Arqueología en la ACEGA 2: el área arqueológica de O Peto (Vedra, A Coruña)

Cadernos de Arqueoloxía e Patrimonio (CAPA)[EN] The archaeological area of O Peto was discovered during the construction of the highway Santiago-Alto de Santo de Domingo. This site shows the existence of archaeological structures near of the galician Iron Age hillforts. In fact O Peto is an artificialized space (where a set of several structures was exhumed) that belongs to a prerroman iron mining complex that suffered several changes in the beginning of Romanization.[ES] El área arqueológica de O Peto se descubrió durante el control arqueológico de la construcción de la Autopista Santiago-Alto de Santo Domingo. Se trata de un ejemplo significativo de la existencia de estructuras anejas en el entorno inmediato del recinto habitacional de los castros de la Edad del Hierro. Constituye un espacio claramente artificializado en el que se superponen estructuras excavadas en la roca, de naturaleza y finalidad diferentes. A este respecto se configura un espacio construido –aparentemente multifuncional- que experimentó sucesivos procesos de ampliación, redefinición, sellado intencionado y abandono entre la Edad del Hierro y Época Romana. La información aportada por la excavación indica que este yacimiento albergó un complejo minerometalúrgico caracterizado tecnológicamente por la utilización de hornos bajos prerromanos sin sangrado de escoria que procesarían el mineral extraído en el propio yacimiento.Proyecto financiado por la Dirección Xeral de Investigación e Desenvolvemento da Consellería de Innovación, Industria e Comercio (Xunta de Galicia) con cargo a la convocatoria Programa de Tecnoloxías para a Innovación- Tecnoloxías da Construcción e da Conservación do Patrimonio do ano 2004. Código de Proxecto: PGIDIT04CCP606003PRPeer reviewe

Arqueología en la ACEGA 2: el área arqueológica de O Peto (Vedra, A Coruña)

Cadernos de Arqueoloxía e Patrimonio (CAPA)[EN] The archaeological area of O Peto was discovered during the construction of the highway Santiago-Alto de Santo de Domingo. This site shows the existence of archaeological structures near of the galician Iron Age hillforts. In fact O Peto is an artificialized space (where a set of several structures was exhumed) that belongs to a prerroman iron mining complex that suffered several changes in the beginning of Romanization.[ES] El área arqueológica de O Peto se descubrió durante el control arqueológico de la construcción de la Autopista Santiago-Alto de Santo Domingo. Se trata de un ejemplo significativo de la existencia de estructuras anejas en el entorno inmediato del recinto habitacional de los castros de la Edad del Hierro. Constituye un espacio claramente artificializado en el que se superponen estructuras excavadas en la roca, de naturaleza y finalidad diferentes. A este respecto se configura un espacio construido –aparentemente multifuncional- que experimentó sucesivos procesos de ampliación, redefinición, sellado intencionado y abandono entre la Edad del Hierro y Época Romana. La información aportada por la excavación indica que este yacimiento albergó un complejo minerometalúrgico caracterizado tecnológicamente por la utilización de hornos bajos prerromanos sin sangrado de escoria que procesarían el mineral extraído en el propio yacimiento.Proyecto financiado por la Dirección Xeral de Investigación e Desenvolvemento da Consellería de Innovación, Industria e Comercio (Xunta de Galicia) con cargo a la convocatoria Programa de Tecnoloxías para a Innovación- Tecnoloxías da Construcción e da Conservación do Patrimonio do ano 2004. Código de Proxecto: PGIDIT04CCP606003PRPeer reviewe

The Florida pancreas collaborative next-generation biobank: Infrastructure to reduce disparities and improve survival for a diverse cohort of patients with pancreatic cancer

Background: Well-annotated, high-quality biorepositories provide a valuable platform to support translational research. However, most biorepositories have poor representation of minority groups, limiting the ability to address health disparities. Methods: We describe the establishment of the Florida Pancreas Collaborative (FPC), the first state-wide prospective cohort study and biorepository designed to address the higher burden of pancreatic cancer (PaCa) in African Americans (AA) compared to Non-Hispanic Whites (NHW) and Hispanic/Latinx (H/L). We provide an overview of stakeholders; study eligibility and design; recruitment strategies; standard operating procedures to collect, process, store, and transfer biospecimens, medical images, and data; our cloud-based data management platform; and progress regarding recruitment and biobanking. Results: The FPC consists of multidisciplinary teams from fifteen Florida medical institutions. From March 2019 through August 2020, 350 patients were assessed for eligibility, 323 met inclusion/exclusion criteria, and 305 (94%) enrolled, including 228 NHW, 30 AA, and 47 H/L, with 94%, 100%, and 94% participation rates, respectively. A high percentage of participants have donated blood (87%), pancreatic tumor tissue (41%), computed tomography scans (76%), and questionnaires (62%). Conclusions: This biorepository addresses a critical gap in PaCa research and has potential to advance translational studies intended to minimize disparities and reduce PaCa-related morbidity and mortality

Cyclin-Dependent Kinases Regulate Ig Class Switching by Controlling Access of AID to the Switch Region

Ig class switching requires cell proliferation and is division linked, but the detailed mechanism is unknown. By analyzing the first switching cells early in the kinetics, our analysis suggested that proliferating B cells had a very short G(1) phase (<3.5 h), a total cell cycle time of ∼11 h, and that Ig class switching preferentially occurred in the late G(1) or early S phase. Inhibition of cyclin-dependent kinases (CDKs) caused dramatic reduction of switching rate within 6 h. This was associated with less targeting of activation-induced cytidine deaminase (AID) to the Igh locus. Interestingly, ectopically expressed nuclear AID in HeLa cells was preferentially found in the early S phase. Furthermore, in CDK2 hypomorphic cells there was reduced nuclear AID accumulation. Thus, our data are compatible with the idea that division-linked Ig class switching is in part due to CDK2-regulated AID nuclear access at the G(1)/S border

Novel Role of LMO2 in DNA Repair Control in Diffuse Large B Cell Lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of Non-Hodgkin Lymphoma (NHL). However, about 40% of patients still do not respond to the current therapy and die. Therefore, there is an urgent need for developing new strategies to improve their outcome. We have shown that LIM domain Only 2 (LMO2) expression is increased in germinal center (GC) B-lymphocytes and GC-derived lymphomas. LMO2 protein is the best single prognostic biomarker which high expression is associated with longer survival of DLBCL patients treated with R-CHOP therapy, irrespective of the cell of origin of these tumors. LMO2 is indirectly involved in gene regulation by forming multipartite DNA-binding complexes with other transcription factors. LMO2 plays an important role in the normal development of hematopoietic and endothelial system, in erythropoiesis, and functions as an oncogene in T-cell acute lymphoblastic leukemia. However, its role in B cell biology as well as B-cell lymphoma is still unclear. We have recently shown that LMO2 over-expression in DLBCL results in genomic instability (Cubedo et al Blood 2012). These studies suggested that LMO2 expression could compromise proper DNA repair. To address whether LMO2 has a role in DNA repair, we evaluated the capacity of DLBCL cells with high expression of LMO2 (DLBCLLMO2+) to repair DNA double-strand breaks (DSBs). DNA DSBs are repaired by two major pathways: Non-homologous end joining (NHEJ) and Homologous recombination (HR). Therefore, next we evaluated whether LMO2 affects the activity of these DSBs repair pathways. To this end, we analyzed the core factors: 53BP1 for NHEJ and Rad51 for HR. We exposed DLBCLLMO2+ and controlcells to genotoxic agents used in DLBCL therapy to produce DSBs and evaluated 53BP1 and Rad51 recruitment to DSBs site via immunofluorescence (IF) studies. Phosphorylated form of H2AX, known as gammaH2AX was used as a DSB molecular marker. In these studies we found a similar frequency of 53BP1 foci in both DLBCLLMO2+ and control cells. These results suggested that NHEJ is active irrespective of LMO2 protein levels. However, DLBCLLMO2+ cells showed a decreased frequency of Rad51 foci after DNA damage. Importantly, DLBCLLMO2+ cells showed similar Rad51 protein levels and cell-cycle distribution when compared to control cells. These results showed a deficient HR pathway in DLBCLLMO2+ cells and suggested a role for LMO2 in the control of HR. In support of this, overexpression of LMO2 in control cells produced a similar HR-deficiency as observed in DLBCLLMO2+ cells. In addition, knockdown of LMO2 in DLBCLLMO2+ cells restored Rad51 foci formation to the levels observed in control cells. These results showed that HR activity in DLBCL cells depends on LMO2 protein levels. It is well known that deficiency in HR can be rescued by deleting 53BP1, which is a HR inhibitor. Hence, in order to address the question whether deletion of 53BP1 in DLBCLLMO2+ cells can rescue HR pathway, we knockdown 53BP1 in DLBCLLMO2+ cells via shRNA and examined the frequency of Rad51 foci after exposure to different genotoxic agents. Interestingly, we found an increase in Rad51 foci formation in DLBCLLMO2+sh53BP1 cells to near control cells levels. This result shows that LMO2-dependent inhibition of HR depends on 53BP1 and suggests that LMO2 and 53BP1 form a complex at the DSB site. Two observations support this hypothesis: 1) LMO2 co-localize with 53BP1 at the DSB site; and 2) LMO2 expression increases the frequency of 53BP1-Rif1 complex at the DSB site. Since Rif1 (Rap1-interacting factor 1) is one of the 53BP1 effector proteins that inhibits HR, here we propose that LMO2 inhibition of HR is through the stabilization of the 53BP1-Rif1 complex. Our study reveals a novel role for LMO2 in the control of genome stability by controlling homologous recombination repair activity. Defect in HR pathway in LMO2 expressing cells could explains why DLBCL patients with higher level of LMO2 are more sensitive to chemotherapy, and its significance as the best prognostic marker for survival. Our studies can potentially give new insight for the treatment of DLBCL patient and improve their outcome. Disclosures No relevant conflicts of interest to declare

