Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform

Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent

ERF deletion rescues RAS deficiency in mouse embryonic stem cells

MEK inhibition in combination with a glycogen synthase kinase-3β (GSK3β) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs.We thank Cian Lynch, Jorge Monsech, and Diego Megias for their help with microarray, ChIP-seq, and high-throughput microscopy analyses. We also thank Dr. Manuel Serrano and Dr. André Nussenzweig for their input on the manuscript, and Dr. Diego Sanz for his support throughout the project. C.M.-R. was funded by a PhD fellowship from La Caixa Foundation, T.O. was funded by a PhD fellowship from the Boehringer Ingelheim Fonds, and S.R. was funded by a Ramon y Cajal contract (RYC-2011-09242). Research was funded by Fundación Botín and Banco Santander through its Santander Universities Global Division; grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-23753 and SAF2014- 57791-REDC; these projects were cofinanced with European Fonds Européen de Développement Économique et Régional [FEDER] funds), Fundació La Marato de TV3, Howard Hughes Medical Institute, and the European Research Council (ERC- 617840) to O.F.-C.; and grants from the Spanish Ministryof Economy and Competitiveness (SAF2013-49147-P and SAF2016-80874-P; these projects were cofinanced with European FEDER funds) to S.R. Author contributions: C.M.-R. and S.R. conducted most of the experiments; T.O. helped with the characterization of RASlox/lox mESCs and with ERF localization studies; E.L. helped with ChIP-seq experiments; M.D., S.O., and M.B. contributed to the work on RAS-deficient cells; M.V.-S. provided technical help; O.D. helped with genomics experiments and bioinformatics analysis; and S.R. and O.F.-C. coordinated the study and wrote the manuscript.S

NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity.

The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2-dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2-deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation. Our work reveals that SUMO- and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.The authors want to thank Jordi Torres and Mark O'Driscoll for comments on the manuscript. Work in OF laboratory related to this project was supported by Fundacion Botin, by Banco Santander through its Santander Universities Global Division and by grants from MINECO (SAF2011-23753 and SAF2014-57791-REDC), Howard Hughes Medical Institute, and the European Research Council (ERC-617840). Work in JM laboratory was funded by a grant from MINECO (BFU2013-49153P).S

POLD3 Is Haploinsufficient for DNA Replication in Mice

The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3(+/-) mice were born at sub-Mendelian ratios, and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by the expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.Research was funded by Fundacion Botin, Banco Santander, through its Santander Universities Global Division, and by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2014-59498-R; SAF2014-57791-REDC), Fundacio La Marato de TV3, the Howard Hughes Medical Institute, and the European Research Council (ERC-617840) to O.F.-C.; by a Marie Curie International Outgoing Fellowshp (IOF) from the FP7 Marie Curie Actions and a grant from MINECO (BFU2014-55168-JIN) that was co-funded by European Regional Development Funds (FEDER) to E.L.; by a grant from MINECO (BFU2013-49153) to J.M.; and by the European Commission (ERC grant ONIDDAC) to T.D.H.S

Efficacy of ATR inhibitors as single agents in Ewing sarcoma

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.We would want to thank Enrique de Alava for providing ES lines. Work in O.F. laboratory was supported by Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from MINECO (SAF2014-57791-REDC and SAF2014-59498-R), Fundació La Marato de TV3, Howard Hughes Medical Institute and the European Research Council (ERC-617840). The A.N. laboratory was supported by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research, an Ellison Medical Foundation Senior Scholar in Aging, and the Alex Lemonade Stand Foundation Award. J.A. laboratory is supported by Asociación Pablo Ugarte, ASION-La Hucha de Tomás, Fundación La Sonrisa de Alex and Instituto de Salud Carlos III (PI12/00816 and Spanish Cancer Network RTICC RD12/0036/0027). A.L. laboratory was supported by the Danish National Research Foundation (DNRF115), Danish Council for Independent Research (Sapere Aude, DFF-Starting Grant 2014) and Danish Cancer Society (KBVU-2014).S

