Publications by authors named "Tobias Schmelzle"

27 Publications

  • Page 1 of 1

PAX8 and MECOM are interaction partners driving ovarian cancer.

Nat Commun 2021 04 26;12(1):2442. Epub 2021 Apr 26.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Basel, Switzerland.

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.
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http://dx.doi.org/10.1038/s41467-021-22708-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076227PMC
April 2021

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors.

Sci Rep 2020 10 15;10(1):17442. Epub 2020 Oct 15.

Disease Area Oncology, Novartis Institutes for Biomedical Research, Novartis, WSJ 386 4.02.01, 4002, Basel, Switzerland.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.
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http://dx.doi.org/10.1038/s41598-020-74584-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566471PMC
October 2020

Identification of FAM181A and FAM181B as new interactors with the TEAD transcription factors.

Protein Sci 2020 02 20;29(2):509-520. Epub 2019 Nov 20.

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

The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and VGLL1-4 (Vestigial-like members 1-4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω-loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full-length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.
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http://dx.doi.org/10.1002/pro.3775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954708PMC
February 2020

Structure-based design of potent linear peptide inhibitors of the YAP-TEAD protein-protein interaction derived from the YAP omega-loop sequence.

Bioorg Med Chem Lett 2019 08 18;29(16):2316-2319. Epub 2019 Jun 18.

Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.

The YAP-TEAD protein-protein interaction is a potential therapeutic target to treat cancers in which the Hippo signaling pathway is deregulated. However, the extremely large surface of interaction between the two proteins presents a formidable challenge for a small molecule interaction disrupter approach. We have accomplished progress towards showing the feasibility of this approach by the identification of a 15-mer peptide able to potently (nanomolar range) disrupt the YAP-TEAD interaction by targeting only one of the two important sites of interaction. This peptide, incorporating non-natural amino acids selected by structure-based design, is derived from the Ω-loop sequence 85-99 of YAP.
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http://dx.doi.org/10.1016/j.bmcl.2019.06.022DOI Listing
August 2019

Molecular and structural characterization of a TEAD mutation at the origin of Sveinsson's chorioretinal atrophy.

FEBS J 2019 06 11;286(12):2381-2398. Epub 2019 Apr 11.

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

Four TEAD transcription factors (TEAD1-4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson's chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol . We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H-bonds and the loss of van der Waals contacts, which explains why the Tyr421His mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex. DATABASE: The crystallographic data have been deposited at the RSCB Protein Data Bank (PDB, www.pdb.org) with the access codes: 6HIL (wt :Tyr421His ), 6HIK (wt :Tyr429His ).
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http://dx.doi.org/10.1111/febs.14817DOI Listing
June 2019

Adaptation of the bound intrinsically disordered protein YAP to mutations at the YAP:TEAD interface.

Protein Sci 2018 10;27(10):1810-1820

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

Many interactions between proteins are mediated by intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) do not adopt a stable three-dimensional structure in their unbound form, but they become more structured upon binding to their partners. In this communication, we study how a bound IDR adapts to mutations, preventing the formation of hydrogen bonds at the binding interface that needs a precise positioning of the interacting residues to be formed. We use as a model the YAP:TEAD interface, where one YAP (IDP) and two TEAD residues form hydrogen bonds via their side chain. Our study shows that the conformational flexibility of bound YAP and the reorganization of water molecules at the interface help to reduce the energetic constraints created by the loss of H-bonds at the interface. The residual flexibility/dynamic of bound IDRs and water might, therefore, be a key for the adaptation of IDPs to different interface landscapes and to mutations occurring at binding interfaces.
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http://dx.doi.org/10.1002/pro.3493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199158PMC
October 2018

Correction of copy number induced false positives in CRISPR screens.

