Germline mutations of breast cancer susceptibility genes through expanded genetic analysis in unselected Colombian patients.

BACKGROUND: In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted. RESULTS: We ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure. CONCLUSION: This study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.

Fast screening using attenuated total reflectance- fourier transform infrared (ATR-FTIR) spectroscopy of patients based on D-dimer threshold value.

Attenuated Total Reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy is an emerging technology in the medical field. Blood D-dimer was initially studied as a marker of the activation of coagulation and fibrinolysis. It is mainly used as a potential diagnosis screening test for pulmonary embolism or deep vein thrombosis but was recently associated with COVID-19 severity. This study aimed to evaluate the use of ATR-FTIR spectroscopy with machine learning to classify plasma D-dimer concentrations. The plasma ATR-FTIR spectra from 100 patients were studied through principal component analysis (PCA) and two supervised approaches: genetic algorithm with linear discriminant analysis (GA-LDA) and partial least squares with linear discriminant (PLS-DA). The spectra were truncated to the fingerprint region (1800-1000 cm-1). The GA-LDA method effectively classified patients according to D-dimer cutoff (≤0.5 µg/mL and >0.5 µg/mL) with 87.5 % specificity and 100 % sensitivity on the training set, and 85.7 % specificity, and 95.6 % sensitivity on the test set. Thus, we demonstrate that ATR-FTIR spectroscopy might be an important additional tool for classifying patients according to D-dimer values. ATR-FTIR spectral analyses associated with clinical evidence can contribute to a faster and more accurate medical diagnosis, reduce patient morbidity, and save resources and demand for professionals.

Somatic and germline ATM variants in non-small-cell lung cancer: Therapeutic implications.

ATM is an apical kinase of the DNA damage response involved in the repair of DNA double-strand breaks. Germline ATM variants (gATM) have been associated with an increased risk of developing lung adenocarcinoma (LUAD), and approximately 9% of LUAD tumors harbor somatic ATM mutations (sATM). Biallelic carriers of pathogenic gATM exhibit a plethora of immunological abnormalities, but few studies have evaluated the contribution of immune dysfunction to lung cancer susceptibility. Indeed, little is known about the clinicopathological characteristics of lung cancer patients with sATM or gATM alterations. The introduction of targeted therapies and immunotherapies, and the increasing number of clinical trials evaluating treatment combinations, warrants a careful reexamination of the benefits and harms that different therapeutic approaches have had in lung cancer patients with sATM or gATM. This review will discuss the role of ATM in the pathogenesis of lung cancer, highlighting potential therapeutic approaches to manage ATM-deficient lung cancers.

ATM and ATR, two central players of the DNA damage response, are involved in the induction of systemic acquired resistance by extracellular DNA, but not the plant wound response.

Background: The plant immune response to DNA is highly self/nonself-specific. Self-DNA triggered stronger responses by early immune signals such as H2O2 formation than nonself-DNA from closely related plant species. Plants lack known DNA receptors. Therefore, we aimed to investigate whether a differential sensing of self-versus nonself DNA fragments as damage- versus pathogen-associated molecular patterns (DAMPs/PAMPs) or an activation of the DNA-damage response (DDR) represents the more promising framework to understand this phenomenon. Results: We treated Arabidopsis thaliana Col-0 plants with sonicated self-DNA from other individuals of the same ecotype, nonself-DNA from another A. thaliana ecotype, or nonself-DNA from broccoli. We observed a highly self/nonself-DNA-specific induction of H2O2 formation and of jasmonic acid (JA, the hormone controlling the wound response to chewing herbivores) and salicylic acid (SA, the hormone controlling systemic acquired resistance, SAR, to biotrophic pathogens). Mutant lines lacking Ataxia Telangiectasia Mutated (ATM) or ATM AND RAD3-RELATED (ATR) - the two DDR master kinases - retained the differential induction of JA in response to DNA treatments but completely failed to induce H2O2 or SA. Moreover, we observed H2O2 formation in response to in situ-damaged self-DNA from plants that had been treated with bleomycin or SA or infected with virulent bacteria Pseudomonas syringae pv. tomato DC3000 or pv. glycinea carrying effector avrRpt2, but not to DNA from H2O2-treated plants or challenged with non-virulent P. syringae pv. glycinea lacking avrRpt2. Conclusion: We conclude that both ATM and ATR are required for the complete activation of the plant immune response to extracellular DNA whereas an as-yet unknown mechanism allows for the self/nonself-differential activation of the JA-dependent wound response.

Adagrasib (Krasati®) en cáncer colorectal metastásico / Adagrasib (Krasati®) in colorectal cancer metastatic

INTRODUÇÃO: El cáncer colorrectal (CCR) representa la segunda causa de mortalidad por tumores en la Argentina.1 Según la Agencia Internacional de Investigación sobre el Cáncer la incidencia en nuestro país durante el año 2020 se calculó en aproximadamente 15.600 casos, registrándose en el mismo período de tiempo unas 8.600 muertes. Aproximadamente el 20% de los individuos se presentan con metástasis a distancia, mientras que otros pacientes diagnosticados en estadios iniciales progresarán en el transcurso de la enfermedad requiriendo quimioterapia sistémica. Sin tratamiento, el promedio de sobrevida de estos pacientes es de unos 6 meses. Los esquemas de quimioterapia utilizados frecuentemente están basados en la combinación de fluoropirimidinas con oxaliplatino o irinotecan asociados con anticuerpos monoclonales contra el factor de crecimiento endotelial vascular (bevacizumab) o el receptor del factor de crecimiento epidérmico (cetuximab, panitunumab) en tumores sin mutaciones en la vía RAS (wildtipe o "salvajes"). En aquellos tumores con deficiencia del sistema de reparación de apareamientos erróneos o alta inestabilidad de microsatélites (por sus siglas en inglés, dMMR o MSI-H, respectivamente) se listan dentro de las opciones terapéuticas el uso de inmunoterapia. En aquellos pacientes en los cuales la enfermedad progresa a pesar de los esquemas mencionados se pueden utilizar regorafenib o trifluridina/tipiracilo y en ciertos subtipos moleculares encorafenib (mutación V600E en el gen BRAF); trastuzumab +/-pertuzumab/lapatinib/tucatinib (HER2 amplificado y sin mutaciones en los genes RAS y BRAF), entre otras. Se debe tener en cuenta que alguna de estas opciones no cuentan con aprobación o no se encuentran disponibles aún en nuestro país. En este documento se plantea evaluar la eficacia y seguridad del uso de adagrasib en pacientes con carcinoma colorectal metastásico portadores de la mutación G12C en el gen KRAS. TECNOLOGIA: Adagrasib (Krasati®) es un inhibidor irreversible y selectivo de la proteína mutante KRAS G12C (homólogo del oncogén vírico de sarcoma de rata Kirsten). La proteína pertenece a la subfamilia de proteínas RAS (KRAS, HRAS y NRAS) que actúan como GTPasas y se desempeñan como reguladores moleculares, controlando un amplio espectro de actividades celulares, como la proliferación y la sobrevida celular. Su inactivación por parte de adagrasib bloquea la transmisión de señales inhibiendo el crecimiento celular y favoreciendo la apoptosis de manera selectiva en tumores portadores de la mutación KRAS G12C. OBJETIVO: El objetivo del presente informe es evaluar rápidamente los parámetros de eficacia, seguridad, costos y recomendaciones disponibles acerca del empleo de adagrasib en pacientes con diagnóstico de carcinoma colorectal metastásico. MÉTODOS: Se realizó una búsqueda bibliográfica en las principales bases de datos tales como PUBMED, LILACS, BRISA, COCHRANE, SCIELO, EMBASE, TRIPDATABASE como así también en sociedades científicas, agencias reguladoras, financiadores de salud y agencias de evaluación de tecnologías sanitarias. Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, evaluación de tecnología sanitaria y guías de práctica clínica de alta calidad metodológica. La fecha de búsqueda de información fue hasta el 19 de mayo de 2023. Para la búsqueda en Pubmed se utilizó la siguiente estrategia de búsqueda: (adagrasib [Supplementary Concept] OR adagrasib [tiab] OR MRTX849 [tiab]) AND ("Colonic Neoplasms"[Mesh] OR ¨Colorectal Cancer¨ [tiab]). EVIDENCIA CLÍNICA: La evaluación de eficacia y seguridad del uso de adagrasib provienen de un ensayo clínico de fase I-II (tipo básquet) no aleatorizado, abierto y multicéntrico cuyo objetivo fue evaluar el uso de adagrasib en pacientes con neoplasias avanzadas portadoras de la mutación G12C en el gen KRAS (estudio denominado KRYSTAL-1, NCT03785249). Los resultados sobre las cohortes de pacientes con carcinoma colorectal avanzado fue publicada en el año 2023 por Yaeger y col.7 Los pacientes enrolados debían tener el diagnóstico de carcinoma colorectal metastásico progresado a múltiples líneas de tratamiento y tener documentada la mutación G12C del gen KRAS. Los pacientes enrolados recibieron 600 mg de adagrasib por vía oral dos veces al día, solo o combinado con cetuximab en pacientes, de manera continua hasta la progresión de la enfermedad, toxicidad inaceptable o retirada del consentimiento. El objetivo principal del estudio fue la tasa de respuesta objetiva, mientras que la sobrevida global y libre de progresión se encontraban entre los secundarios. Un total de 76 pacientes (mediana de edad 59-60 años) fueron enrolados y recibieron adagrasib monoterapia (n:44) o combinado con cetuximab (n:32). RECOMENDACIONES: Las guías de la Red Nacional de Centros para el Tratamiento Integral del cáncer de los Estados Unidos (NCCN, su sigla del inglés National Comprehensive Cáncer Network), la Sociedad Europea de Oncología Médica (ESMO, su sigla del inglés European Society for Medical Oncology) no mencionan el uso de adagrasib solo o combinado con cetuximab dentro de las opciones terapéuticas para el tratamiento del cáncer colorectal avanzado. En Argentina, la Asociación Argentina de Oncología Clínica tampoco menciona su utilización. CONCLUSIONES: La evidencia sobre el uso de adagrasib para el tratamiento de pacientes con diagnóstico de carcinoma colorectal metastásico progresados a múltiples líneas de tratamiento se basa en un único ensayo clínico no aleatorizado de fase I-II. Este estudio mostró que aquellos pacientes, progresados generalmente a tres líneas de tratamiento y con tumores que presentan mutaciones G12C en el gen KRAS, que utilizaron adagrasib con cetuximab reportaron una mediana de sobrevida global de 13,4 meses y libre de progresión de 6,9 meses, y una tasa de respuesta del 46%. Los eventos adversos severos fueron reportados en el 16% de los pacientes. Actualmente se encuentra en curso un ensayo clínico aleatorizado de fase III que cuenta con centros en Argentina y tratará de establecer la seguridad y eficacia de la adición de adagrasib a un esquema de poliquimioterapia como segunda línea de tratamento. La Administración de Alimentos y Medicamentos de Estados Unidos y la Agencia Europea de Medicamentos aún no ha autorizado la comercialización del medicamento en la indicación antes mencionada. En referencias a las recomendaciones relevadas, no se hallaron guías que mencionen los esquemas de adagrasib combinado con cetuximab o en monoterapia como una opción para el tratamiento del carcinoma colorectal avanzado. Utilizando precios de referencia internacionales, el costo de adquisición para un ciclo de tratamiento fue estimado en aproximadamente 4,8 millones de pesos argentinos.