UNG protects B cells from AID-induced telomere loss

Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG). In contrast, in the absence of UNG activity, deleterious processing by mismatch repair leads to telomere loss and defective cell proliferation. Indeed, we show that UNG deficiency reduces B cell clonal expansion in the germinal center in mice and blocks the proliferation of tumor B cells expressing AID. We propose that AID-induced damage at telomeres acts as a fail-safe mechanism to limit the tumor promoting activity of AID when it overwhelms uracil excision repair

Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR

A Novel Mouse Model for the Hyper-IgM Syndrome: A Spontaneous Activation-Induced Cytidine Deaminase Mutation Leading to Complete Loss of Ig Class Switching and Reduced Somatic Hypermutation

We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G→A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies

LMO2 Facilitates Synthetic Lethality after PARP Inhibition (PARPi) in Diffuse Large B-Cell Lymphoma (DLBCL)

Abstract Novel therapies for DLBCL are needed to improve patients' outcomes. LIM domain only 2 (LMO2) protein is ubiquitously expressed and plays important roles in endothelial and hematopoietic cell development. LMO2 protein expression is upregulated in germinal center B (GCB) cells, the cell of origin of DLBCL. 73% of GCB and 45% of ABC DLBCLs express LMO2 protein at levels of reactive GCB cells. Although the function of LMO2 in B cells and DLBCL is unknown, expression of LMO2 serves as one of the best prognostic markers of longer survival following R-CHOP therapy. Additionally, LMO2 expression in DLBCL cells results in genomic instability. These observations suggest that LMO2 may decrease DNA repair efficiency. Indeed, here we demonstrate that primary DLBCL tumors and cell lines expressing high levels of LMO2 protein (LMO2HIGH) are defective in DNA double-strand break (DSB) repair via the homologous recombination (HR) pathway. We found that LMO2HIGH DLBCL cells and LMO2LOW DLBCL cells expressing a GFP-LMO2 fusion protein via a doxycycline-inducible system have a defective accumulation of the HR proteins BRCA1 and RAD51 to damaged chromosomes as visualized by immunofluorescence (IF) and Western blot assays. Furthermore, LMO2HIGH DLBCL exposed to ionizing radiation showed decreased levels of phosphorylated (S4 and S8) replication protein A (RPA32 subunit), a marker of DSB end-resection activity and an essential step for HR-dependent DSB repair. Consequently, LMO2HIGH DLBCL showed decreased HR activity as assessed via a DR-GFP reporter system and scoring the frequency of HR-dependent sister chromatid exchanges. Also, in LMO2HIGH vs LMO2LOW DLBCLs, we observed higher levels of ionizing radiation-induced foci (IRIF) for 53BP1 and RIF1 - non-homologous end-joining (NHEJ) core factors playing a critical role in defining DSB repair pathway choice. Similarly, we found a higher accumulation of 53BP1 and RIF1 chromatin-enriched fraction after DNA damage in LMO2HIGH than LMO2LOW DLBCLs. Furthermore, we show that LMO2 forms a complex with 53BP1 but not BRCA1 after DNA damage, as demonstrated by Co-IP, GST pull-down assays and spatial co-localization in IRIFs. This suggests that LMO2 functionally interacts