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From a structural point of view, there are many types of DNA damage including single or double strand breaks, base modifications and losses or base-pair mismatches. The amount of lesions that we face is enormous with estimates suggesting that each of our 1013 cells has to deal with around 10.000 lesions per day [1]. While the majority of these events are properly resolved by specialized mechanisms, a deficient response to DNA damage, and particularly to DSB, harbors a serious threat to human health [2].

Summary In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19 Arf and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.

Dear Editor, The purpose of this letter is to address the activity of CGK733, a small molecule that was reported to inhibit both ATM and ATR kinase activities and block checkpoint signaling with great selectivity [1]. This manuscript was retracted [2]. Nevertheless, CGK733 is marketed as an ATM and ATR kinase inhibitor and several manuscripts have reported that CGK733 has effects on human cells [3–7]. We have investigated the effects of CGK733 in cells exposed to either ionizing radiation (IR) or ultraviolet radiation (UV). To determine whether CGK733 inhibits ATM kinase activity we examined the well-characterized IR-inducible ATM kinase-dependent phosphorylations on ATM serine 1981 [8] and CHK2 threonine 68 [9]. We treated H460 human lung cancer cells with 10 μM CGK733, 10 μM KU55933 or 1 μM KU60019 and exposed these to 5 Gy γ-rays from a 137Cs source. KU55933 and KU60019 are inhibitors of ATM kinase activity [10,11]. We selected H460 because we previously documented the ATM-dependent effects of both KU55933 and KU60019 in these cells [12,13] and we selected a concentration of 10 μM CGK733 because this is the highest dose reported to inhibit the kinase activities of ATM and ATR in human cells. We observed that 10 μM CGK733 did not inhibit the IR-induced phosphorylation of either ATM serine 1981 or CHK2 threonine 68 one hour following irradiation whereas both 10 μM KU55933 and 1 μM KU60019, which served as positive controls for the experiment, inhibited the IR-induced phosphorylation of both ATM serine 1981 and CHK2 threonine 68 (Fig. 1A). Similar results were obtained using CGK733 purchased from Tocris Bioscience and Sigma–Aldrich. Fig. 1 (A) Immunoblots of IR-inducible ATM kinase-dependent phosphorylations on ATM serine 1981 and CHK2 threonine 68. H460 human lung cancer cells were treated with 10 μM CGK733 (Tocris Bioscience), 10 μM KU55933 or 1 μM KU60019 (KuDOS ... To determine whether CGK733 inhibits ATR kinase activity we examined the phosphorylation of ATM on serine 1981 and the well-characterized UV-induced phosphorylation on CHK1 serine 317 [14]. We treated H460 cells with 10 μM CGK733 or 10 μM ETP-46464 and exposed these to 10 J/m2 UV. ETP-46464 is an inhibitor of ATR kinase activity [15]. We observed that 10 μM CGK733 did not inhibit the UV-induced phosphorylation of CHK1 serine 317 six hours following irradiation whereas 10 μM ETP-46464, which served as a positive control for the experiment, inhibited the UV-induced phosphorylation of CHK1 serine 317 (Fig. 1B). Further, the phosphorylation of ATM serine 1981 was increased in cells incubated in 10 μM ETP-46464 and exposed to 10 J/m2 UV. This has been observed previously [15] and is consistent with a model in which inhibition of the kinase activity of ATR causes an accumulation of double-strand DNA breaks, perhaps as a result of the disruption of protein complexes that protect stalled replication from endonucleases. We conclude that CGK733 does not inhibit ATM or ATR kinase activity in H460 human lung cancer cells.

One limitation for the study of chromosomal fragile sites is that they must be studied on metaphase spreads, after the breakage. We show here that bacterial lac operator (lacO) repeats are prone to spontaneous breakage, which when combined with a fluorescent lac repressor (lacR) has allowed us to track a fragile site through the cell cycle. By using this system, we show that Plk1-interacting checkpoint helicase (PICH) is already present at fragile sites during interphase, suggesting roles for this helicase beyond mitosis. In addition, we report that the oncogene Myc promotes the formation of anaphase bridges and micronuclei containing fragile-site sequences.

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.

