po 288 replication stress response as a target for eradicating colorectal cancer stem cells

Introduction Cancer stem cells (CSCs) are subsets of multipotent SCs responsible for tumour development, propagation and evolution, whose targeting is required for tumour eradication. There is (pre)clinical evidence on a role of CSCs in therapeutic resistance and intra-tumour heterogeneity, which limits the efficacy of antineoplastic regimens. In this context, CSCs reportedly share with embryonic/adult SCs a very robust DNA damage response, which favours the survival and resistance to genotoxins, and can be exploited for therapeutic purposes. Material and methods We generated a panel of ~30 CRC patient-derived tumorspheres enriched for CSCs (CRC-SCs) and characterised them at the genetic level. To discover potential monotherapeutic anti-CSC agents, we performed high-throughput screenings on multiple CRC-SCs with a library of clinically-relevant drugs. Flow cytometry, fluorescence microscopy and epistatic analyses were conducted to uncover the mechanism of action of identified compound(s), while genetic, cytogenetic and phosphoproteomic studies were carried out to identify predictive biomarkers. DNA replication stress (RS) levels were evaluated by analysing phosphorylated ATM/RPA foci and by performing COMET and DNA fibre assays, and were modulated by single administration of genome destabilising agents or by prolonged exposure to increased doses of compounds targeting the replication stress response (RSR). Results and discussions We demonstrated that the CHK1 inhibitor LY2606368 is a potent anti-CSC agent able to kill more than one third of CRC-SCs, both in vitro and in vivo. Moreover, we provided evidence of high but heterogeneous RS levels in CRC-SCs, showing that, in CRC-SCs, RS is mainly boosted endogenously by p53 deficiency, supernumerary chromosomes and DNA replication abnormalities, which results in high dependency on CHK1-mediated RSR. Accordingly, formerly LY2606368-resistant CRC-SCs were sensitised by boosting DNA replication errors or inducing whole-genome doubling, while formerly LY2606368-sensitive CRC-SCs made resistant by the continuous in vitro or in vivo administration of LY2606368 displayed diminished RS levels due to RSR rewiring, and became independent on CHK1. Conclusion Our results demonstrate that RSR is efficient and rewirable in CSCs thereby constituting a prominent therapeutic target. In particular, we designed dedicated RS-modulating or RSR-targeting strategies for long-term CSC depletion in CRC

Replication stress in colorectal cancer stem cells

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NA61/SHINE facility at the CERN SPS: beams and detector system

NA61/SHINE (SPS Heavy Ion and Neutrino Experiment) is a multi-purpose experimental facility to study hadron production in hadron-proton, hadron-nucleus and nucleus-nucleus collisions at the CERN Super Proton Synchrotron. It recorded the first physics data with hadron beams in 2009 and with ion beams (secondary 7Be beams) in 2011. NA61/SHINE has greatly profited from the long development of the CERN proton and ion sources and the accelerator chain as well as the H2 beamline of the CERN North Area. The latter has recently been modified to also serve as a fragment separator as needed to produce the Be beams for NA61/SHINE. Numerous components of the NA61/SHINE set-up were inherited from its predecessors, in particular, the last one, the NA49 experiment. Important new detectors and upgrades of the legacy equipment were introduced by the NA61/SHINE Collaboration. This paper describes the state of the NA61/SHINE facility - the beams and the detector system - before the CERN Long Shutdown I, which started in March 2013

Multiplicity and transverse momentum fluctuations in inelastic proton-proton interactions at the CERN Super Proton Synchrotron

Measurements of multiplicity and transverse momentum fluctuations of charged particles were performed in inelastic p+p interactions at 20, 31, 40, 80 and 158 GeV/c beam momentum. Results for the scaled variance of the multiplicity distribution and for three strongly intensive measures of multiplicity and transverse momentum fluctuations \$\Delta[P_{T},N]\$, \$\Sigma[P_{T},N]\$ and \$\Phi_{p_T}\$ are presented. For the first time the results on fluctuations are fully corrected for experimental biases. The results on multiplicity and transverse momentum fluctuations significantly deviate from expectations for the independent particle production. They also depend on charges of selected hadrons. The string-resonance Monte Carlo models EPOS and UrQMD do not describe the data. The scaled variance of multiplicity fluctuations is significantly higher in inelastic p+p interactions than in central Pb+Pb collisions measured by NA49 at the same energy per nucleon. This is in qualitative disagreement with the predictions of the Wounded Nucleon Model. Within the statistical framework the enhanced multiplicity fluctuations in inelastic p+p interactions can be interpreted as due to event-by-event fluctuations of the fireball energy and/or volume.Comment: 18 pages, 12 figure

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations

Enhanced repair of DNA interstrand crosslinking in ovarian cancer cells from patients following treatment with platinum-based chemotherapy

Despite high tumour response rates to platinum-based chemotherapy in ovarian cancer survival is poor due to the emergence of drug resistance. Mechanistic studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. A modification of the single cell gel electrophoresis (comet) assay allows the sensitive detection of DNA interstrand crosslinking in both tumour and normal cells derived directly from clinical material. Tumour cells isolated from 50 ovarian cancer patients were treated ex vivo with 100 μM cisplatin for 1 h and crosslink formation and repair (unhooking) measured. No significant difference in the peak level of crosslinking in tumour cells was observed between patients who were either newly diagnosed or previously treated with platinum-based therapy, or between tumour and mesothelial cells from an individual patient. This indicates no difference in cellular mechanisms such as drug transport or detoxification. In contrast, the percentage repair (unhooking) of DNA interstrand crosslinks was much greater in the group of treated patients. At 24 h in the 36 newly diagnosed patient tumour samples, only one gave >50% repair and 23 gave <10% repair; however, 19 out of 22 treated patient samples gave >10% repair and 14 showed >50% repair. The estimated median difference (newly diagnosed minus treated) was −52 (95% CI −67 to −28), and the P-value from a Mann–Whitney test was <0.001. In eight patients, it was possible to obtain tumour samples prior to any chemotherapy, and also on relapse or at interval debulking surgery following platinum-based chemotherapy. In these patients, the mean % repair prior to therapy was 2.85 rising to 71.23 following treatment. These data demonstrate increased repair of DNA interstrand crosslinks in ovarian tumour cells following platinum therapy which may contribute to clinical acquired resistance

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation