Exogenous and endogenous factors that modify telomere length in hereditary breast and ovarian cancer patients

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 1-06-2017Esta tesis tiene embargado el acceso al texto completo hasta el 01-12-2018The role of telomere shortening on cancer risk is a complex issue, and likely depending on several factors. In a previous study published by our group, we showed that patients harboring mutations in the BRCA1 and/or BRCA2 genes presented an accelerated telomeric shortening and an anticipation in the onset of cancer across successive generations in families affected. This suggested, that in the context of hereditary breast and ovarian cancer, mutations in BRCA1 and BRCA2 modified the risk of developing cancer through an accelerated telomere shortening. Subsequently, two independent studies using a larger number of families found very different results, compared to those reported by our group; however, the three studies were retrospective, and did not consider the possible existence of telomere length modifiers as confounding factors. In this way, the main objective of this Thesis has been focused on the identification of genetic and environmental factors modifying the telomere length, mainly in the context of hereditary breast and ovary cancer. To this end, we proposed a prospective study involving families affected by hereditary breast and ovarian cancer, with or without mutation in the BRCA1 and BRCA2 genes. Participants, have been cited in medical consultancy for the extraction of biological samples and to complete a questionnaire about their treatment and evolution of the disease. Regarding the exogenous factors, in a first study we could confirm that the chemotherapy shortened the telomeres in the patients who were being treated, although this effect was transitory. After correction for the treatment status, both the results and the conclusions were different in relation to the effect of the BRCA1 and BRCA2 mutations on telomere shortening as a causal risk factor for developing of cancer. Suggesting that chemotherapy was indeed a confounding factor for this type of studies. We continued studying genetic factors as potential telomere length modifiers: In this case, two “cancer risk polymorphisms”, which modify breast and ovarian cancer risk susceptibility, for BRCA1 and BRCA2 mutation carriers. These polymorphisms were found in transcriptional regulatory regions of OGG1 and NEIL2 genes. Both genes, encoding for DNA glycosylases which are key enzymes, involved in the base excision repair pathway (BER) which has an important role on repairing oxidative DNA damage at telomeres. Hence, we wanted to study molecular bases responsible for this association and its role on the telomere biology. Hence, we evaluated the role of these SNPs in relation to OGG1 and NEIL2 transcriptional regulation, its contribution on genomic and / or telomere instability, as well as its possible relation with systemic oxidative stress. Thus, we identified that these SNPs have a functional effect at the transcriptional level and can affect the enzyme efficiency of repairing oxidative DNA damage at the telomere, as well as telomere length for BRCA1 and BRCA2 mutations carriers. Based on a possible synthetic lethality relationship between OGG1 (BER) and BRCA1 (HR) DNA repair pathways, we tested the antiproliferative properties of inhibiting pharmacologically OGG1 enzyme using a panel of OGG1 inhibitors, in a set of BRCA1 and BRCA2 deficient cancer cell lines. We have identified that in cells with the same genetic background, inactivation of BRCA1 make cells very sensitive to OGG1 inhibitors. This may open a new framework for the treatment of hereditary breast cancer. In addition, we present two examples of hereditary diseases, hereditary cardiac angiosarcoma and aplastic anemia, in which the molecular mechanism of the disease initiation is telomere dysregulation, caused by mutations in genes associated with the shelterin complex and the telomerase respectively. This examples illustrate the complexity of telomere biology in relation to cancer and / or other diseases.Las siguientes becas, ayudas y proyectos han permitido la realización de esta Tesis Doctoral: • Proyecto PI12/00070 Fondo de Investigación Sanitaria (FIS) del Instituto de Salud Carlos III dirigido por el Dr. Javier Benítez. Periodo: 2013-2017 • Proyecto HORIZON 2020-BRIDGES (2015-2019) • FEBS Short-term fellowship (2016

A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li-Fraumeni-like families

Cardiac angiosarcoma (CAS) is a rare malignant tumour whose genetic basis is unknown. Here we show, by whole-exome sequencing of a TP53-negative Li-Fraumeni-like (LFL) family including CAS cases, that a missense variant (p.R117C) in POT1 (protection of telomeres 1) gene is responsible for CAS. The same gene alteration is found in two other LFL families with CAS, supporting the causal effect of the identified mutation. We extend the analysis to TP53-negative LFL families with no CAS and find the same mutation in a breast AS family. The mutation is recently found once in 121,324 studied alleles in ExAC server but it is not described in any other database or found in 1,520 Spanish controls. In silico structural analysis suggests how the mutation disrupts POT1 structure. Functional and in vitro studies demonstrate that carriers of the mutation show reduced telomere-bound POT1 levels, abnormally long telomeres and increased telomere fragility

Small molecule inhibitor of OGG1 blocks oxidative DNA damage repair at telomeres and potentiates methotrexate anticancer effects

The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment

Molecular insights into the OGG1 gene, a cancer risk modifier in BRCA1 and BRCA2 mutations carriers.

We have recently shown that rs2304277 variant in the OGG1 glycosidase gene of the Base Excision Repair pathway can increase ovarian cancer risk in BRCA1 mutation carriers. In the present study, we aimed to explore the role of this genetic variant on different genome instability hallmarks to explain its association with cancer risk.We have evaluated the effect of this polymorphism on OGG1 transcriptional regulation and its contribution to telomere shortening and DNA damage accumulation. For that, we have used a series of 89 BRCA1 and BRCA2 mutation carriers, 74 BRCAX cases, 60 non-carrier controls and 23 lymphoblastoid cell lines (LCL) derived from BRCA1 mutation carriers and non-carriers.We have identified that this SNP is associated to a significant OGG1 transcriptional down regulation independently of the BRCA mutational status and that the variant may exert a synergistic effect together with BRCA1 or BRCA2 mutations on DNA damage and telomere shortening.These results suggest that this variant, could be associated to a higher cancer risk in BRCA1 mutation carriers, due to an OGG1 transcriptional down regulation and its effect on genome instability.J.B.'s laboratory is partially funded by the Spanish Ministry of Health PI12/00070 supported by FEDER funds, and the Spanish Research Network on Rare diseases (CIBERER). C.B-B is granted by the PI12/00070. M.A.B.'s laboratory is funded with the Spanish Ministry of Science and Innovation, projects SAF2008-05384 and 2007-A-200950 (TELOMARKER), European Research Council Advanced grant GA#232854, the Korber Foundation, Fundacion Botin and Fundacion Lilly. MU is supported by the Spanish Ministry of Health PI14/00459 with FEDER funds.S

Impact of chemotherapy on telomere length in sporadic and familial breast cancer patients

et al.Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.This work was partially funded by project FIS PI12/00070 from the Carlos III Health Institute and the Spanish Network on Rare Diseases (CIBERER) and by project SAF2012-35779 (Spanish Ministry of Economy and Competiveness). M.A.B.’s Laboratory is funded with the Spanish Ministry of Science and Innovation, Projects SAF2008-05384 and 2007-A-200950 (TELOMARKER), European Research Council Advanced Grant GA#232854, the Köber Foundation, Botín Foundation, and Lilly Foundation.Peer Reviewe

A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li-Fraumeni-like families

This work is licensed under a Creative Commons Attribution 4.0 International License.-- et al.Cardiac angiosarcoma (CAS) is a rare malignant tumour whose genetic basis is unknown. Here we show, by whole-exome sequencing of a TP53-negative Li-Fraumeni-like (LFL) family including CAS cases, that a missense variant (p.R117C) in POT1 (protection of telomeres 1) gene is responsible for CAS. The same gene alteration is found in two other LFL families with CAS, supporting the causal effect of the identified mutation. We extend the analysis to TP53-negative LFL families with no CAS and find the same mutation in a breast AS family. The mutation is recently found once in 121,324 studied alleles in ExAC server but it is not described in any other database or found in 1,520 Spanish controls. In silico structural analysis suggests how the mutation disrupts POT1 structure. Functional and in vitro studies demonstrate that carriers of the mutation show reduced telomere-bound POT1 levels, abnormally long telomeres and increased telomere fragility.We are grateful to T. de Lange (The Rockefeller University) and K. Collins (The University of California) for providing POT1 and TPP1 plasmids, respectively. J.B.’s laboratory is partially funded by the Spanish Ministry of Health PI12/00070, the Spanish Ministry of Science and Innovation (INNPRONTA 2012) and the Spanish Research Network on Rare diseases (CIBERER). O.C. is granted by the CIBERER and C.B.-B. by the PI12/00070 supported by FEDER funds. P.G.-P. is partially supported by the Spanish Ministry of Health PI11/0699, PI12/01941 and RD12/0042/0066. M.A.B.’s laboratory is funded with the Spanish Ministry of Science and Innovation, projects SAF2008-05384 and 2007-A-200950 (TELOMARKER), European Research Council Advanced grant GA#232854, the Körber Foundation, Fundación Botín and Fundación Lilly. R.P.’ lab is partially funded by PI11/0949 Supported by FEDER funds.Peer Reviewe

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

This study was funded by the Spanish Ministry of Economy and Competitiveness, the Fundacion Botin, and the Roche Extending the Innovation Network Academia Partnering Program (M.A.B.).Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres.S

A common SNP in the UNG gene decreases ovarian cancer risk in BRCA2 mutation carriers

Single nucleotide polymorphisms (SNPs) in DNA glycosylase genes involved in the base excision repair (BER) pathway can modify breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. We previously found that SNP rs34259 in the uracil-DNA glycosylase gene (UNG) might decrease ovarian cancer risk in BRCA2 mutation carriers. In the present study, we validated this finding in a larger series of familial breast and ovarian cancer patients to gain insights into how this UNG variant exerts its protective effect. We found that rs34259 is associated with significant UNG downregulation and with lower levels of DNA damage at telomeres. In addition, we found that this SNP is associated with significantly lower oxidative stress susceptibility and lower uracil accumulation at telomeres in BRCA2 mutation carriers. Our findings help to explain the association of this variant with a lower cancer risk in BRCA2 mutation carriers and highlight the importance of genetic changes in BER pathway genes as modifiers of cancer susceptibility for BRCA1 and BRCA2 mutation carriers.JMB is supported by grant FPU15/01978 from the Spanish Ministry of Education, Culture, and Sport. CB-B is supported by the Spanish Ministry of Health FIS PI12/00070. This study was partially funded by the Spanish Ministry of Economy and Competitiveness (MINECO) SAF2014-57680-R. JB’s laboratory is partially funded by FIS PI16/00440 supported by FEDER funds and the Spanish Network on Rare Diseases (CIBERER). MU is supported by grant PI14/00459 from the European Regional Development Fund (ERDF). RP’s laboratory is funded by grant P14-01495 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain) supported by FEDER funds. JLG-G thanks the Instituto de Salud Carlos III for grant number PI16/01031, cofinanced by the European Regional Development Fund (ERDF).Peer reviewe

Small molecule inhibitor of OGG1 blocks oxidative DNA damage repair at telomeres and potentiates methotrexate anticancer effects

The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment

A mutation in the POT1 gene is responsible for cardiac angiosarcoma in TP53-negative Li-Fraumeni-like families

Cardiac angiosarcoma (CAS) is a rare malignant tumour whose genetic basis is unknown. Here we show, by whole-exome sequencing of a TP53-negative Li-Fraumeni-like (LFL) family including CAS cases, that a missense variant (p.R117C) in POT1 (protection of telomeres 1) gene is responsible for CAS. The same gene alteration is found in two other LFL families with CAS, supporting the causal effect of the identified mutation. We extend the analysis to TP53-negative LFL families with no CAS and find the same mutation in a breast AS family. The mutation is recently found once in 121,324 studied alleles in ExAC server but it is not described in any other database or found in 1,520 Spanish controls. In silico structural analysis suggests how the mutation disrupts POT1 structure. Functional and in vitro studies demonstrate that carriers of the mutation show reduced telomere-bound POT1 levels, abnormally long telomeres and increased telomere fragility