Biology subjects

Lopez, B. S.

Publications and source records attributed to Lopez, B. S..

10 recordsLinked to original sources

bioRxiv2025

Dysregulation of replication stress-induced ROS in transformed cell lines: a vicious circle at cancer initiation

The canonical DNA damage response (DDR) maintains genome stability, involving DNA synthesis/cell cycle arrest. However, unchallenged cells proliferate when they are continually exposed to low-level/endogenous replication stress. We previously discovered and characterized a noncanonical cellular response that is specific to nonblocking replication stress, i.e., low-level stress (LoL-DDR), in primary cells. Although this response generates replication stress-induced reactive oxygen species (RIR), it triggers a program that prevents the accumulation of premutagenic 8-oxo-guanine (8-oxoG). Primary cells control RIR production via NADPH oxidases. Increasing the severity of replication stress above a precise threshold triggers the canonical DDR, leading to cell cycle arrest and RIR suppression, resulting in a peak-shaped dose response for RIR production. Here, we show that the LoL-DDR is dysregulated in cancer cell lines, which exhibit the following differences compared with primary cells: 1-RIR are not detoxified under high-level stress conditions, resulting in a continuous increase in the dose-response curve of RIR production; 2-RIR are not produced by NADPH oxidases; and 3-replication stress favors the accumulation of the premutagenic 8-oxoG. Moreover, using an in vitro breast cancer progression model, we show that LoL-DDR dysregulation occurs at an early stage of cancer progression. Since, conversely, ROS trigger replication stress this establishes a "vicious circle" involving replication-stress and ROS that continuously jeopardizes genome integrity that should fuel and amplify tumorigenesis.

cell biology↗

bioRxiv2024

The inflammasome sensor NLRP3 interacts with REV7 to maintain genome integrity through homologous recombination

DNA double strand break (DSB) is a highly toxic lesion that can generate genome instability, a major source of tumorigenesis. DSBs are mainly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The selection of the DSB repair pathway primarily depends on the DNA resection of the DSB ends. Indeed, HR is initiated by resection at the DSB generating 3 single stranded extension. The shieldin complex prevents resection fostering DSB repair toward NHEJ. Here, we reveal that the inflammasome sensor NLRP3 facilitates DNA end resection to promote the HR pathway in an inflammasome-independent manner. Strikingly, NLRP3 silencing decreases HR efficiency, as evidenced by RAD51 foci and functional HR assays. Mechanistically, we describe that NLRP3 interacts with REV7, a subunit of the shieldin complex, and its depletion increases REV7 recruitment to IR-induced DSBs. Similar to cancer cells harboring HR mutated genes, we find that NLRP3 deficient cells are sensitive to PARP inhibitors (PARPi) and exhibit an epistatic relationship with BRCA1 deficiency. Remarkably, loss of REV7 in NLRP3-depleted cells induces PARPi resistance by restoring HR. This study unravels the crucial role of the innate immune receptor NLRP3 in regulating the selection of DSB repair pathways to maintain genome integrity.

molecular biology↗

bioRxiv2024

PKA prevents resection of DNA double strand breaks and favor non-homologous end-joining

Genome rearrangement is a hallmark of cancer and ageing. DNA double-strand breaks (DSBs) are highly toxic lesions that can generate genome rearrangements. Several pathways compete for DSB repair, and selection of the appropriate repair process is decisive for genetic stability. DSB repair act according to two steps: 1-the choice between nonhomologous end-joining (NHEJ) versus resection of DNA ends, generating single-stranded DNA (ssDNA); 2-on ssDNA, homologous recombination (HR) and sub-processes. Here we show that 53BP1, which plays a prime role at the first step by protecting against resection and fostering NHEJ, physically interacts with the catalytic subunits of the cAMP dependant protein kinase A (PKAcs). PKA favors the recruitment of 53BP1 at DNA damages sites, and consistently, prevents resection, favoring NHEJ. Inhibition of PKA stimulated resection and coherently reduces NHEJ. Reversely, activation of PKA with 8-Bromo-cAMP stimulate NHEJ and reduces HR. These data open new avenues for potential anticancer strategies. More generally, they underline the high complexity of the regulation of DSB repair, identifying PKA as a novel actor of the DNA damage response, acting at the choice of the DSB repair process, which might be essential for genome integrity maintenance.

molecular biology↗

bioRxiv2024

Influenza A Virus Disruption of Dendritic Cell-Natural Killer Cell Crosstalk Impacts Activation of Helper and Cytotoxic T cell Subsets

Dendritic cells (DC) and Natural killer (NK) cells engage in reciprocal interactions to trigger an efficient innate immune response while governing the adaptive immune response. Here we used an ex vivo autologous human primary immune cell co-culture of DCs and NK cells to investigate the impact of DC-NK cell crosstalk on activation of CD4+ and CD8+ naive T cell responses to influenza A viral (IAV) infection. Using multiparameter flow cytometry, we observed that culturing T cells with DC and NK cells led to enhanced expression of CD69 and CD25 activation markers and increased proliferative ability of both CD4+ and CD8+ T cell subsets. Exposure of DCs to the pandemic A/California/07/2009 (H1N1) strain in NK cell co-culture led to a reduced frequency of CD4+CD69+, CD8+CD69+, CD4+CD25+, CD8+CD25+ T cell subsets and a reduced expansion of CD4+ T cells. The IAV-mediated curtailment of T cell activation was dependent on the ability of A/California/07/2009 (H1N1) to replicate as inactivation of the virus rescued expression of CD69, CD25 on both CD4+ and CD8+ T cell subsets and triggered expansion of CD4+ T cells. Further, we discovered exposure of DCs to the A/Victoria/361/2011 (H3N2) IAV strain also significantly impaired expression of CD69 on CD4+ and CD8+ T cells and CD25 on CD8+ T cells. In contrast with the A/California/07/2009 (H1N1 strain), inactivation of A/Victoria/361/2011 (H3N2) failed to fully restore T cell expression of CD69 and CD25 and proliferation. Collectively, these data demonstrate that IAV partially usurps the ability of DC-NK cell crosstalk to activate naive CD4+ and CD8+ T cells in a strain-dependent manner. These data may inform the immunological signals required to trigger a potent cellular immune response to IAV, which may elicit broader and more durable protection than current inactivated vaccine platforms.

immunology↗

bioRxiv2022

In vivo inactivation of RAD51-mediated homologous recombination leads to premature aging, but not to tumorigenesis

Genetic instability is a hallmark of both cancer and aging. Homologous recombination (HR) is a prominent DNA repair pathway maintaining genomic integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the consequences of mutations in the pivotal HR player, RAD51, on cancer development remain puzzling. Moreover, in contrast with other HR genes, RAD51 mouse models are not available to experimentally address the role of RAD51 on aging and carcinogenesis, in vivo. Here, we engineered a mouse model with an inducible dominant negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative non-conservative repair pathways. We found that, in vivo expression of SMRad51 did not trigger tumorigenesis, but instead induced premature aging. We propose that these in vivo phenotypes result from the exhaustion of proliferating progenitors submitted to chronic endogenous replication stress resulting from RAD51-mediated HR suppression. Our data underline the importance of the RAD51 activity for progenitors homeostasis, preventing aging, and more generally for the balance between cancer and aging.

cancer biology↗

bioRxiv2020

An adaptive pre-DNA-damage-response protects genome integrity

The DNA damage response (DDR) interrupts cell cycle progression to restore genome integrity. However, unchallenged proliferating cells are continually exposed to endogenous stress, raising the question of a stress-threshold for DDR activation. Here, we identified a stress threshold below which primary human fibroblasts, activate a cell-autonomous response that not activates full DDR and not arrests cell cycle progression. We characterized this "pre-DDR" response showing that it triggers the production of reactive oxygen species (ROS) by the NADPH oxidases DUOX1 and DUOX2, under the control of NF-{kappa}B and PARP1. Then, replication stress-induced ROS (RIR) activates the FOXO1 detoxifying pathway, preventing the nuclear accumulation of the pre-mutagenic 8-oxoGuanine lesion, upon endogenous as well as exogenous pro-oxidant stress. Increasing the replication stress severity above the threshold triggers the canonical DDR, leading to cell cycle progression arrest, but also to RIR suppression. These data reveal that cells adapt their response to stress severity, unveiling a tightly regulated "pre-DDR" adaptive response that protects genome integrity without arresting cell cycle progression.

cell biology↗

bioRxiv2020

ATRIP protects progenitor cells against DNA damage in vivo

The maintenance of genomic stability during the cell cycle of progenitor cells is essential for the faithful transmission of genetic information. Mutations in genes that ensure genome stability lead to human developmental syndromes. Mutations in Ataxia Telangiectasia and Rad3-related (ATR) or in ATR-interacting protein (ATRIP) lead to Seckel syndrome, which is characterized by developmental malformations and short life expectancy. While the roles of ATR in replicative stress response and chromosomal segregation are well established, it is unknown how ATRIP contributes to maintaining genomic stability in progenitor cells in vivo. Here, we generated the first mouse model to investigate ATRIP function. Conditional inactivation of Atrip in progenitor cells of the CNS and eye led to microcephaly, microphthalmia and postnatal lethality. To understand the mechanisms underlying these malformations, we used lens progenitor cells as a model and found that ATRIP loss promotes replicative stress and TP53-dependent cell death. Trp53 inactivation in Atrip-deficient progenitor cells rescued apoptosis but increased mitotic DNA damage and mitotic defects. Our findings demonstrate an essential role of ATRIP in preventing DNA damage accumulation during unchallenged replication.

cell biology↗

bioRxiv2019

Mammalian RAD51 prevents non-conservative alternative end-joining and single strand annealing through non-catalytic mechanismssms

The selection of the DNA double-strand breaks (DSBs) repair pathway is decisive for genetic stability/instability. We proposed that it acts according to two successive steps: 1-canonical non-homologous end-joining (C-NHEJ) versus single-strand DNA (ssDNA) resection; 2- on ssDNA, gene conversion (GC) versus non-conservative single-strand annealing (SSA) or alternative end-joining (A-EJ).\n\nUsing intramolecular substrates, we systematically analysed the equilibrium between the different DSB repair pathways. We show that ablation of RAD51 stimulated both SSA and A-EJ but did not stimulate C-NHEJ, validating the two-step model. Moreover, we found that two ATP-mutant dominant-negative forms of RAD51 that stimulated non-conservative repair, failed to load into damaged chromatin, clarifying the role of ATP in RAD51-mediated HR, also. In contrast, another dominant-negative form of RAD51, which retains its DNA binding capacities, repressed SSA and A-EJ, revealing two separable functions of RAD51 i.e. GC and non-conservative repair inhibition. In vitro assays show that the binding of RAD51 on both complementary ssDNA is required to block both spontaneous and RAD52-induced strand annealing. Therefore, RAD51 represses non-conservative repair (SSA and A-EJ), by inhibiting the annealing step through ssDNA occupancy, independently of the catalytic strand-exchange activity required for GC.

molecular biology↗

bioRxiv2019

RAD51 promotes non-homologous genetic rearrangements that are prevented by 53BP1

Homologous recombination (HR), which requires long sequence homologies, is considered a high fidelity mechanism, preserving genome stability. In contrast, we show here that the central HR players RAD51 or BRCA2, promote genetic instability, fostering translocations and capture of ectopic chromosomal sequences when joining distant DNA breaks. Surprisingly, these events do not involve sequence homologies. Moreover, our data reveal that 53BP1 protects against RAD51-mediated non-homologous genetic rearrangements. Finally, analysis of a large panel of breast tumors revealed that BRCA2 proficiency is associated with increased frequency of capture of non-homologous sequences at junctions of structural variants (translocations, duplications, inversions, deletions). These data reveal that HR proteins (RAD51, BRCA2) possess the intrinsic capacity to generate genetic instability through sequence homology-independent processes, and that 53BP1 protects against it. We propose that BRCA2/RAD51-mediated genome instability occurs in the course of sequence homology search for HR.

molecular biology↗

bioRxiv2019

A homozygous hypomorphic BRCA2 variant causes primary ovarian insufficiency without cancer or Fanconi anemia traits.

Primary Ovarian insufficiency (POI) affects 1% of women under forty. We studied a patient with a non-syndromic POI, from a consanguineous Turkish family. Exome sequencing identified a homozygous missense variant c.8524C>T/p.R2842C in BRCA2. BRCA2 is a major player in homologous recombination (HR). BRCA2 deficiency induces cancer predisposition and Fanconi Anemia (FA). Remarkably, neither the patient nor her family exhibit somatic pathologies. The patients somatic cells presented intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation when compared to controls, the heterozygous mothers and FA cells. R2842C-BRCA2 partially complemented BRCA2 depletion for double-strand break-induced HR. The residual HR function in patients cells could explain the absence of somatic pathology. BRCA2 is expressed in human fetal ovaries in pachytene stage oocytes, when meiotic HR occurs. This study has a major impact on the understanding of genome maintenance in somatic and meiotic cells and on the management of POI patients.

genetics↗