Publications by authors named "Dominique Kranz"

11 Publications

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eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled.

J Biol Chem 2020 06 7;295(26):8759-8774. Epub 2020 May 7.

Institute of Biological and Chemical Systems-Functional Molecular Systems (IBCS-FMS), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted ()/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.
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http://dx.doi.org/10.1074/jbc.RA120.012892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324525PMC
June 2020

A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment.

Cell Death Differ 2018 12 7;25(12):2053-2070. Epub 2018 Mar 7.

Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany.

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.
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http://dx.doi.org/10.1038/s41418-018-0080-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261943PMC
December 2018

β-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon cancer cells.

Sci Rep 2018 02 16;8(1):3178. Epub 2018 Feb 16.

German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Medical Faculty Mannheim, 69120, Heidelberg, Germany.

Wnt signaling is an evolutionarily conserved signaling route required for development and homeostasis. While canonical, β-catenin-dependent Wnt signaling is well studied and has been linked to many forms of cancer, much less is known about the role of non-canonical, β-catenin-independent Wnt signaling. Here, we aimed at identifying a β-catenin-independent Wnt target gene signature in order to understand the functional significance of non-canonical signaling in colon cancer cells. Gene expression profiling was performed after silencing of key components of Wnt signaling pathway and an iterative signature algorithm was applied to predict pathway-dependent gene signatures. Independent experiments confirmed several target genes, including PLOD2, HADH, LCOR and REEP1 as non-canonical target genes in various colon cancer cells. Moreover, non-canonical Wnt target genes are regulated via RoR2, Dvl2, ATF2 and ATF4. Furthermore, we show that the ligands Wnt5a/b are upstream regulators of the non-canonical signature and moreover regulate proliferation of cancer cells in a β-catenin-independent manner. Our experiments indicate that colon cancer cells are dependent on both β-catenin-dependent and -independent Wnt signaling routes for growth and proliferation.
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http://dx.doi.org/10.1038/s41598-018-20641-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816634PMC
February 2018

ERAD-dependent control of the Wnt secretory factor Evi.

EMBO J 2018 02 29;37(4). Epub 2018 Jan 29.

Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.
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http://dx.doi.org/10.15252/embj.201797311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813261PMC
February 2018

Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

FASEB J 2017 11 21;31(11):4832-4844. Epub 2017 Jul 21.

Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany; and Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany

Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of , , and and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand-receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.-Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.
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http://dx.doi.org/10.1096/fj.201700144RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636703PMC
November 2017

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis.

EMBO J 2014 Feb 17;33(3):181-97. Epub 2014 Jan 17.

German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Department for Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg, Germany.

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.
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http://dx.doi.org/10.1002/embj.201385686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983683PMC
February 2014

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

Proc Natl Acad Sci U S A 2013 Oct 30;110(42):16856-61. Epub 2013 Sep 30.

Institute of Molecular Oncology and Göttingen Centre of Molecular Biosciences, Faculty of Medicine, University of Göttingen, 37077 Göttingen, Germany.

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.
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http://dx.doi.org/10.1073/pnas.1304355110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801042PMC
October 2013

A killer promoting survival: p53 as a selective means to avoid side effects of chemotherapy.

Cell Cycle 2012 Jun 1;11(11):2053-4. Epub 2012 Jun 1.

German Cancer Research Center, Heidelberg, Germany.

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http://dx.doi.org/10.4161/cc.20698DOI Listing
June 2012

Clustering phenotype populations by genome-wide RNAi and multiparametric imaging.

Mol Syst Biol 2010 Jun;6:370

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.
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http://dx.doi.org/10.1038/msb.2010.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913390PMC
June 2010

BRCA1 and Tip60 determine the cellular response to ultraviolet irradiation through distinct pathways.

J Cell Biol 2008 Jul;182(1):197-213

Medical Biotechnology Center, Institute for Medical Biology, University of Southern Denmark, 5000 Odense C, Denmark.

The histone acetyltransferase Tip60 regulates the apoptotic response to ultraviolet (UV) irradiation. A previously suggested mechanism for this regulation consists of the ability of Tip60 to coactivate transcription by the tumor suppressor p53. In this study, we show that Tip60 is required for the early DNA damage response (DDR) to UV, including the phosphorylation of histone 2AX, c-Jun N-terminal kinases (JNKs), and ataxia telangiectasia-related substrates. In contrast, p53 was not required for UV-induced DDR. Rather, p53 accumulation by either knockdown of Mdm2 or addition of an Mdm2 inhibitor, Nutlin-3, before irradiation strongly attenuated the UV-induced DDR and increased cell survival. This protective effect of preaccumulated p53 was mediated, at least in part, by the increased expression of CDKN1A/p21, subsequent down-regulation of BRCA1, and impaired JNK activation accompanied by decreased association of replication protein A with chromatin. We conclude that Tip60 enables UV-induced DDR signaling even in the absence of p53, whereas preaccumulated p53 suppresses UV-induced DDR by reducing the levels of BRCA1.
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http://dx.doi.org/10.1083/jcb.200712014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447902PMC
July 2008

Nongenotoxic p53 activation protects cells against S-phase-specific chemotherapy.

Cancer Res 2006 Nov;66(21):10274-80

Medical Biotechnology Center, Institute for Medical Biology, University of Southern Denmark, Odense, Denmark.

Mutations in the tumor suppressor gene TP53 represent the most frequent genetic difference between tumor cells and normal cells. Here, we have attempted to turn this difference into an advantage for normal cells during therapy. Using the Mdm2 antagonist nutlin-3, we first activated p53 in U2OS and HCT116 cells to induce cell cycle arrest. These arrested cells were found to be resistant to subsequent transient treatment with the nucleoside analogue gemcitabine, as revealed by clonogenic assays following drug removal. In contrast, isogenic cells lacking functional p53 continued to enter S phase regardless of nutlin-3 pretreatment and remained highly susceptible to gemcitabine-mediated cytotoxicity. The sequential treatment with nutlin-3 alone, followed by transient exposure to nutlin-3 plus gemcitabine, efficiently compromised the clonogenicity of tumor cells with deletions or mutations of p53 but largely spared the proliferation of nontransformed human keratinocytes. Nutlin-3 pretreatment also conferred protection of p53-proficient cells against cytosine arabinoside but not against doxorubicin or cisplatin. We propose that the cell cycle arrest function of p53 can be used to convert p53 from a killer to a protector of cells, with the potential to reduce unwanted side effects of chemotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-1527DOI Listing
November 2006