Discrimination of dyslipidemia types with ATR-FTIR spectroscopy and chemometrics associated with multivariate analysis of the lipid profile, anthropometric, and pro-inflammatory biomarkers.

BACKGROUND: Obesity, dyslipidemia, and low-grade inflammatory state form a triad of self-sustaining metabolic dysfunction. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a simple, rapid, and non-destructive technique that generates spectral fingerprints of biomolecules that can be correlated with metabolic changes. We verified the efficiency of ATR-FTIR spectroscopy in blood plasma (n = 74) to discriminate the types of dyslipidemias and suggest metabolic inflammatory changes. METHODS: Principal Component Analysis (PCA) was performed on the biochemical and anthropometric data to verify whether the dyslipidemia types share a similar biochemical profile plausible of discrimination in chemometric modeling. To discriminate the types of dyslipidemias based on spectral data, Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA) was used and validated with leave-one-out cross-validation. RESULTS: Although no significant difference was obtained between the types of dyslipidemia and normal subjects by CRP, leptin, and cfDNA, there was a significant difference between normal subjects vs combined hyperlipidemia (CH) + hypercholesterolemia (HCL) + hypertriglyceridemia (HTG) (p < 0.05) by the 1245 cm-1 peak [νas(PO2-)] (possible indication of chronic inflammation by increased cfDNA). The area under the curve of the region between 1770 and 1720 cm-1 was significantly increased for CH in relation to other dyslipidemias and normal subjects. Furthermore, there were significant differences for the main representative peaks of lipids, proteins, carbohydrates, and nucleic acids between the types of dyslipidemias and between the types of dyslipidemias and normal subjects. The OPLS-DA model achieved 100 % accuracy with 1 latent variable and Standard Error of Cross-Validation (SECV) < 0.004 for all types of dyslipidemia  and the control group. CONCLUSIONS: Our results suggest that ATR-FTIR spectroscopy associated with chemometric modeling is a plausible applicant for screening the types of dyslipidemias. However, more extensive studies should be conducted to verify the real applicability in clinical analysis laboratories or medical clinics.