PLoS Comput Biol 2018 07 19;14(7):e1006279. Epub 2018 Jul 19.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

Cell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function viability screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70-80% decrease of false positive hits with either method in regions of high copy number compared to no correction.
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http://dx.doi.org/10.1371/journal.pcbi.1006279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067744PMC
July 2018

Effect of the acylation of TEAD4 on its interaction with co-activators YAP and TAZ.

Protein Sci 2017 Dec 11;26(12):2399-2409. Epub 2017 Nov 11.

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

The Hippo pathway is deregulated in various cancers, and the discovery of molecules that modulate this pathway may open new therapeutic avenues in oncology. TEA/ATTS domain (TEAD) transcription factors are the most distal elements of the Hippo pathway and their transcriptional activity is regulated by the Yes-associated protein (YAP). Amongst the various possibilities for targeting this pathway, inhibition of the YAP:TEAD interaction is an attractive strategy. It has been shown recently that TEAD proteins are covalently linked via a conserved cysteine to a fatty acid molecule (palmitate) that binds to a deep hydrophobic cavity present in these proteins. This acylation of TEAD seems to be required for efficient binding to YAP, and understanding how it modulates the YAP:TEAD interaction may provide useful information on the regulation of TEAD function. In this report we have studied the effect of TEAD4 acylation on its interaction with YAP and the other co-activator transcriptional co-activator with PDZ-binding motif (TAZ). We show in our biochemical and cellular assays that YAP and TAZ bind in a similar manner to acylated and non-acylated TEAD4. This indicates that TEAD4 acylation is not a prerequisite for its interaction with YAP or TAZ. However, we observed that TEAD4 acylation significantly enhances its stability, suggesting that it may help this transcription factor to acquire and/or maintain its active conformation.
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http://dx.doi.org/10.1002/pro.3312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699492PMC
December 2017

Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Cell 2017 Jul;170(3):577-592.e10

Novartis Institutes for Biomedical Research, Oncology Disease Area, Basel 4002, Switzerland; Cambridge, MA 02139, USA; and Emeryville, CA 94608, USA.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
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http://dx.doi.org/10.1016/j.cell.2017.07.005DOI Listing
July 2017

Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD.

Elife 2017 04 21;6. Epub 2017 Apr 21.

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

TEAD (/ATTS omain) transcription factors are the most distal effectors of the Hippo pathway. YAP (es-ssociated rotein) is a coactivator protein which, upon binding to TEAD proteins, stimulates their transcriptional activity. Since the Hippo pathway is deregulated in various cancers, designing inhibitors of the YAP:TEAD interaction is an attractive therapeutic strategy for oncology. Understanding the molecular events that take place at the YAP:TEAD interface is therefore important not only to devise drug discovery approaches, but also to gain knowledge on TEAD regulation. In this report, combining single site-directed mutagenesis and double mutant analyses, we conduct a detailed analysis on the role of several residues located at the YAP:TEAD interface. Our results provide quantitative understanding of the interactions taking place at the YAP:TEAD interface and give insights into the formation of the YAP:TEAD complex and more particularly on the interaction between TEAD and the Ω-loop found in YAP.
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http://dx.doi.org/10.7554/eLife.25068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400505PMC
April 2017

CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions.

Cancer Discov 2016 08 3;6(8):900-13. Epub 2016 Jun 3.

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

Unlabelled: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines.

Significance: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.
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http://dx.doi.org/10.1158/2159-8290.CD-16-0178DOI Listing
August 2016

Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5.

Science 2016 Mar 11;351(6278):1208-13. Epub 2016 Feb 11.

Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.
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http://dx.doi.org/10.1126/science.aad5944DOI Listing
March 2016

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers.

PLoS Genet 2015 Aug 21;11(8):e1005465. Epub 2015 Aug 21.

Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.

YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or YAP1 amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.
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http://dx.doi.org/10.1371/journal.pgen.1005465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546604PMC
August 2015

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

Hepatology 2015 Nov 25;62(5):1497-510. Epub 2015 Aug 25.

Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, Novartis Pharma AG, Basel, Switzerland.

Unlabelled: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature.

Conclusions: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.
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http://dx.doi.org/10.1002/hep.27992DOI Listing
November 2015

The surprising features of the TEAD4-Vgll1 protein-protein interaction.

Chembiochem 2014 Mar 6;15(4):537-42. Epub 2014 Feb 6.

Disease Area Oncology, Novartis Institutes for Biomedical Research, 141 Klybeckstrasse, 4057 Basel (Switzerland).

The Hippo signaling pathway, which controls organ size in animals, is altered in various human cancers. The TEAD transcription factors, the most downstream elements in this pathway, are regulated by different cofactors, such as the Vgll (vestigial-like) proteins. Having studied the interaction between Vgll1-derived peptides and human TEAD4, we show that, although it lacks a key secondary structure element required for tight binding by two other TEAD cofactors (YAP and TAZ), Vgll1-derived peptides bind to TEAD with nanomolar affinity. We identify a β-strand:loop:α-helix motif as the minimal Vgll binding site. Finally, we reveal an unexpected difference between mouse and human Vgll1-derived peptides.
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http://dx.doi.org/10.1002/cbic.201300715DOI Listing
March 2014

The TEAD4-YAP/TAZ protein-protein interaction: expected similarities and unexpected differences.

Chembiochem 2013 Jul 18;14(10):1218-25. Epub 2013 Jun 18.

Disease Area Oncology, Novartis Institutes for Biomedical Research, 141 Klybeckstrasse, 4057 Basel, Switzerland.

The Hippo pathway controls cell homeostasis, and its deregulation can lead to human diseases. In this pathway, the YAP and TAZ transcriptional cofactors play a key role in stimulating gene transcription through their interaction with the TEAD transcriptional factors. Our study of YAP and TAZ peptides in biochemical and biophysical assays shows that both proteins have essentially the same affinity for TEAD. Molecular modeling and structural biology data suggest that they also bind to the same site on TEAD. However, this apparent similarity hides differences in the ways in which the two proteins interact with TEAD. The secondary structure elements of their TEAD binding site do not contribute equally to the overall affinity, and critical interactions with TEAD are made through different residues. This convergent optimization of the YAP/TAZ TEAD binding site suggests that the similarity in the affinities of binding of YAP to TEAD and of TAZ to TEAD is important for Hippo pathway functionality.
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http://dx.doi.org/10.1002/cbic.201300163DOI Listing
July 2013

The tyrosine phosphatase PTPN14 is a negative regulator of YAP activity.

PLoS One 2013 16;8(4):e61916. Epub 2013 Apr 16.

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

The Hippo (Hpo) pathway is a novel signaling pathway that controls organ size in Drosophila and mammals and is deregulated in a variety of human cancers. It consists of a set of kinases that, through a number of phosphorylation events, inactivate YAP, a transcriptional co-activator that controls cellular proliferation and apoptosis. We have identified PTPN14 as a YAP-binding protein that negatively regulates YAP activity by controlling its localization. Mechanistically, we find that the interaction of ectopic YAP with PTPN14 can be mediated by the respective WW and PPxY motifs. However, the PTPN14 PPxY motif and phosphatase activity appear to be dispensable for the negative regulation of endogenous YAP, likely suggesting more complex mechanisms of interaction and modulation. Finally, we demonstrate that PTPN14 downregulation can phenocopy YAP activation in mammary epithelial cells and synergize with YAP to induce oncogenic transformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061916PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628344PMC
November 2013

The path to oncology drug target validation: an industry perspective.

