Publications by authors named "Grainne Kerr"

20 Publications

  • Page 1 of 1

Therapeutic Assessment of Targeting ASNS Combined with l-Asparaginase Treatment in Solid Tumors and Investigation of Resistance Mechanisms.

ACS Pharmacol Transl Sci 2021 Feb 13;4(1):327-337. Epub 2021 Jan 13.

Disease area Oncology, Novartis Institute for Biomedical Research, Basel, Switzerland.

Asparagine deprivation by l-asparaginase (L-ASNase) is an effective therapeutic strategy in acute lymphoblastic leukemia, with resistance occurring due to upregulation of ASNS, the only human enzyme synthetizing asparagine (, (1), 629-654). l-Asparaginase efficacy in solid tumors is limited by dose-related toxicities ( 2017, pp 1413-1422). Large-scale loss of function genetic screens identified ASNS as a cancer dependency in several solid malignancies (, (3), 564-576.e16. , (3), 577-592.e10). Here we evaluate the therapeutic potential of targeting ASNS in melanoma cells. While we confirm dependency on ASNS silencing, this is largely dispensable for tumor growth, even in the face of asparagine deprivation, prompting us to characterize such a resistance mechanism to devise novel therapeutic strategies. Using quantitative proteome and transcriptome profiling, we characterize the compensatory mechanism elicited by ASNS knockout melanoma cells allowing their survival. Mechanistically, a genome-wide CRISPR screen revealed that such a resistance mechanism is elicited by a dual axis: GCN2-ATF4 aimed at restoring amino acid levels and MAPK-BCLXL to promote survival. Importantly, pharmacological inhibition of such nodes synergizes with l-asparaginase-mediated asparagine deprivation in ASNS deficient cells suggesting novel potential therapeutic combinations in melanoma.
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http://dx.doi.org/10.1021/acsptsci.0c00196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887857PMC
February 2021

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen.

Sci Rep 2020 11 18;10(1):20044. Epub 2020 Nov 18.

NIBR Chemical Biology and Therapeutics, Novartis, Cambridge, USA.

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.
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http://dx.doi.org/10.1038/s41598-020-76960-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676242PMC
November 2020

The Discovery of SWI/SNF Chromatin Remodeling Activity as a Novel and Targetable Dependency in Uveal Melanoma.

Mol Cancer Ther 2020 10 3;19(10):2186-2195. Epub 2020 Aug 3.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no approved targeted therapies and very few effective treatments for this cancer. Although activating mutations in the G protein alpha subunits, and , are key genetic drivers of the disease, few additional drug targets have been identified. Recently, studies have identified context-specific roles for the mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer lineages. Here, we find evidence that the SWI/SNF complex is essential through analysis of functional genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools and small-molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage-specific transcription factor Microphthalmia-associated Transcription Factor, suggesting that these two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the treatment of this cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1013DOI Listing
October 2020

DOT1L inhibition is lethal for multiple myeloma due to perturbation of the endoplasmic reticulum stress pathway.

Oncotarget 2020 Mar 17;11(11):956-968. Epub 2020 Mar 17.

Novartis Institutes for BioMedical Research (NIBR) Oncology, Basel, Switzerland.

The histone 3 lysine 79 (H3K79) methyltransferase (HMT) DOT1L is known to play a critical role for growth and survival of -rearranged leukemia. Serendipitous observations during high-throughput drug screens indicated that the use of DOT1L inhibitors might be expandable to multiple myeloma (MM). Through pharmacologic and genetic experiments, we could validate that DOT1L is essential for growth and viability of a subset of MM cell lines, in line with a recent report from another team. activity against established MM xenografts was observed with a novel DOT1L inhibitor. In order to understand the molecular mechanism of the dependency in MM, we examined gene expression changes upon DOT1L inhibition in sensitive and insensitive cell lines and discovered that genes belonging to the endoplasmic reticulum (ER) stress pathway and protein synthesis machinery were specifically suppressed in sensitive cells. Whole-genome CRISPR screens in the presence or absence of a DOT1L inhibitor revealed that concomitant targeting of the H3K4me3 methyltransferase SETD1B increases the effect of DOT1L inhibition. Our results provide a strong basis for further investigating DOT1L and SETD1B as targets in MM.
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http://dx.doi.org/10.18632/oncotarget.27493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082114PMC
March 2020

Genetic heterogeneity and clonal evolution during metastasis in breast cancer patient-derived tumor xenograft models.

Comput Struct Biotechnol J 2020 31;18:323-331. Epub 2020 Jan 31.

