The evaluation of the DNA damage response, particularly the DNA damage foci formation, is crucial for understanding tumor biology and assessing the impacts of various drugs. We have developed a sophisticated semi-automated image analysis pipeline which generates quantitative map of the spatiotemporal distribution of DNA damage foci within live tumor spheroids. Our framework seamlessly integrates live imaging of tumor spheroids via Light Sheet Fluorescence Microscopy with a DNA damage foci formation assay using a genetically encoded fluorescently labeled DNA damage sensor, the mCherry-tagged minimun domain of 53BP1. By combining advanced imaging techniques with computational tools, our framework offers a powerful tool for studying DNA damage response mechanisms in complex 3D cellular environments.

Supplementary Figure 2 from Identification of N-Terminally Truncated Stable Nuclear Isoforms of CDC25B That Are Specifically Involved in G2/M Checkpoint Recovery

Supplementary Table 1 from Identification of N-Terminally Truncated Stable Nuclear Isoforms of CDC25B That Are Specifically Involved in G2/M Checkpoint Recovery

Melanoma is the most aggressive form of skin cancer. Targeted antimelanoma therapies such as BRAF V600 inhibitors have shown spectacular results. However, these therapies are restricted to BRAF V600 mutated melanoma and display both adverse effects and resistance outbreaks. A high-content screening campaign identified compounds that displayed antimelanoma activities that can potentiate anti–BRAF V600 inhibitors. We identified ivermectin, a US Food and Drug Administration (FDA)-approved macrocyclic lactone widely used as an anthelmintic and insecticidal agent. Macrocyclic lactones possess in vitro cytotoxicity against either BRAF wild type (wt) or BRAF V600 mutated melanoma cells lines. Daily intraperitoneal injections of ivermectin strongly reduce pulmonary metastasis implantation in vivo in murine and melanoma models. Interestingly, these macrocyclic lactones are also able to trigger vemurafenib-dependent cytotoxicity in vitro in BRAF-wt melanoma cells, although vemurafenib action was thought to be restricted to melanoma bearing the BRAF V600 mutation. These macrocyclic lactones are also able to increase the in vitro antimelanoma activity of another BRAF V600 clinical inhibitor, dabrafenib. Serine/threonine p21-activated protein kinase 1 (PAK1) is the key target of ivermectin responsible for its antimelanoma activity. Ivermectin is a promising agent that could strongly increase melanoma therapy efficiency because it is cytotoxic for BRAF-wt and BRAF mutated melanomas and because it increases the efficiency of antimutated BRAF inhibitors independent of the BRAF status of tumors. Because ivermectin is FDA approved and a study of ivermectin would be able to include all patients in the same protocol, this combination treatment should be next investigated in clinical studies.

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

The approval of substantial numbers of targeted biologic therapies (e.g., monoclonal antibodies, fusion proteins) for treatment of inflammatory diseases has positioned these drugs as important to fight chronic disorders such as psoriasis, rheumatoid arthritis and Crohn's disease. One of the concerns raised with the administration of biologic therapies is that because most of them are immunogenic glycoproteins they induce undesirable immune response leading to the generation of specific anti-drug antibodies (ADA). The development of "self" derived protein therapeutics (comprised of human germline sequence), such as recombinant "human" antibodies, helped to reduce the production of ADA but did not avoid all immunogenicity. Reduced efficacy and safety issues such as anaphylaxis or vasculitis accompany the development of ADA. In addition to immune reactions directed against the biologic therapies as a whole, some of them such as anti-TNFα are able to induce auto-immune response, notably antinuclear antibody (ANA). ANA development was associated with induced lupus and in psoriasis it was suggested that it may act as a marker of treatment failure to anti-TNFα. With a focus on psoriasis, this paper makes a current point on the consequences and challenges of the development of anti-drug antibodies and auto-immunity in patients who receive biologic therapies.

