The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

• MeSH
• DNA-Binding Proteins genetics   MeSH
• DNA Repair Enzymes genetics   MeSH
• Genetic Predisposition to Disease * MeSH
• MRE11 Homologue Protein * genetics   MeSH
• Acid Anhydride Hydrolases * genetics   MeSH
• Nuclear Proteins * genetics   MeSH
• Humans MeSH
• Neoplasms * genetics   MeSH
• Cell Cycle Proteins * genetics   MeSH
• Germ-Line Mutation * MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Meta-Analysis MeSH
• Systematic Review MeSH

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

• MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 MeSH
• DNA Repair MeSH
• Polynucleotide 5'-Hydroxyl-Kinase * MeSH
• DNA Damage MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

• MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• DNA Repair Enzymes genetics   metabolism   MeSH
• MRE11 Homologue Protein genetics   metabolism   MeSH
• Acid Anhydride Hydrolases genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Proteins * genetics   MeSH
• DNA Repair genetics   MeSH
• Cell Cycle Proteins * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Hepatocellular carcinoma (HCC) mainly stems from liver cirrhosis and its genetic predisposition is believed to be rare. However, two recent studies describe pathogenic/likely pathogenic germline variants (PV) in cancer-predisposition genes (CPG). As the risk of de novo tumors might be increased in PV carriers, especially in immunosuppressed patients after a liver transplantation, we analyzed the prevalence of germline CPG variants in HCC patients considered for liver transplantation. Using the panel NGS targeting 226 CPGs, we analyzed germline DNA from 334 Czech HCC patients and 1662 population-matched controls. We identified 48 PVs in 35 genes in 47/334 patients (14.1%). However, only 7/334 (2.1%) patients carried a PV in an established CPG (PMS2, 4×NBN, FH or RET). Only the PV carriers in two MRN complex genes (NBN and RAD50) were significantly more frequent among patients over controls. We found no differences in clinicopathological characteristics between carriers and non-carriers. Our study indicated that the genetic component of HCC is rare. The HCC diagnosis itself does not meet criteria for routine germline CPG genetic testing. However, a low proportion of PV carriers may benefit from a tailored follow-up or targeted therapy and germline testing could be considered in liver transplant recipients.

• Publication type
• Journal Article MeSH

Cancer therapy failure is a fundamental challenge in cancer treatment. One of the most common reasons for therapy failure is the development of acquired resistance of cancer cells. DNA-damaging agents are frequently used in first-line chemotherapy regimens and DNA damage response, and DNA repair pathways are significantly involved in the mechanisms of chemoresistance. MRE11, a part of the MRN complex involved in double-strand break (DSB) repair, is connected to colorectal cancer (CRC) patients' prognosis. Our previous results showed that single-nucleotide polymorphisms (SNPs) in the 3' untranslated region (3'UTR) microRNA (miRNA) binding sites of MRE11 gene are associated with decreased cancer risk but with shorter survival of CRC patients, which implies the role of miRNA regulation in CRC. The therapy of colorectal cancer utilizes oxaliplatin (oxalato(trans-l-1,2-diaminocyclohexane)platinum), which is often compromised by chemoresistance development. There is, therefore, a crucial clinical need to understand the cellular processes associated with drug resistance and improve treatment responses by applying efficient combination therapies. The main aim of this study was to investigate the effect of miRNAs on the oxaliplatin therapy response of CRC patients. By the in silico analysis, miR-140 was predicted to target MRE11 and modulate CRC prognosis. The lower expression of miR-140 was associated with the metastatic phenotype (p < 0.05) and poor progression-free survival (odds ratio (OR) = 0.4, p < 0.05). In the in vitro analysis, we used miRNA mimics to increase the level of miR-140 in the CRC cell line. This resulted in decreased proliferation of CRC cells (p < 0.05). Increased levels of miR-140 also led to increased sensitivity of cancer cells to oxaliplatin (p < 0.05) and to the accumulation of DNA damage. Our results, both in vitro and in vivo, suggest that miR-140 may act as a tumor suppressor and plays an important role in DSB DNA repair and, consequently, CRC therapy response.