Trying to kill cancer cells by generating DNA damage is by no means a new idea. Radiotherapy and genotoxic drugs are routinely used in cancer therapy. More recent developments also explored the potential of targeting the DNA damage response (DDR) in order to increase the toxicity of radio- and chemo- therapy. Chk1 inhibitors have pioneered studies in this regard. Interestingly, early studies noted that Chk1 inhibitors were particularly toxic for p53-deficient cells. The model proposed for this observation was that this effect was due to the simultaneous abrogation of the G2 (Chk1) and G1 (p53) checkpoints. We here challenge this view, and propose a model where the toxicity of Chk1 inhibitors is rather due to the fact that these compounds generate high loads of replicative stress (RS) during S-phase, which are further boosted by the less restrictive S-phase entry found in p53-deficient cells. This new model implies that the particular toxicity of Chk1 inhibitors might not be restricted to p53-deficient cells, but could be extended to other mutations that promote a promiscuous S-phase entry. In addition, this rationale also implies that the same effect should also be observed for other molecules that target the RS-response (RSR), such as inhibitors of the Chk1-activating kinase ATR.

This ‘Have you seen?' features important insights into genomic aberrations of pluripotent stem cells from studies recently published in Nature, Cell Stem Cell and Cell Death & Differentiation. These findings highlight necessary caution before translating this technology into actual therapies.

Fanconi anemia is characterized by a higher sensitivity to DNA crosslinking agents, including aldehydes. In this issue of Molecular Cell, Oberbeck et al. (2014) reveal that detoxification of aldehydes by pregnant mothers contributes to limit the severity of the disease.

Replication comes with a price. The molecular gymnastics that occur on DNA during its duplication frequently derive to a wide spectrum of abnormalities which are still far from understood. These are brought together under the unifying term "replicative stress" (RS) which likely stands for large and unprotected regions of single-stranded DNA (ssDNA). In addition to RS, recombinogenic stretches of ssDNA are also formed at resected DNA double strand breaks (DSBs). Both situations converge on a ssDNA intermediate, which is the triggering signal for a damage situation. The cellular response in both cases is coordinated by a phosphorylation-based signaling cascade that starts with the activation of the ATR (ATM and Rad3-related) kinase. Given that ATR is essential for replicating cells, understanding the consequences of a defective ATR response for a mammalian organism has been limited until recent years. We here discuss on the topic and review the findings that connect ATR to ageing and cancer.

Genetic overexpression of the protein deacetylase Sir2 increases longevity in a variety of lower organisms, and this has prompted a great interest on the effects of its closest mammalian homologue, Sirt1, on aging and cancer. We have generated transgenic mice moderately overexpressing Sirt1 under its own regulatory elements (Sirt1-tg). Old Sirt1-tg mice present lower levels of DNA damage, decreased expression of the aging-associated gene p16Ink4a, a better general health and fewer spontaneous carcinomas and sarcomas. These effects, however, were not sufficiently potent to affect longevity. To further extend these observations, we developed a metabolic syndrome-associated liver cancer model in which wild-type mice develop multiple carcinomas. Sirt1-tg mice show a reduced susceptibility to liver cancer and exhibited improved hepatic protection from both DNA damage and metabolic damage. Together, these results provide direct proof for the anti-aging activity of Sirt1 in mammals and for its tumour suppression activity in aging- and metabolic syndrome-associated cancer.

ATR recognizes critically short telomeres as fragile sites and protects them from chromosomal fusions., Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

Ageing is an unavoidable corollary to being alive; the most intuitive interpretation of ageing being that it is the consequence of progressive body degeneration. In agreement with this, current models propose that ageing occurs through a stepwise accumulation of DNA damage, which ultimately limits the regenerative capacity of tissues. On the other hand, there is increasing evidence that fetal distress can influence the development of disease in adult life, a phenomenon known as ‘intrauterine programming'. The extent to which an intrauterine exposure to DNA damage can compromise lifespan remains unclear. My group has recently generated a murine model of a human syndrome linked to defective DNA repair and observed that these animals age prematurely, but the accumulation of DNA damage is restricted mostly to the embryonic period. Here, I discuss the implications of this finding and propose that ageing can be influenced by fetal distress.

One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27Kip1 and retinoblastoma tumor suppressors and is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance, but not the initiation, of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ataxia telangiectasia mutated–dependent and ataxia telangiectasia mutated and Rad3-related–dependent DDR but downstream of p38 mitogen-activated protein kinase. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important tumor suppressors in human cancer. [Cancer Res 2009;69(22):8726–32]

The progressive accumulation of DNA damage is thought to be one of the driving forces that initiates ageing. However, the nature of the damage that arises endogenously is still ill-defined. A known source of endogenous damage is replicative stress (RS), which is intrinsically associated to DNA replication and prevented mainly by the ATR kinase. Here, we have developed a murine model of the human Seckel Syndrome characterized by a severe deficiency in ATR. Seckel mice suffer high levels of RS during embryogenesis when proliferation is widespread, but which decrease to marginal levels in postnatal life. In spite of this decrease, adult Seckel mice present accelerated ageing, which is further aggravated in the absence of p53 due to a further increase of RS. Together, these results support the concept that endogenous RS, particularly in utero, contributes to the onset of ageing in postnatal life and this is counterbalanced by the RS-limiting role of the checkpoint proteins ATR and p53.

Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

Neuropilin-1 (NRP-1) is a type 1 membrane protein that binds the axon guidance factors belonging to the class-3 semaforin family. In endothelial cells, NRP-1 serves as a co-receptor for vascular endothelial growth factor (VEGF) and regulates VEGF receptor 2 (VEGFR-2)–dependent angiogenesis. Although gene-targeting studies documenting embryonic lethality in NRP-1 null mice have demonstrated a critical role for NRP-1 in vascular development, the activities of NRP-1 in mature endothelial cells have been incompletely defined. Using RNA interference-mediated silencing of NRP-1 or VEGFR-2 in primary human endothelial cells, we confirm that NRP-1 modulates VEGFR-2 signaling-dependent mitogenic functions of VEGF. Importantly, we now show that NRP-1 regulates endothelial cell adhesion to extracellular matrix proteins independently of VEGFR-2. Based on its dual role as an enhancer of VEGF activity and a mediator of endothelial cell adhesiveness described here, NRP-1 emerges as a promising molecular target for the development of antiangiogenic drugs.

At close hand to one's genomic material are the histones that make up the nucleosome. Standing guard, one variant stays hidden doubling as one of the core histones. But, thanks to its prime positioning, a variation in the tail of H2AX enables rapid modification of the histone code in response to DNA damage. A role for H2AX phosphorylation has been demonstrated in DNA repair, cell cycle checkpoints, regulated gene recombination events, and tumor suppression. In this review, we summarize what we have learned about this marker of DNA breaks, and highlight some of the questions that remain to be elucidated about the physiological role of H2AX. We also suggest a model in which chromatin restructuring mediated by H2AX phosphorylation serves to concentrate DNA repair/signaling factors and/or tether DNA ends together, which could explain the pleotropic phenotypes observed in its absence.

In Neurospora, DNA unpaired in meiosis both is silenced and induces silencing of all DNA homologous to it. This process, called meiotic silencing by unpaired DNA, is thought to protect the host genome from invasion by transposable elements. We now show that silencing of unpaired (unsynapsed) chromosome regions also takes place in the mouse during both male and female meiosis. The tumor suppressor protein BRCA1 is implicated in this silencing, mirroring its role in the meiotic silencing of the X and Y chromosomes in normal male meiosis. These findings impact on the interpretation of the relationship between synaptic errors and sterility in mammals and extend our understanding of the biology of Brca1.

Posttranslational modifications of histone tails regulate numerous biological processes including transcription, DNA repair, and apoptosis. Although recent studies suggest that structural alterations in chromatin are critical for triggering the DNA damage response, very little is known about the nature of DNA damage-induced chromatin perturbations. Here we show that the serine 14 residue in the NH2-terminal tail of histone H2B is rapidly phosphorylated at sites of DNA double-strand breaks. At late time points after irradiation, the phosphorylated form of H2B, H2B-Ser14P, accumulates into irradiation-induced foci. H2B-Ser14P foci formation is not associated with the apoptotic phosphorylation of H2B but is strictly dependent on the phosphorylated isoform of H2AX. Our results broaden the spectrum of histone modifications that constitute the DNA damage “histone code” and suggest a model for the underlying chromatin structure within damage-induced foci.

Living organisms are continually exposed to DNA damage arising from reactive species inside the cell and from environmental sources. Probably the most dangerous form of damage is the DNA doublestrand break (DSB), which interrupts both strands of the molecule. If it is not rapidly resealed, a DSB can cause aberrant chromosomal rearrangements, mutations, or cell death. To protect themselves, organisms from bacteria to humans have developed two major pathways to heal DSBs, homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR utilizes an intact copy (homolog or sister chromatid) of the broken chromosome as a template for repair, whereas the NHEJ pathway joins the two ends of a DSB directly, with little or no requirement for sequence homology. Defects in either of these pathways can compromise genomic integrity and increase the potential for tumorigenesis. Recent studies indicate that, in addition to proteins that directly mediate enzymatic DNA repair, factors that organize specialized chromatin structures surrounding a DSB may facilitate DNA damage signaling and repair. It is well known that histone acetylases and histone deacetylases (HDACs) are important modifiers of chromatin and that they play a central role as transcriptional regulators. The work of Jazayeri et al. (1) in this issue of PNAS demonstrates that the Sin3p/Rpd3p deacetylase complex is required for efficient repair by NHEJ in Saccharomyces cerevisiae . This study sheds light on how chromatin, traditionally viewed as a barrier to DNA-templated processes, can be modified in a manner that increases the faithful transmission of genetic information. Highly localized changes (expansion and contraction) in chromatin structure are necessary to drive many DNA-directed processes, including transcription, replication, recombination, and repair. There are at least two classes of enzymes that promote regional changes in higher-order chromatin structure. The so-called chromatin-remodeling complexes use ATPase activity to catalyze nucleosome mobility. Chromatin structure is also …

A transcription-independent role is identified for the human Polycomb group protein SCML2B in regulating the cell cycle. SCML2B cooperates with p21 to inhibit CDK2/CYCE complexes during early G1, resulting in delayed entry into S phase., Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals., Author Summary The processes of development and differentiation require an exquisite coordination of the gene expression program with the proliferation of the cells. The Polycomb group of proteins are important development regulators and most research to date has focused on their involvement in maintaining epigenetic silencing of genes during development and the self-renewal and differentiation of stem cells. Up to now, we've seen that Polycomb proteins influence the transcriptional status of cell-cycle regulators via chromatin modifications. Here we describe a transcription-independent function for a human Polycomb group protein in regulating the cell cycle. We show that the Polycomb group protein SCML2 directly regulates the progression of cells from G1 into S phase by cooperating with p21 to restrain the activation of CDK2/CYCE complexes in early G1. This function is carried out by the “B” isoform of SCML2 that does not interact with the Polycomb complex PRC1. Further, SCML2B phosphorylation is regulated through the cell cycle and is partly dependent on CDK1 and CDK2. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and cell-cycle machinery in mammals, providing insight into novel functions of the mammalian Polycomb system.

The histone H2A variant H2AX is phosphorylated in response to DNA double-strand breaks originating from diverse origins, including dysfunctional telomeres. Here, we show that normal mitotic telomere maintenance does not require H2AX. Moreover, H2AX is dispensable for the chromosome fusions arising from either critically shortened or deprotected telomeres. However, H2AX has an essential role in controlling the proper topological distribution of telomeres during meiotic prophase I. Our results suggest that H2AX is a downstream effector of the ataxia telangiectasia–mutated kinase in controlling telomere movement during meiosis.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

Heterochromatin is refractory to DNA transactions, including repair. In a recent issue of Molecular Cell, Goodarzi et al. (2008) reveal how the central transducing kinase of the DNA damage response relieves this natural barrier by increasing heterochromatic DNA accessibility.

Our laboratory has focused much of its research in trying to understand how cells respond to "replicative stress" (RS), a type of DNA damage which arises unavoidably every time that a cell replicates its DNA, and which is mainly prevented by a RS-Response (RSR) coordinated by ATR and Chk1 kinases. Given that certain oncogenes can generate substantial amounts of RS, we hypothesized that cells carrying these oncogenes might be “addicted” to a proficient RSR. To explore these ideas, we have generated several cellular, animal and chemical tools for the study of ATR function in mammals. These include (1) a cell system in which ATR can be selectively activated at will; (2) mice with a reduced or increased RSR, and (3) chemical inhibitors of ATR. Our early works suggested that ATR inhibitors would indeed be particularly deleterious for tumors with high levels of RS, such as those induced by the Myc oncogene. Our ideas and new results in this area will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN05-03. Citation Format: Oscar Fernandez-Capetillo. Targeting oncogene-induced DNA damage for cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN05-03.

One of the most discussed recent models for cancer development is based on the finding of an activated DNA damage response in early stages of cancer development. We now know that this DNA damage occurs due to replication stress (RS) induced by oncogenes. In mammalian cells, RS is chiefly limited by the action of ATR and Chk1 kinases. In what regards to cancer, several Chk1 inhibitors have been (and are being) tested in clinical trials, so far with modest results. However, we believe that whereas Chk1 (or ATR) inhibitors might fail as generic anti-cancer therapies, they might be much more efficient for the treatment of tumours with high levels of oncogene-induced RS. To address this hypothesis, we have (1) provided genetic proof-of-principle to show that limited ATR levels are indeed particularly toxic for tumours with high levels of RS (Murga et al Nat Struct Mol Biol, 2011), and (2) generated ATR inhibitors which show synthetic lethal interactions with cancer-associated mutations (Toledo et al Nat Struct Mol Biol, 2011). In what regards to the genetics, and with the use of an ATR hypomorphic mouse strain previously developed in our lab (Murga et al Nat Genet, 2009), we now know that limited ATR levels fully prevent the development of Myc-induced lymphomas or pancreatic tumours. Moreover, we also showed that Chk1 inhibitors are very effective for the treatment of Myc-induced lymphomas. In contrast, these inhibitors were largely ineffective in the treatment of Ras-induced pancreatic tumours, which had no detectable evidences of RS. Hence, these data strongly suggest that the use of Chk1 (or ATR) inhibitors would be particularly beneficial for the treatment of tumours harbouring high levels of RS. Importantly, the levels of RS in tumours can be analyzed in tumour biopsies. Hence, we have provided a rationale that could be readily used in the clinic for a more efficient use of ATR and Chk1 inhibitors in cancer chemotherapy. Until recently, no potent inhibitors of ATR existed. One of the limitations for the discovery of ATR inhibitors is that the activity of the kinase is restricted to replicating cells. This hindered cell-based screenings due to the large number of false positives that would derive from an indirect effect of the tested compound on the cell cycle. Overcoming this limitation, we previously developed a cellular system in which ATR activity can be unleashed at will, throughout the cell cycle and in the absence of any actual DNA damage (Toledo et al Genes & Dev 2008). After adapting this system for a High-Throughput Imaging pipeline, and with the help of the Experimental Therapeutics Programme of the CNIO, we have now identified several compounds that can inhibit ATR in the nanomolar range (Toledo et al Nat Struct Mol Biol, 2011). We are now at the early stages of characterizing these inhibitors and their potential uses, and have shared these reagents with many investigators around the world. Besides the published ones, we have now also identified some compounds with good pharmacological properties in vivo, so that we are now ready to test the effect of ATR inhibitors in mouse preclinical models of cancer. References 1. Toledo, L.I., Murga, M., Gutierrez-Martinez-P, R. Soria and Fernandez-Capetillo, O. (2008). ATR signaling can drive cells into senescence in the absence of DNA breaks. Genes and Dev 22:297-302. 2. Murga, M., Buenting, S., Montaña, M.F., Soria, R, Lee, Y.S., Mulero, F., Cañamero, M., McKinnon, P.J., Nussenzweig, A. and Fernandez-Capetillo, O. (2009). A mouse model of the ATR-Seckel Syndrome reveals that intrauterine exposure to replicative stress limits mammalian lifespan. Nat Genet 41(8):891-8. 3. Toledo, L.I., Murga, M., Zur, R. Oyarzabal, J., Bischoff, J.R., Fernandez-Capetillo, O. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. (2011) Nat Struct Mol Biol. 18(6):721-7. 4. Murga, M., Campaner, S., Lopez-Contreras, A.J., Toledo, L.I., Soria, R., Montaña, M.F., D'Artista, L., Scheckler, T., Guerra, C., Garcia, E., Barbacid, M., Hidalgo, M., Amati, B., Fernandez-Capetillo, O. Exploiting oncogene-induced replicative stress for the selective elimination of Myc-driven tumors. Nat Struct Mol Biol. doi: 10.1038/nsmb.2189 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY23-03. doi:1538-7445.AM2012-SY23-03

Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with γ-irradiation, meiotic recombination, DNA replication, and antigen receptor rearrangements. Here, we show that loss of a single H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and H2AX −/− p53 −/− lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c- myc oncogene to antigen receptor loci. Restoration of the H2AX null allele with wild-type H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

As part of Cell’s 40th anniversary celebration, we are spotlighting 40 principal investigators under the age of 40, and we asked each of them to describe their biggest personal challenges while working in science. See the full profiles of all our “40 under 40” scientists and their responses to this and other questions at http://www.cell.com/40/under40.

Stalled replication forks occasionally collapse, leading to potentially catastrophic DNA double-strand breaks. Now, Toledo et al. (2013) reveal that fork breakage occurs when the pool of the single-strand DNA-binding protein RPA becomes exhausted. This study has important implications for the origin and treatment of cancers with high levels of replicative stress.