Ataxia-telangiectasia: una revisión desde la etiopatogenia al manejo actual con descripción de casos reportados en Perú / Ataxia telangiectasia: A review from etiopathogenesis to current management with a description of reported cases in Peru

La Ataxia-Telangiectasia (AT) es una rara enfermedad de herencia autosómica recesiva y de afección multisistémica, caracterizada por ataxia progresiva, inmunodeficiencia variable con infecciones recurrentes, riesgo incrementado de neoplasias con o sin telangiectasias óculo-cutáneas. La AT es causada por variantes patogénicas bialélicas en el gen ATM. Su diagnóstico se basa en la sospecha de un cuadro clínico compatible, niveles elevados de alfafetoproteína, atrofia cerebelosa y estudios genéticos. No existe tratamiento curativo de AT y su manejo se basa en medidas de soporte y prevención de complicaciones y asesoramiento genético. En esta revisión, actualizamos la epidemiología, manifestaciones clínicas, diagnóstico y tratamiento de AT incluyendo una búsqueda de casos publicados en el Perú.

Ataxia-Telangiectasia (AT) is a rare autosomal recessive disease with multisystemic involvement, characterized by slowly progressive ataxia, variable immunodeficiency with recurrent infections, increased risk of neoplasms with or without oculocutaneous telangiectasias. AT is caused by biallelic pathogenic variants within the ATM gene. Its diagnosis is based on suspicion of a compatible clinical symptomatology, increased levels of alpha-fetoprotein, cerebellar atrophy, and genetic testing. There is no curative treatment for AT and its management is based on supportive and preventive measures of eventual complications and genetic counseling. This review updates the epidemiology, clinical manifestations, diagnosis, and treatment of AT, including a search for cases published in Peru.

ATM inhibition drives metabolic adaptation via induction of macropinocytosis.

Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.

Alternative end-joining originates stable chromosome aberrations induced by etoposide during targeted inhibition of DNA-PKcs in ATM-deficient tumor cells.

ATM and DNA-PKcs coordinate the DNA damage response at multiple levels following the exposure to chemotherapy. The Topoisomerase II poison etoposide (ETO) is an effective chemotherapeutic agent that induces DNA double-strand breaks (DSB), but it is responsible from the chromosomal rearrangements frequently found in therapy-related secondary tumors. Targeted inhibition of DNA-PKcs in ATM-defective tumors combined with radio- or chemotherapy has been proposed as relevant therapies. Here, we explored the DNA repair mechanisms and the genetic consequences of targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumor cells after exposure to ETO. We demonstrated that chemical inhibition of DNA-PKcs followed by treatment with ETO resulted in the accumulation of chromatid breaks and decreased mitotic index in both A-T cells and ATM-knocked-down (ATMkd) tumor cells. The HR repair process in DNA-PKcs-inhibited ATMkd cells amplified the RAD51 foci number, with no correlated increase in sister chromatid exchanges. The analysis of post-mitotic DNA lesions presented an augmented number of persistent unresolved DSB, without alterations in the cell cycle progression. Long-term examination of chromosome aberrations revealed a strikingly high number of chromatid and chromosome exchanges. By using genetic and pharmacological abrogation of PARP-1, we demonstrated that alternative end-joining (alt-EJ) repair pathway is responsible for those chromosome abnormalities generated by limiting c-NHEJ activities during directed inhibition of DNA-PKcs in ATM-deficient cells. Targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumors stimulates the DSB repair by alt-EJ, which is liable for the origin of cells carrying stable chromosome aberrations that may eventually restrict the therapeutic strategy.

Double heterozygous mutation in RAD50 and ATM genes in a Peruvian family with five cancer types: a case report.

INTRODUCTION: Cancer is the second leading cause of death worldwide, with 70% of cancer deaths occurring in low- or middle- income countries. To mitigate the mortality of this disease, it is recommended the evaluation of multiple high-penetrance genes. METHODS: We used a multi-gene panel testing to identify germline variants in a unique case of a breast cancer patient with a family history of five different neoplasm types. The patient, at the age of 50 years, was diagnosed with a high-grade cribriform ductal carcinoma in situ in her left breast. RESULTS: We identified two heterozygous mutations, one classified as pathogenic/likely pathogenic in RAD50 and the other classified as a variant of uncertain significance (VUS) in ATM. CONCLUSION: In conclusion, the use of the multi-gene panel leads to the identification of a double heterozygous mutation in RAD50 and ATM in a breast cancer patient from a Peruvian family with several cancer types. This data helps our physician team and the patient to choose a treatment following the post-test genetic counseling.

New insights on familial colorectal cancer type X syndrome.

Familial colorectal cancer type X (FCCTX) is a heterogeneous colorectal cancer predisposition syndrome that, although displays a cancer pattern similar to Lynch syndrome, is mismatch repair proficient and does not exhibit microsatellite instability. Besides, its genetic etiology remains to be elucidated. In this study we performed germline exome sequencing of 39 cancer-affected patients from 34 families at risk for FCCTX. Variant classification followed the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic/likely pathogenic variants were identified in 17.65% of the families. Rare and potentially pathogenic alterations were identified in known hereditary cancer genes (CHEK2), in putative FCCTX candidate genes (OGG1 and FAN1) and in other cancer-related genes such as ATR, ASXL1, PARK2, SLX4 and TREX1. This study provides novel important clues that can contribute to the understanding of FCCTX genetic basis.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

A novel 1-((3-(2-toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)pyrimidin-2(1H)-one activates intrinsic mitochondria-dependent pathway and decreases angiogenesis in PC-3 cells.

Prostate cancer is among the most common cancer diagnoses in men, and the best treatment for patients with metastatic disease in advanced stages is still unclear. Previously, we have demonstrated that the three 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2- ones derivatives (8a, 8e and 9c) present important cytotoxicity and selectivity for tumoral cells. Considering that various cytotoxic drugs have been assessed in patients with prostate cancer, but few drugs show survival advantage, we decided to study these three compounds (8a, 8e and 9c) in prostate cancer cells, androgen receptor (AR)-positive 22Rv-1 and AR-negative PC-3 cells. We obtained the half maximal inhibitory concentration (IC50) of 8a, 8e and 9c in prostate cancer cells and based on high selectivity of 9c to PC-3 cells, we determined the mechanism of this compound to induce cell death through different methods. We show here that 9c compound induces cell cycle arrest in G2/M, increasing the levels of reactive oxygen species and DNA damage, and triggers DNA damage response by ataxia-telangiectasia mutated (ATM) and histone H2AX phosphorylation induction. The compound also led PC-3 to lipid peroxidation and mitochondrial depolarization which triggered the activation of intrinsic pathway, confirmed by increase of cleaved caspase-9 and 3. In this work we also show the ability of 9c in reducing vascular endothelial growth factor expression (VEGF) and inhibiting topoisomerase I enzyme, therefore indicating a potential new molecule to be further investigated for prostate cancer management.

The association of three DNA repair genes polymorphisms on the frequency of chromosomal alterations detected by fluorescence in situ hybridization.

PURPOSE: Gas station workers (GSWs) are exposed to carcinogenic agents. The aim was to study the association of high somatic chromosome alterations (CAs) rates in the blood of GSWs and the polymorphisms of three genes playing a role in DNA double-strand break repair. METHODS: This is a cross-sectional study with 114 GSWs and 115 age-matched controls. Cytogenetic analyses, blood exams, medical interviews and genotypes for RAD51/G135C (rs1801320), ATM/P1054R (rs1800057) and CHEK2/T470C (rs17879961) genes were performed. RESULTS: The CA rate in GSWs was 9.8 CAs/1000 metaphases, and 19.1% of the workers had > 10 CAs per 1000 metaphases (group two). GSWs had decreased levels of monocytes (P = 0.024) in their blood exams. The number of variant alleles of the RAD51/G135C polymorphism was higher in GSWs (P = 0.011) compared to the controls, and were associated with enhanced number of CAs per worker (P = 0.008). No allele variant was found for CHEK2/T470C in this study. CONCLUSION: The RAD51/G135C polymorphism appears to be related to genome instability in gas station workers. Increasing the knowledge of DNA repair gene variations involved in maintaining genomic stability in GSWs may be crucial for future cancer prevention.