Disease Area Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Genetic heterogeneity within a tumor arises by clonal evolution, and patients with highly heterogeneous tumors are more likely to be resistant to therapy and have reduced survival. Clonal evolution also occurs when a subset of cells leave the primary tumor to form metastases, which leads to reduced genetic heterogeneity at the metastatic site. Although this process has been observed in human cancer, experimental models which recapitulate this process are lacking. Patient-derived tumor xenografts (PDX) have been shown to recapitulate the patient's original tumor's intra-tumor genetic heterogeneity, as well as its genomics and response to treatment, but whether they can be used to model clonal evolution in the metastatic process is currently unknown. Here, we address this question by following genetic changes in two breast cancer PDX models during metastasis. First, we discovered that mouse stroma can be a confounding factor in assessing intra-tumor heterogeneity by whole exome sequencing, thus we developed a new bioinformatic approach to correct for this. Finally, in a spontaneous, but not experimental (tail-vein) metastasis model we observed a loss of heterogeneity in PDX metastases compared to their orthotopic "primary" tumors, confirming that PDX models can faithfully mimic the clonal evolution process undergone in human patients during metastatic spreading.
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http://dx.doi.org/10.1016/j.csbj.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026725PMC
January 2020

Tumor-derived IFN triggers chronic pathway agonism and sensitivity to ADAR loss.

Nat Med 2019 01 17;25(1):95-102. Epub 2018 Dec 17.

Novartis Institutes for Biomedical Research, Oncology Disease Area, Cambridge, MA, USA.

Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases . Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade . Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.
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http://dx.doi.org/10.1038/s41591-018-0302-5DOI Listing
January 2019

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Biochem Biophys Res Commun 2019 01 4;508(1):109-116. Epub 2018 Dec 4.

Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA. Electronic address:

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.009DOI Listing
January 2019

Dose and Schedule Determine Distinct Molecular Mechanisms Underlying the Efficacy of the p53-MDM2 Inhibitor HDM201.

Cancer Res 2018 11 22;78(21):6257-6267. Epub 2018 Aug 22.

Disease Area Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 , and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2-p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure-response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53-MDM2 inhibitors. Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0338DOI Listing
November 2018

SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors.

Nat Med 2018 05 5;24(4):512-517. Epub 2018 Mar 5.

Cancer Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.
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http://dx.doi.org/10.1038/nm.4497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343825PMC
May 2018

Autocrine Wnt regulates the survival and genomic stability of embryonic stem cells.

Sci Signal 2017 01 10;10(461). Epub 2017 Jan 10.

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

Wnt signaling plays an important role in the self-renewal and differentiation of stem cells. The secretion of Wnt ligands requires Evi (also known as Wls). Genetically ablating Evi provides an experimental approach to studying the consequence of depleting all redundant Wnt proteins, and overexpressing Evi enables a nonspecific means of increasing Wnt signaling. We generated Evi-deficient and Evi-overexpressing mouse embryonic stem cells (ESCs) to analyze the role of autocrine Wnt production in self-renewal and differentiation. Self-renewal was reduced in Evi-deficient ESCs and increased in Evi-overexpressing ESCs in the absence of leukemia inhibitory factor, which supports the self-renewal of ESCs. The differentiation of ESCs into cardiomyocytes was enhanced when Evi was overexpressed and teratoma formation and growth of Evi-deficient ESCs in vivo were impaired, indicating that autocrine Wnt ligands were necessary for ESC differentiation and survival. ESCs lacking autocrine Wnt signaling had mitotic defects and showed genomic instability. Together, our study demonstrates that autocrine Wnt secretion is important for the survival, chromosomal stability, differentiation, and tumorigenic potential of ESCs.
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http://dx.doi.org/10.1126/scisignal.aah6829DOI Listing
January 2017

Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples.

PLoS One 2015 26;10(5):e0127146. Epub 2015 May 26.

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.

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127146PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444292PMC
April 2016

Dpp/Gbb signaling is required for normal intestinal regeneration during infection.

Dev Biol 2015 Mar 29;399(2):189-203. Epub 2014 Dec 29.

German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Department for Cell and Molecular Biology, Medical Faculty Mannheim, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Electronic address:

Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues with a high intrinsic turnover, such as the intestinal epithelium, must launch a rapid response to infections while simultaneously coordinating cell proliferation and differentiation decisions. In this study, we searched for genes required for regeneration of the Drosophila intestine, and thereby affecting overall organism survival after infection with pathogenic bacteria. We found that Dpp/Gbb (BMP) signaling is essential for normal midgut regeneration, and that infection induces the BMP signaling ligands Dpp and Gbb. We demonstrate that Dpp is induced in visceral muscle and required for signaling activation. Subsequently, Gbb is induced in enterocytes after oral infection. Loss-of Dpp signaling in ISCs and transient committed progenitors called enteroblasts (EBs), or in EBs alone, led to a blockage in EC differentiation or maturation. Furthermore, our data show that down-regulation of Dpp signaling in the precursor cells including EBs also resulted in an increased number of abnormally small Pdm1-positive cells, suggesting a role of Dpp/Gbb signaling in EC growth. In addition, we show that Dpp/Gbb signaling acted downstream or in parallel to the Notch pathway to promote EC differentiation and growth. Our results suggest that Dpp/BMP signaling plays an important role in EBs to maintain tissue integrity and homeostasis during pathogenic infections.
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http://dx.doi.org/10.1016/j.ydbio.2014.12.017DOI Listing
March 2015

Effect of a High-Intensity, Intermittent-Exercise Protocol on Neurocognitive Function in Healthy Adults: Implications for Return-to-Play Management After Sport-Related Concussion.

J Sport Rehabil 2015 11 3;24(4). Epub 2015 Dec 3.

School of Health and Human Performance, Dublin City University, Dublin, Ireland.

Context: Determination of return to play (RTP) after sport-related concussion (SRC) is critical given the potential consequences of premature RTP. Current RTP guidelines may not identify persistent exercise-induced neurocognitive deficits in asymptomatic athletes after SRC. Therefore, postexercise neurocognitive testing has been recommended to further inform RTP determination. To implement this recommendation, the effect of exercise on neurocognitive function in healthy athletes should be understood.

Objective: To examine the acute effects of a high-intensity intermittent-exercise protocol (HIIP) on neurocognitive function assessed by the Symbol Digits Modality Test (SDMT) and Stroop Interference Test.

Design: Cohort study.

Setting: University laboratory.

Participants: 40 healthy male athletes (age 21.25 ± 1.29 y, education 16.95 ± 1.37 y).

Intervention: Each participant completed the SDMT and Stroop Interference Test at baseline and after random allocation to a condition (HIIP vs control). A mixed between-within-subjects ANOVA assessed time- (pre- vs postcondition) -by-condition interaction effects.

Main Outcome Measures: SDMT and Stroop Interference Test scores.

Results: There was a significant time-by-condition interaction effect (P < .001, η2 = .364) for the Stroop Interference Test scores, indicating that the HIIP group scored significantly lower (56.05 ± 9.34) postcondition than the control group (66.39 ± 19.6). There was no significant time-by-condition effect (P = .997, η2 < .001) for the SDMT, indicating that there was no difference between SDMT scores for the HIIP and control groups (59.95 ± 10.7 vs 58.56 ± 14.02).

Conclusions: In healthy athletes, the HIIP results in a reduction in neurocognitive function as assessed by the Stroop Interference Test, with no effect on function as assessed by the SDMT. Testing should also be considered after high-intensity exercise in determining RTP decisions for athletes after SRC in conjunction with the existing recommended RTP protocol. These results may provide an initial reference point for future research investigating the effects of an HIIP on the neurocognitive function of athletes recovering from SRC.
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http://dx.doi.org/10.1123/jsr.2014-0201DOI Listing
November 2015

E-CRISP: fast CRISPR target site identification.

Nat Methods 2014 Feb;11(2):122-3

1] Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany. [2] Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.

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http://dx.doi.org/10.1038/nmeth.2812DOI Listing
February 2014

Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells.

Nat Commun 2013 ;4:2610

1] German Cancer Research Center (DKFZ), Div. Signalling and Functional Genomics, and Heidelberg University, Dept. Cell and Molecular Biology, Faculty of Medicine Mannheim, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany [2].

Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.
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http://dx.doi.org/10.1038/ncomms3610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826636PMC
June 2014

E-TALEN: a web tool to design TALENs for genome engineering.

Nucleic Acids Res 2013 Nov 3;41(20):e190. Epub 2013 Sep 3.

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

Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.
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http://dx.doi.org/10.1093/nar/gkt789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814377PMC
November 2013

Loss of epidermal Evi/Wls results in a phenotype resembling psoriasiform dermatitis.

J Exp Med 2013 Aug 5;210(9):1761-77. Epub 2013 Aug 5.

Division of Signaling and Functional Genomics and 2 Division of Vascular Oncology and Metastasis, German Cancer Research Center, Heidelberg, Germany.

Cells of the epidermis renew constantly from germinal layer stem cells. Although epithelial cell differentiation has been studied in great detail and the role of Wnt signaling in this process is well described, the contribution of epidermal Wnt secretion in epithelial cell homeostasis remains poorly understood. To analyze the role of Wnt proteins in this process, we created a conditional knockout allele of the Wnt cargo receptor Evi/Gpr177/Wntless and studied mice that lacked Evi expression in the epidermis. We found that K14-Cre, Evi-LOF mice lost their hair during the first hair cycle, showing a reddish skin with impaired skin barrier function. Expression profiling of mutant and wild-type skin revealed up-regulation of inflammation-associated genes. Furthermore, we found that Evi expression in psoriatic skin biopsies is down-regulated, suggesting that Evi-deficient mice developed skin lesions that resemble human psoriasis. Immune cell infiltration was detected in Evi-LOF skin. Interestingly, an age-dependent depletion of dendritic epidermal T cells (DETCs) and an infiltration of γδ(low) T cells in Evi mutant epidermis was observed. Collectively, the described inflammatory skin phenotype in Evi-deficient mice revealed an essential role of Wnt secretion in maintaining normal skin homeostasis by enabling a balanced epidermal-dermal cross talk, which affects immune cell recruitment and DETC survival.
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http://dx.doi.org/10.1084/jem.20121871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754868PMC
August 2013

RNA-Seq vs dual- and single-channel microarray data: sensitivity analysis for differential expression and clustering.

PLoS One 2012 10;7(12):e50986. Epub 2012 Dec 10.

Centre for Scientific Computing and Complex Systems Modelling, Dublin City University, Dublin, Ireland.

With the fast development of high-throughput sequencing technologies, a new generation of genome-wide gene expression measurements is under way. This is based on mRNA sequencing (RNA-seq), which complements the already mature technology of microarrays, and is expected to overcome some of the latter's disadvantages. These RNA-seq data pose new challenges, however, as strengths and weaknesses have yet to be fully identified. Ideally, Next (or Second) Generation Sequencing measures can be integrated for more comprehensive gene expression investigation to facilitate analysis of whole regulatory networks. At present, however, the nature of these data is not very well understood. In this paper we study three alternative gene expression time series datasets for the Drosophila melanogaster embryo development, in order to compare three measurement techniques: RNA-seq, single-channel and dual-channel microarrays. The aim is to study the state of the art for the three technologies, with a view of assessing overlapping features, data compatibility and integration potential, in the context of time series measurements. This involves using established tools for each of the three different technologies, and technical and biological replicates (for RNA-seq and microarrays, respectively), due to the limited availability of biological RNA-seq replicates for time series data. The approach consists of a sensitivity analysis for differential expression and clustering. In general, the RNA-seq dataset displayed highest sensitivity to differential expression. The single-channel data performed similarly for the differentially expressed genes common to gene sets considered. Cluster analysis was used to identify different features of the gene space for the three datasets, with higher similarities found for the RNA-seq and single-channel microarray dataset.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050986PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518479PMC
June 2013

GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update.

Nucleic Acids Res 2013 Jan 27;41(Database issue):D1021-6. Epub 2012 Nov 27.

Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany.

RNA interference (RNAi) represents a powerful method to systematically study loss-of-function phenotypes on a large scale with a wide variety of biological assays, constituting a rich source for the assignment of gene function. The GenomeRNAi database (http://www.genomernai.org) makes available RNAi phenotype data extracted from the literature for human and Drosophila. It also provides RNAi reagent information, along with an assessment as to their efficiency and specificity. This manuscript describes an update of the database previously featured in the NAR Database Issue. The new version has undergone a complete re-design of the user interface, providing an intuitive, flexible framework for additional functionalities. Screen information and gene-reagent-phenotype associations are now available for download. The integration with other resources has been improved by allowing in-links via GenomeRNAi screen IDs, or external gene or reagent identifiers. A distributed annotation system (DAS) server enables the visualization of the phenotypes and reagents in the context of a genome browser. We have added a page listing 'frequent hitters', i.e. genes that show a phenotype in many screens, which might guide on-going RNAi studies. Structured annotation guidelines have been established to facilitate consistent curation, and a submission template for direct submission by data producers is available for download.
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http://dx.doi.org/10.1093/nar/gks1170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531141PMC
January 2013

The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis.

EMBO Mol Med 2012 Jan 7;4(1):38-51. Epub 2011 Dec 7.

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

Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.
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http://dx.doi.org/10.1002/emmm.201100186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306557PMC
January 2012