• Publication type
• Journal Article MeSH

V současnosti jsme svědky zapojení imunoterapie využívající anti-CD3 konstrukty (blinatumomab) nebo CAR-T buňky do léčby relabované nebo rezistentní B prekurzorové akutní lymfoblastické leukémie (BCP ALL), ale zatím ne T ALL. Naše laboratoř provádí diagnostiku minimální reziduální nemoci (MRN) pro země EU, Švýcarsko a Izrael. Součástí nového protokolu AIEOP BFM 2017 pro léčbu nově diagnostikovanou BCP ALL bude terapie blinatumomabem pro pacienty s pomalou odpovědí na léčbu. Pro efekt jsou důležité komponenty imunitního systému, zejména T lymfocytů. Rezistence na tento typ cílené léčby může být způsobená různou kombinací dysfunkce T lymfocytů (případně i dalších složek imunitního systému), exprese inhibičních molekul na blastech a změny imunofenotypu blastů, zejména ztráty antigenu CD19 nebo liniového přesmyku (obvykle do monocytární linie, jak jsme nedávno popsali). Ztráta antigenu CD19 představuje výzvu pro detekci MRN, která je důležitá pro stanovení intenzity léčby. Plánujeme detailně popsat mechanismy rezistence na imunoterapii a navrhnout cesty, jak rezistenci obejít.; Immunotherapeutic approaches using anti-CD3 antibody constructs (blinatumomab) or CAR-T cells newly became part of the treatment of the relapsed or resistant B precursor acute lymphoblastic leukemia (ALL) but so far not in T ALL. Our lab serves as a reference center for minimal residual disease (MRD) diagnostics in a blinatumomab trial for the EU, Switzerland and Israel. The next frontline protocol AIEOP BFM 2017 will use blinatumomab for patients with slow response to chemotherapy. Resistance to the blinatumomab therapy is caused by a variable combination of a T cell dysfunction, an expression of inhibitory molecules on blasts and a shift of the immunophenotype of the blasts (a loss of CD19 expression or a lineage switch to the monocytic lineage as we recently described). Loss of CD19 antigen is a challenge for the flow cytometric MRD assessment, which is important for setting the treatment intensity. We plan to describe the detailed mechanisms of immunotherapy resistance and to design ways to circumvent them.

• Keywords
• blinatumomab,
• MeSH
• CD3 Complex MeSH
• Drug Resistance, Neoplasm MeSH
• Molecular Targeted Therapy MeSH
• Immunotherapy MeSH
• Monitoring, Physiologic MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma immunology   therapy   MeSH
• Antineoplastic Agents, Immunological MeSH
• Neoplasm, Residual diagnosis   MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• hematologie a transfuzní lékařství
• alergologie a imunologie
• onkologie
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

• Publication type
• Journal Article MeSH

A growing body of evidence supports the notion that cancer resistance is driven by a small subset of cancer stem cells (CSC), responsible for tumor initiation, growth, and metastasis. Both CSC and chemoresistant cancer cells may share common qualities to activate a series of self-defense mechanisms against chemotherapeutic drugs. Here, we aimed to identify proteins in chemoresistant triple-negative breast cancer (TNBC) cells and corresponding CSC-like spheroid cells that may contribute to their resistance. We have identified several candidate proteins representing the subfamilies of DNA damage response (DDR) system, the ATP-binding cassette, and the 26S proteasome degradation machinery. We have also demonstrated that both cell types exhibit enhanced DDR when compared to corresponding parental counterparts, and identified RAD50 as one of the major contributors in the resistance phenotype. Finally, we have provided evidence that depleting or blocking RAD50 within the Mre11-Rad50-NBS1 (MRN) complex resensitizes CSC and chemoresistant TNBC cells to chemotherapeutic drugs.

• MeSH
• Drug Resistance, Neoplasm drug effects   genetics   MeSH
• Cisplatin administration & dosage   MeSH
• Cyclophosphamide administration & dosage   MeSH
• DNA-Binding Proteins genetics   MeSH
• Doxorubicin administration & dosage   MeSH
• DNA Repair Enzymes genetics   MeSH
• MRE11 Homologue Protein genetics   MeSH
• Acid Anhydride Hydrolases genetics   MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Neoplastic Stem Cells drug effects   metabolism   MeSH
• DNA Damage drug effects   MeSH
• Disease-Free Survival MeSH
• Cell Cycle Proteins genetics   MeSH
• Triple Negative Breast Neoplasms drug therapy   genetics   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Masa aberantních buněk mnohočetného myelomu je v době diagnózy složena ze směsi několika nezávislých klonů s proměnlivou velikostí v čase. Populace aberantních buněk minimální reziduální nemoci (MRN) představuje pouze podmnožinu klonů z původního množství, které přežily léčbu. Zmenšení počtu klonů mnohočetného myelomu je spojeno se snížením celkové genetické variability. Snížení heterogenity uvnitř nádoru po aplikaci primární terapie dovoluje zařazení individualizované léčby zacílené na zbytkový klon. Genomové sekvenování spolu s profilováním genové exprese ve zbytkovém klonu umožní identifikovat sadu klíčových mutací a/nebo poškozených metabolických drah, které mohou být odstraněny některou z nových cílených terapeutických látek.; Mass of multiple myeloma aberrant cells represents at the time of diagnosis a mixture of multiple independent clones that vary in size with time. Population of aberrant cells of minimal residual disease (MRD) represents only a subset of clones from their complex pool, which survived the treatment. The reduction of the number of multiple myeloma clones is associated with decreased genetic variability. Decrease in intratumor heterogeneity after frontline therapy allows implementation of individualized therapy targeting residual clone. Genome wide sequencing combined with gene expression profiling of residual clone will enable to identify key set of mutations and/or affected pathways that can be eliminated by some of the novel targeted therapeutic agents.

• MeSH
• Clone Cells MeSH
• Molecular Targeted Therapy methods   MeSH
• Genome, Human MeSH
• Genomics methods   MeSH
• Precision Medicine methods   MeSH
• Humans MeSH
• Multiple Myeloma drug therapy   genetics   MeSH
• Mutation MeSH
• Flow Cytometry methods   MeSH
• Neoplasm, Residual drug therapy   genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Check Tag
• Humans MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• onkologie
• genetika, lékařská genetika
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

Krátce po celosvětovém zahájení očkování proti SARS-CoV-2 se objevily obavy z možné souvislosti mezi vakcinací, závažnou trombocytopenií a vznikem trombózy v atypických lokalizacích. Obavy se primárně týkaly vakcíny AstraZeneca (ChAdOx1 nCov-19), později vakcíny Johnson&Johnson (Ad26.COV2.S), ale také po aplikaci mRNA vakcín Pfizer-BioNTech a Moderna byla popsána akutní imunitní trombocytopenická purpura (ITP) provázená krvácením bez trombózy a žilní trombózy v atypických lokalizacích. Zkoumáním okolností vzniku těchto komplikací bylo zjištěno, že se jedná o obdobný mechanismus jako u heparinem indukované trombocytopenie 2. typu (HIT 2. typu), vzácného a závažného hyperkoagulačního stavu s žilními a tepennými trombózami. Příčinou HIT jsou IgG protilátky aktivující trombocyty, namířené proti antigenu, kterým je makromolekulární komplex tvořený destičkovým faktorem 4 (PF4) a heparinem. Pro odlišení obou stavů se začal razit nový termín vakcínou indukovaná (imunitní) trombotická trombocytopenie (VITT). Postižení pacienti měli vysoké hladiny protilátek proti imunitnímu komplexu tvořenému PF 4 a polyaniontovou složkou vakcíny (dvojvláknovou DNA). U pacientů, kteří mají po očkování trombózu v jakékoliv cévní lokalizaci, provázenou absolutní nebo relativní trombocytopenií a systémovými projevy, lze využít ELISA test k průkazu protilátek proti PF4 a funkční testy aktivace destiček k rychlé a spolehlivé diagnostice VITT a k jeho odlišení od žilní tromboembolické nemoci. Podání imunoglobulinu zvyšuje počet trombocytů, snižuje krevní srážlivost a inhibuje aktivaci trombocytů IgG protilátkami, podobně jako u HIT. Pro léčbu trombózy jsou vhodné přímé orální inhibitory F. Xa, přímé inhibitory trombinu a nepřímé inhibitory F.Xa.

Shortly after the worldwide initiation of vaccination against SARS-CoV-2, concerns emerged about a possible link between vaccination, severe thrombocytopenia, and the development of atypical venous thrombosis. Concerns were primarily about AstraZeneca (ChAdOx1 nCov-19), later Johnson & Johnson (Ad26.COV2.S), but cases of acute immune thrombocytopenic purpura (ITP) and bleeding without thrombosis and also atypical venous thrombosis after exposure to the messenger RNA-based vaccines produced by Pfizer-BioNTech and Moderna have been reported. Examination of the circumstances of these complications revealed that this is a similar mechanism to heparin-induced thrombocytopenia (HIT), a prothrombotic thrombocytopenic hypercoagulable disorder with venous and arterial thrombosis. HIT is caused by platelet-activating IgG antibodies directed against an antigen that is a macromolecular complex consisting of platelet factor 4 (PF4) and heparin. Naming this new entity vaccine-induced immune thrombotic thrombocytopenia (VITT) was suggested to avoid confusion with HIT. Patients had high levels of antibodies to the immune complex formed by PF 4 and the polyanionic component of the vaccine (double-stranded DNA). In patients with thrombosis at any vascular site after vaccination, accompanied by absolute or relative thrombocytopenia and systemic manifestations, HIT Ig ELISA assay to detect antibodies against PF4 and platelet-activating functional tests may be used for VITT recognition and differentiation from venous thromboembolic disease. Immune globulin impedes antibody-mediated platelet clearance and down-regulate platelet activation by immune complexes, as in HIT. It is prudent to choose from among the nonheparin antithrombotic agents - direct oral F.Xa inhibitors, direct thrombin inhibitors and indirect F.Xa inhibitors for the treatment of thrombosis.

• MeSH
• Platelet Activation drug effects   MeSH
• Anticoagulants therapeutic use   MeSH
• Immunoglobulins, Intravenous therapeutic use   MeSH
• Humans MeSH
• Thrombocytopenia * chemically induced   physiopathology   therapy   MeSH
• Platelet Factor 4 physiology   adverse effects   MeSH
• Thrombophilia chemically induced   physiopathology   prevention & control   MeSH
• Thrombosis chemically induced   physiopathology   MeSH
• Vaccination MeSH
• COVID-19 Vaccines * blood   adverse effects   MeSH
• Check Tag
• Humans MeSH