármacosRESUMEN
BACKGROUND AND AIM: Colorectal cancer (CRC) originates from pre-existing polyps in the colon. The development of different subtypes of CRC is influenced by various genetic and epigenetic characteristics. CpG island methylator phenotype (CIMP) is found in about 15-20% of sporadic CRCs and is associated with hypermethylation of certain gene promoters. This study aims to find prognostic genes and compare their expression and methylation status as potential biomarkers in patients with serrated sessile adenomas/polyps (SSAP) and CRC, in order to evaluate which, one is a better predictor of disease. METHOD: This study employed a multi-phase approach to investigate genes associated with CRC and SSAP. Initially, two gene expression datasets were analyzed using R and Limma package to identify differentially expressed genes (DEGs). Venn diagram analysis further refined the selection, revealing four genes from the Weissenberg panel with significant changes. These genes, underwent thorough in silico evaluations. Once confirmed, they proceeded to wet lab experimentation, focusing on expression and methylation status. This comprehensive methodology ensured a robust examination of the genes involved in CRC and SSAP. RESULT: This study identified cancer-specific genes, with 8,351 and 1,769 genes specifically down-regulated in SSAP and CRC tissues, respectively. The down-regulated genes were associated with cell adhesion, negative regulation of cell proliferation, and drug response. Four highly downregulated genes in the Weissenberg panel, including CACNA1G, IGF2, MLH1, and SOCS1. In vitro analysis showed that they are hypermethylated in both SSAP and CRC samples while their expressions decreased only in CRC samples. CONCLUSION: This suggests that the decrease in gene expression could help determine whether a polyp will become cancerous. Using both methylation status and gene expression status of genes in the Weissenberg panel in prognostic tests may lead to better prognoses for patients.
Asunto(s)
Neoplasias Colorrectales , Islas de CpG , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina , Homólogo 1 de la Proteína MutL , Proteína 1 Supresora de la Señalización de Citocinas , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Metilación de ADN/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Islas de CpG/genética , Femenino , Pólipos del Colon/genética , Pólipos del Colon/metabolismo , Pólipos del Colon/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Masculino , Regulación hacia Abajo/genética , Simulación por Computador , Persona de Mediana Edad , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Regiones Promotoras Genéticas/genética , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Perfilación de la Expresión Génica/métodos , Anciano , PronósticoRESUMEN
The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1koALKBH3ko cells showed enhanced resistance toward SN1- and SN2-type methylating agents, whereas MLH1koALKBH2ko cells were only resistant to SN1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH2ko3ko) rendered cells sensitive to SN1- and SN2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH2ko3ko cells have an ATR-dependent arrest at the G2/M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1koALKBH2ko3ko cells. Finally, in MLH1koALKBH2ko3ko cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Reparación de la Incompatibilidad de ADN , Homólogo 1 de la Proteína MutL , Humanos , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , DesmetilaciónRESUMEN
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.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN , Homólogo 1 de la Proteína MutL , Unión Proteica , Humanos , Proteínas de Unión al ADN/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Unión Proteica/efectos de los fármacos , Línea Celular Tumoral , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Neuronas/metabolismo , Persona de Mediana Edad , MasculinoRESUMEN
Prime editing (PE), a recent progression in CRISPR-based technologies, holds promise for precise genome editing without the risks associated with double-strand breaks. It can introduce a wide range of changes, including single-nucleotide variants, insertions, and small deletions. Despite these advancements, there is a need for further optimization to overcome certain limitations to increase efficiency. One such approach to enhance PE efficiency involves the inhibition of the DNA mismatch repair (MMR) system, specifically MLH1. The rationale behind this approach lies in the MMR system's role in correcting mismatched nucleotides during DNA replication. Inhibiting this repair pathway creates a window of opportunity for the PE machinery to incorporate the desired edits before permanent DNA repair actions. However, as the MMR system plays a crucial role in various cellular processes, it is important to consider the potential risks associated with manipulating this system. The new versions of PE with enhanced efficiency while blocking MLH1 are called PE4 and PE5. Here, we explore the potential risks associated with manipulating the MMR system. We pay special attention to the possible implications for human health, particularly the development of cancer.