Specific Human ATR and ATM Inhibitors Modulate Single Strand DNA Formation in Leishmania major Exposed to Oxidative Agent.

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA.

Germline variants of Brazilian women with breast cancer and detection of a novel pathogenic ATM deletion in early-onset breast cancer.

BACKGROUND: It is estimated that 5-10% of breast cancer cases are hereditary. The identification of pathogenic germline variants allows individualized preventive health care, improvement of clinical management and genetic counseling. Studies in ethnically admixed Latin American populations have identified regions with increased frequency of deleterious variants in breast cancer predisposing genes. In this context, the Brazilian population exhibits great genetic heterogeneity, and is not well represented in international databases, which makes it difficult to interpret the clinical relevance of germline variants. METHODS: We evaluated the frequency of pathogenic/likely pathogenic (P/LP) germline variants in up to 37 breast cancer predisposing genes, in a cohort of 105 breast and/or ovarian cancer Brazilian women referred to two research centers between 2014 and 2019. RESULTS: A total of 22 patients (21%) were found to carry P/LP variants, and 16 VUS were detected in 15 patients (14.3%). Additionally, a novel pathogenic ATM intragenic deletion was identified in an early-onset breast cancer. We also detected a BRCA1 pathogenic variant (c.5074+2T>C) in higher frequency (10×) than in other studies with similar cohorts. CONCLUSIONS: Our findings contribute to the characterization of the genetic background of breast cancer predisposition in the Brazilian population as a useful resource to discriminate between deleterious variants and VUS, thus enabling improvement in the preventive health care and clinical management of carriers.

PARP Inhibitor Olaparib Causes No Potentiation of the Bleomycin Effect in VERO Cells, Even in the Presence of Pooled ATM, DNA-PK, and LigIV Inhibitors.

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes' assembly. Nuclear PAR affects chromatin's structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polß), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.

Hydrogen peroxide and Helicobacter pylori extract treatment combined with APE1 knockdown induce DNA damage, G2/M arrest and cell death in gastric cancer cell line.

Chronic inflammation resulting from Helicobacter pylori (H. pylori) infection, the major risk factor for gastric cancer, results in increased release of reactive oxygen species (ROS), promoting oxidative stress and DNA damage. APE1 endonuclease, a key component of the base excision repair (BER) pathway, is responsible for the repair of damage induced by ROS. However, the APE1 gene and other DNA damage response (DDR) genes are still poorly understood in gastric cancer. Thus, we aimed to investigate whether the silencing of APE1 by shRNA can interfere with the survival of AGS gastric cancer cells after treatment with hydrogen peroxide (H2O2) and/or H. pylori extract (HPE) and its relation with the expression of DDR genes (ATM, ATR, and H2AX) and miRNAs that target DDR genes. In the AGS cells expressing APE1, isolated or combined treatment with H2O2 and HPE promoted a slight increase in the cell proliferation and increased the levels of intracellular ROS and DNA double strand breaks (DSBs) indicated by ©H2AX foci, a reduction in the proportion of cells in the G0/G1 phase and an increase in the initial apoptosis rate. Moreover, upregulation of APE1, ATR, miR-15a, miR-21, miR-24 and miR-421 and downregulation of ATM and H2AX was observed. In silenced AGS cells after treatment with H2O2 alone or combined with HPE, we observed an increase in the cell proliferation rate and the levels of intracellular ROS and DSBs and a reduction in the proportion of cells in S and G2/M phase arrest, leading to late apoptosis. APE1 knockdown also caused a reduction in the expression of ATM and miR-421, while ATR expression was increased. Based on our results, APE1 knockdown may promote changes in cellular processes by increasing genomic instability, leading to G2/M arrest and cell apoptosis, so it may be a promising strategy for controlling tumor progression.