USP7 and VCP define the SUMO/Ubiquitin landscape at the DNA replication fork

The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatinMINECO (BFU2014-55168-JIN; RTI2018-093485-B-I00) and a Ramo´ n y Cajal Fellowship from MINECO (RYC-2016-20705), co-funded by European Regional Development Funds (FEDER) to E.L.; by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to O.F.-C.; fellowships from Fundacion Ramón Areces-UAM and La Caixa Foundation to P.V. (LCF/BQ/ES18/11670008

A SUMO and ubiquitin code coordinates protein traffic at replication factories

Post-translational modifications regulate each step of DNA replication to ensure the faithful transmission of genetic information. In this context, we recently showed that deubiquitination of SUMO2/3 and SUMOylated proteins by USP7 helps to create a SUMO-rich and ubiquitin-low environment around replisomes that is necessary to maintain the activity of replication forks and for new origin firing. We propose that a two-flag system mediates the collective concentration of factors at sites of DNA replication, whereby SUMO and Ubiquitinated-SUMO would constitute “stay” or “go” signals respectively for replisome and accessory factors. We here discuss the findings that led to this model, which have implications for the potential use of USP7 inhibitors as anticancer agents.Work in OF laboratory is funded by Fundación Botín, Banco Santander,through itsSantander UniversitiesGlobalDivision and by grants from the SpanishMinistry of Economy and Competitive-ness (MINECO) (SAF2014-59498-R;SAF2014-57791-REDC), Fundaci oLa Mar-ato de TV3, Howard Hughes MedicalInstitute, and the European Research Council (ERC-617840) to OF. EL is fundedby a grant from MINECO (BFU2014-55168-JIN) that is co-funded by European Regional Development Funds (FEDER)Peer reviewe

Targeting ATR in cancer

The chemical treatment of cancer started with the realization that DNA damaging agents such as mustard gas present notable antitumoural properties. Consequently, early drug development focused on genotoxic chemicals, some of which are still widely used in the clinic. However, the efficacy of such therapies is often limited by the side effects of these drugs on healthy cells. A refinement to this approach is to use compounds that can exploit the presence of DNA damage in cancer cells. Given that replication stress (RS) is a major source of genomic instability in cancer, targeting the RS-response kinase ataxia telangiectasia and Rad3-related protein (ATR) has emerged as a promising alternative. With ATR inhibitors now entering clinical trials, we here revisit the biology behind this strategy and discuss potential biomarkers that could be used for a better selection of patients who respond to therapy.Research was funded by Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2014-59498-R and SAF2014-57791-REDC); these projects were co-financed with European Regional Development funds, the Swedish Research council, Cancerfonden (CAN 2015/674) and the European Research Council (ERC-617840) to O.F.-C. and by a grant from MINECO (BFU2014-55168-JIN) that is co-funded with European Regional Development funds to E.L.Peer reviewe

Replication stress and cancer: It takes two to tango

Problems arising during DNA replication require the activation of the ATR–CHK1 pathway to ensure the stabilization and repair of the forks, and to prevent the entry into mitosis with unreplicated genomes. Whereas the pathway is essential at the cellular level, limiting its activity is particularly detrimental for some cancer cells. Here we review the links between replication stress (RS) and cancer, which provide a rationale for the use of ATR and Chk1 inhibitors in chemotherapy. First, we describe how the activation of oncogene-induced RS promotes genome rearrangements and chromosome instability, both of which could potentially fuel carcinogenesis. Next, we review the various pathways that contribute to the suppression of RS, and how mutations in these components lead to increased cancer incidence and/or accelerated ageing. Finally, we summarize the evidence showing that tumors with high levels of RS are dependent on a proficient RS-response, and therefore vulnerable to ATR or Chk1 inhibitors.Work in the OF laboratory is supported by the Spanish Ministry of Science (SAF2011-23753), Association for International Cancer Research (12-0229), Fundació LaMarató de TV3 (33/C (2013)), Howard Hughes Medical Institute and the European Research Council (ERC-210520).Peer reviewe