Asunto(s)
Sistemas CRISPR-Cas , Reparación de la Incompatibilidad de ADN , Edición Génica , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Reparación del ADN , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , AnimalesRESUMEN
Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
Asunto(s)
Sistemas CRISPR-Cas , Daño del ADN , Reparación del ADN , Edición Génica , Proteína p53 Supresora de Tumor , Edición Génica/métodos , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Línea Celular , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Citosina/metabolismoRESUMEN
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.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteína Huntingtina , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Expansión de Repetición de Trinucleótido , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Reparación de la Incompatibilidad de ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Expansión de Repetición de Trinucleótido/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Genes Modificadores , Proteína 3 Homóloga de MutS/genética , Proteína 3 Homóloga de MutS/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Sistemas CRISPR-Cas , Estudio de Asociación del Genoma CompletoRESUMEN
BACKGROUND: Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. METHODS: MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. RESULTS: We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. CONCLUSIONS: dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.
Asunto(s)
Neoplasias Colorrectales , Células Asesinas Naturales , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Ratones , Humanos , Línea Celular Tumoral , Reparación de la Incompatibilidad de ADN , Citotoxicidad Inmunológica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/deficiencia , Homólogo 1 de la Proteína MutL/metabolismo , Ratones Endogámicos BALB C , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/inmunología , Síndromes Neoplásicos Hereditarios/patología , Neoplasias EncefálicasRESUMEN
The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's yeast approximately 80% of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. To our surprise we identified genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented.
Asunto(s)
Proteínas de Ciclo Celular , Intercambio Genético , Meiosis , Homólogo 1 de la Proteína MutL , Proteínas MutL , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas MutL/metabolismo , Proteínas MutL/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Unión ProteicaRESUMEN
BACKGROUND: Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. METHODS: Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. RESULTS: Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. CONCLUSION: This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.
Asunto(s)
Fragmentación del ADN , Infertilidad Masculina , MicroARNs , Homólogo 1 de la Proteína MutL , Estrés Oxidativo , Espermatozoides , Varicocele , Humanos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Varicocele/genética , Varicocele/metabolismo , Varicocele/patología , Estrés Oxidativo/genética , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Espermatozoides/metabolismo , Adulto , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Semen/metabolismo , Motilidad Espermática/genética , Antioxidantes/metabolismoRESUMEN
While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.
Asunto(s)
Neoplasias de la Mama , Homólogo 1 de la Proteína MutL , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Inestabilidad Genómica , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismoRESUMEN
MutL homolog 1 (MLH1), a member of the MutL homolog family, is required for normal recombination in most organisms. However, its role in soybean (Glycine max) remains unclear to date. Here, we characterized the Glycine max female and male sterility 1 (Gmfms1) mutation that reduces pollen grain viability and increases embryo sac abortion in soybean. Map-based cloning revealed that the causal gene of Gmfms1 is Glycine max MutL homolog 1 (GmMLH1), and CRISPR/Cas9 knockout approach further validated that disruption of GmMLH1 confers the female-male sterility phenotype in soybean. Loss of GmMLH1 function disrupted bivalent formation, leading to univalent mis-segregation during meiosis and ultimately to female-male sterility. The Gmmlh1 mutant showed about a 78.16% decrease in meiotic crossover frequency compared to the wild type. The residual chiasmata followed a Poisson distribution, suggesting that interference-sensitive crossover formation was affected in the Gmmlh1 mutant. Furthermore, GmMLH1 could interact with GmMLH3A and GmMLH3B both in vivo and in vitro. Overall, our work demonstrates that GmMLH1 participates in interference-sensitive crossover formation in soybean, and provides additional information about the conserved functions of MLH1 across plant species.
Asunto(s)
Intercambio Genético , Glycine max , Meiosis , Proteínas de Plantas , Glycine max/genética , Glycine max/metabolismo , Meiosis/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Infertilidad Vegetal/genética , Mutación/genética , Polen/genética , Polen/crecimiento & desarrolloRESUMEN
Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post-translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2-mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ-resistant cells, and is mainly concentrated in histone H3K9. Combined multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti-epileptic drug, stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment.