An understanding of nuclear reprogramming is fundamental to the use of cells in regenerative medicine. Due to technological obstacles, the time course and extent of reprogramming of cells following ...fusion has not been assessed to date. Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days. This study was made possible by the development of a broadly applicable approach, species-specific transcriptome amplification (SSTA), which enables global resolution of transcripts derived from the nuclei of two species, even when the proportions of species-specific transcripts are highly skewed. Remarkably, either phenotype can be dominant; an excess of primary keratinocytes leads to activation of the keratinocyte program in muscle cells and the converse is true when muscle cells are in excess. We conclude that nuclear reprogramming in heterokaryons is rapid, extensive, bidirectional, and dictated by the balance of regulators contributed by the cell types.--Adam Palermo, Regis Doyonnas, Nidhi Bhutani, Jason Pomerantz, Ozan Alkan, Helen M. Blau. Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional.
Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation ...predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a ...sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Abstract
Introduction.
Ataxia telangiectasia and Rad3-related (ATR) protein kinases have been identified as a key part of the DNA damage repair processes (DDRP) and cell cycle signaling. The DDRP is ...known to stimulate DNA repair, promote cell survival and, consequently, can diminish the therapeutic effect of existing DNA-damaging chemotherapy agents and ionizing radiation (IR) therapy. Therefore, there is a need for the development of potent and selective therapies to deliver ATR inhibitors for the treatment of cancer, as part of rational combination regimens with DNA damaging therapies. In this work, we develop and characterize a novel inhibitor of ATR kinase (BG129) and its nano-liposomal formulation (nLs-BG129).
Methods. In vitro activity of novel ATR inhibitors (BG129) was tested in a broad panel of cancer cell lines as a monotherapy or in combination with DNA-damaging chemotherapeutics (carboplatin, gemcitabine, and SN-38). On-target effects of BG129 in vitro were evaluated by Western-Blot to demonstrate dose-dependent inhibition of pChk1 and pRPA2, and a corresponding increase in γH2AX. The ATR inhibitor was encapsulated into unilamellar liposomal vesicles formed from hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG) at a 3:2:0.15 molar ratio and loaded stably into liposomes employing a triethylammonium sucroseoctasulfategradient. Liposomes were characterized by size and drug loading efficiency. Pharmacokinetic (PK) properties of nLs-BG129 were evaluated in mice. The antitumor activity of nLs- BG129 in combination with nano-liposomal irinotecan (nal-IRI) was tested in vivo in ovarian and various lung cancer xenograft models.
Results.
BG129 synergized with SN-38, gemcitabine, and carboplatin in vitro in cell-based assays. On-target dose-dependent inhibition of pChk1 and pRPA2 and a corresponding increase in γH2AX were observed in cells, following treatment with BG129. Nano-liposomal BG129 showed dramatically improved pharmacokinetics (AUC = 2835 μg/ml*h), compared to either free BG129 or VE-822/M6620/Berzosertib (AUC = 18.18 μg/ml*h). nLs-BG129 also enhanced antitumor activity of nal-IRI in multiple ovarian and SCLC and NSCLC xenograft models, when given in combination and was well tolerated.
Conclusions. Here we introduced a novel nano-liposomal formulation of an ATRi with good in vivo stability and prolonged clearance, with the ability to enhance the anti-tumor activity of DNA-damaging chemotherapeutics in vivo. The nano-liposomal formulation of ATRi can potentially sensitize tumor responses to DNA-damaging agents and improve their activity and tolerability in cancer patients when compared to unencapsulated and fast clearing ATR inhibitors, such as those currently in the clinic.
Citation Format: Alexander Koshkaryev, Ozan Alkan, Bolin Geng, Lia Luus, Andreas Raue, Walid Kamoun, Suresh Tipparaju, Dmitri Kirpotin, Daryl Drummond. Targeting DNA-damage response pathway with a novel nano-liposomal ATR inhibitor in solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 8.
Abstract
Most commonly used anti-cancer therapies involve the systemic administration of anti-proliferative and DNA damaging agents. Deregulation of DNA damage response (DDR) pathways can cause ...resistance to DNA damaging therapies. Synergistic combination strategies of novel DDR pathway modulators promise to re-sensitize to and amplify currently used standard-of-care therapies.
Our goal is to gain mechanistic understanding of the DDR pathway by developing a predictive computational model using single cell microscopy phenotypic data and signaling data from a panel of cancer cell lines. We focus on synergistic and potentiating effects of drug combinations using standard-of-care drugs such as doxorubicin, gemcitabine and irinotecan, and DDR pathway modulators. Our computational model explains the mechanisms of synergistic or additive phenotype that are observed for various modulators and was validated using DDR signaling data.
The presented computational model rationalizes the design of novel combination therapies targeting the DDR pathway, includes heterogeneity of different cell lines and is an important step towards the design of a biomarker strategy.
Citation Format: Andreas Raue, Ozan Alkan, Daryl Drummond, Birgit Schoeberl. Model guided understanding of synergistic combination therapies in the DNA damage response pathway. abstract. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-34.
Abstract
Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a ...multi-scale computational model using the U2OS osteosarcoma cancer cell line that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated based on an extensive data set that comprises cell cycle distribution of the initial cell population, signaling data measured by western blot, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model can predict the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we were able to confirm experimentally. Computational models, like the one presented here, can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death, as well as to rationally identify chemotherapy-potentiating drug combinations.
Citation Format: Ozan Alkan, Birgit Schoeberl, Millie Shah, Alexander Koshkaryev, Tim Heinemann, Daryl Drummond, Michael B. Yaffe, Andreas Raue. Understanding chemotherapy-induced replicative stress to identify rational combination therapies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2830.
Background Ankyrin 3 ( ANK3 ) has been strongly implicated as a risk gene for bipolar disorder (BD) by recent genome-wide association studies of patient populations. However, the genetic variants of ...ANK3 contributing to BD risk and their pathological function are unknown. Methods To gain insight into the potential disease relevance of ANK3 , we examined the function of mouse Ank3 in the regulation of psychiatric-related behaviors using genetic, neurobiological, pharmacological, and gene-environment interaction (G×E) approaches. Ank3 expression was reduced in mouse brain either by viral-mediated RNA interference or through disruption of brain-specific Ank3 in a heterozygous knockout mouse. Results RNA interference of Ank3 in hippocampus dentate gyrus induced a highly specific and consistent phenotype marked by decreased anxiety-related behaviors and increased activity during the light phase, which were attenuated by chronic treatment with the mood stabilizer lithium. Similar behavioral alterations of reduced anxiety and increased motivation for reward were also exhibited by Ank3+/– heterozygous mice compared with wild-type Ank3+/+ mice. Remarkably, the behavioral traits of Ank3 +/– mice transitioned to depression-related features after chronic stress, a trigger of mood episodes in BD. Ank3 +/– mice also exhibited elevated serum corticosterone, suggesting that reduced Ank3 expression is associated with elevated stress reactivity. Conclusions This study defines a new role for Ank3 in the regulation of psychiatric-related behaviors and stress reactivity that lends support for its involvement in BD and establishes a general framework for determining the disease relevance of genes implicated by patient genome-wide association studies.
Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that ...the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and β₁-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.
We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a ...Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.
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