with 53BP1 during DSB repair. Indeed, knockdown of 53BP1 in LMO2HIGH cells increased the levels of BRCA1 and RAD51 IRIF to values similar to LMO2LOW cells without affecting LMO2 levels, revealing that LMO2 depends on 53BP1 to inhibit HR activity. Since LMO2HIGH DLBCL cells exhibit a defective HR-pathway, we next explored the therapeutic potential of PARPi in DLBCL. We found that in LMO2HIGH but not LMO2LOW DLBCL cell lines the PARPi olaparib induced a significant decrease in cell proliferation and colony formation and an increase in cell death via apoptosis. The sensitivity to olaparib directly correlated with LMO2 protein levels. The proliferation defect in LMO2HIGH DLBCL cells was due to the increased DNA damage caused by exposure to PARPi, as observed by an increase in γH2AX foci. Induction of LMO2 expression in LMO2LOW DLBCL cell lines led to sensitivity to olaparib, demonstrating that the proliferation defect induced by olaparib was dependent on LMO2 expression. Silencing of LMO2 via shRNA or CRISP/Cas9 in LMO2HIGH cell lines rescued the proliferation defect induced by olaparib. The proliferation deficiency induced by olaparib was synergistic with doxorubicin in LMO2HIGH but not in LMO2LOW DLBCL cell lines and untreated patient-derived primary DLBCL tumors. We also examined the in vivo efficacy of olaparib in DLBCL mice models. Olaparib treatment prolonged survival of mice harboring LMO2HIGH but not LMO2LOW DLBCL xenograft tumors. Olaparib in combination with RCHOP significantly prolonged survival of mice harboring LMO2HIGH DLBCL tumors compared to cohorts treated with either olaparib or RCHOP alone. Further, analysis of tumors excised from OCI-LY1 bearing animals treated with olaparib for 3 days revealed increased cell death and a higher sensitivity to PARPi in OCI-LY1 LMO2 cells compared to the OCI-LY1 LMO2 negative-GFP control cells, indicating in vivo survival advantage for LMO2LOW cells upon olaparib treatment. In summary, high expression of LMO2 results in HR-dysfunction phenocopying the BRCA1/2 mutations observed in breast and ovarian tumors. In LMO2HIGH DLBCL, PARPi-induced killing is synergistic with doxorubicin, thus providing a clear path for therapeutic development of PARPi in DLBCL. Figure. Figure. Disclosures Lossos: Affimed: Research Funding

LMO2 Confers Synthetic Lethality to PARP Inhibition in DLBCL

Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCLs) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2-positive DLBCLs and T cell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Furthermore, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2-positive tumors. Together, our results reveal that LMO2 expression predicts HR deficiency and the potential therapeutic use of PARP inhibitors in DLBCL and T-ALL.This work was supported by grant 1R01CA233945 from the National Cancer Institute, Florida Health Bankhead-Coley Cancer Research Award AWD-005151 and the Intramural Funding Program from the University of Miami SCCC (to R.E.V. and I.S.L.). I.S.L. is supported by the Dwoskin and Anthony Rizzo Families Foundations and Jaime Erin Follicular Lymphoma Research Consortium. R.E.V. is supported by R01GM121595 from the National Institute of General Medical Sciences and funds from the University of Miami SCCC