There has been considerable interest in targeting cell cycle checkpoints particularly in emerging and alternative anticancer strategies. Here, we show that checkpoint abrogation by AZD7762, a potent and selective CHK1/2 kinase inhibitor enhances genotoxic treatment efficacy in immature KG1a leukemic cell line and in AML patient samples, particularly those with a complex karyotype, which display major genomic instability and chemoresistance. Furthermore, these data suggest that constitutive DNA-damage level might be useful markers to select AML patients susceptible to receive checkpoint inhibitor in combination with conventional chemotherapy. Moreover, this study demonstrates for the first time that AZD7762 inhibitor targets the CD34+CD38-CD123+ primitive leukemic progenitors, which are responsible for the majority of AML patients relapse. Finally, CHK1 inhibition does not seem to affect clonogenic potential of normal hematopoietic progenitors.

Abstract CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage. Cancer Res; 71(5); 1968–77. ©2011 AACR.

Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity in response to DNA injury. The aim of this study was to identify new deubiquitinating enzymes (DUBs) involved in the regulation of the G(2)/M checkpoint. By using an siRNA-based screen to identify DUBs with an inherent ability to enhance a CDC25B-dependent G(2)/M checkpoint bypass, we have identified 11 candidates whose invalidation compromises checkpoint stringency. We subsequently focused our attention on one of these, the previously uncharacterized USP50. Using a TAP-tag approach associated to mass spectrometry, in addition to a yeast-two-hybrid screen, we identified HSP90 as a major interacting partner for USP50. We also demonstrate USP50 depletion causes a loss in accumulation of the HSP90 client Wee1, which is an essential component of the G(2)/M cell cycle arrest. Finally, we show that in response to DNA damaging agents, USP50 accumulates in the nucleus. We propose that USP50 may act through a HSP90-dependent mechanism to counteract CDC25B mitotic inducing activity and prevent Wee1 degradation, thereby repressing entry into mitosis following activation of the DNA damage checkpoint.

Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25B-dependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.

CDC25 inhibition by thiazolopyrimidines: CDC25 is an attractive target for cancer therapy, as it is overexpressed in numerous cancers and is often associated with tumor aggressiveness and poor prognosis. Based on an in silico/in vitro screen, we developed a series of thiazolopyrimidines to further improve inhibitory activity and cytotoxic properties.

CDC25 phosphatases are key actors in cyclin‐dependent kinases activation whose role is essential at various stages of the cell cycle. CDC25 expression is upregulated in a number of human cancers. CDC25 phosphatases are therefore thought to represent promising novel targets in cancer therapy. Here, we report the identification and the characterization of IRC‐083864, an original bis‐quinone moiety that is a potent and selective inhibitor of CDC25 phosphatases in the low nanomolar range. IRC‐083864 inhibits cell proliferation of a number of cell lines, regardless of their resistance to other drugs. It irreversibly inhibits cell proliferation and cell cycle progression and prevents entry into mitosis. In addition, it inhibits the growth of HCT‐116 tumor spheroids with induction of p21 and apoptosis. Finally, IRC‐083864 reduced tumor growth in mice with established human prostatic and pancreatic tumor xenografts. This study describes a novel compound, which merits further study as a potential anticancer agent. © 2008 Wiley‐Liss, Inc.

La dermatite atopique, également appelée eczéma atopique, est une dermatose inflammatoire chronique, s’observant le plus souvent chez le nourrisson, mais pouvant aussi apparaître à l’âge adulte ou chez la personne âgée. Avec une prévalence en hausse, elle constitue aujourd’hui un problème de santé publique, notamment du fait de l’influence de l’environnement sur le génotype.Atopic dermatitis: some general remarks. Atopic dermatitis, also called atopic eczema, is a chronic inflammatory skin disease, usually observed in infants but which can also occur in adults and elderly people. Increasingly prevalent, atopic dermatitis today constitutes a public health issue, notably as a result of the influence of the environment on the genotype.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.