Methods Mol Biol 2013 ;986:3-13

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

The advent of a variety of genomic, proteomic and other system-based scientific approaches has raised the expectations of identifying novel targets for oncology drug discovery. However, the complexity of human genome cancer alterations requires a careful analysis of the function of candidate targets identified by these efforts. The postulation and testing of a hypothesis that modulation of a protein or pathway will result in a therapeutic effect in a preclinical setting is crucial for target validation activities. In this chapter, we provide an overview on target identification and validation approaches to interrogate the functional and therapeutic relevance of a candidate cancer drug target as an essential step towards justifying the subsequent investment in drug discovery efforts.
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http://dx.doi.org/10.1007/978-1-62703-311-4_1DOI Listing
August 2013

K-RAS mutant pancreatic tumors show higher sensitivity to MEK than to PI3K inhibition in vivo.

PLoS One 2012 31;7(8):e44146. Epub 2012 Aug 31.

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

Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044146PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432074PMC
February 2013

Akt and ERK control the proliferative response of mammary epithelial cells to the growth factors IGF-1 and EGF through the cell cycle inhibitor p57Kip2.

Sci Signal 2012 Mar 6;5(214):ra19. Epub 2012 Mar 6.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Epithelial cells respond to growth factors including epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and insulin. Using high-content immunofluorescence microscopy, we quantitated differences in signaling networks downstream of EGF, which stimulated proliferation of mammary epithelial cells, and insulin or IGF-1, which enhanced the proliferative response to EGF but did not stimulate proliferation independently. We found that the abundance of the cyclin-dependent kinase inhibitors p21Cip1 and p57Kip2 increased in response to IGF-1 or insulin but decreased in response to EGF. Depletion of p57Kip2, but not p21Cip1, rendered IGF-1 or insulin sufficient to induce cellular proliferation in the absence of EGF. Signaling through the PI3K (phosphatidylinositol 3-kinase)-Akt-mTOR (mammalian target of rapamycin) pathway was necessary and sufficient for the increase in p57Kip2, whereas MEK [mitogen-activated or extracellular signal-regulated protein kinase (ERK) kinase]-ERK activity suppressed this increase, forming a regulatory circuit that limited proliferation in response to unaccompanied Akt activity. Knockdown of p57Kip2 enhanced the proliferative phenotype induced by tumor-associated PI3K mutant variants and released mammary epithelial acini from growth arrest during morphogenesis in three-dimensional culture. These results provide a potential explanation for the context-dependent proliferative activities of insulin and IGF-1 and for the finding that the CDKN1C locus encoding p57Kip2 is silenced in many breast cancers, which frequently show hyperactivation of the PI3K pathway. The status of p57Kip2 may thus be an important factor to assess when considering targeted therapy against the ERK or PI3K pathways.
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http://dx.doi.org/10.1126/scisignal.2001986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174537PMC
March 2012

Engineering tumors with 3D scaffolds.

Nat Methods 2007 Oct 2;4(10):855-60. Epub 2007 Sep 2.

School of Engineering and Applied Sciences, Harvard University, 40 Oxford Street; Cambridge, Massachusetts 02138, USA.

Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.
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http://dx.doi.org/10.1038/nmeth1085DOI Listing
October 2007

Functional role and oncogene-regulated expression of the BH3-only factor Bmf in mammary epithelial anoikis and morphogenesis.

Proc Natl Acad Sci U S A 2007 Mar 26;104(10):3787-92. Epub 2007 Feb 26.

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

The formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro involves selective apoptosis of centrally localized cells that lack matrix attachment. Similarly, apoptosis is induced by forced detachment of mammary epithelial cells from matrix, a process referred to as anoikis. Through microarray analysis, we found that mRNA levels of the proapoptotic BH3-only protein Bmf are up-regulated during both anoikis and acinar morphogenesis. Importantly, down-regulation of Bmf expression by small interfering RNAs is sufficient to prevent anoikis and acinar cell death and promote anchorage-independent growth to a similar extent as down-regulation of another BH3-only protein, Bim, which was previously shown to be required for these processes. Knockdown of the BH3-only proteins Bad or Bid does not suppress anoikis or luminal apoptosis or promote anchorage-independent growth, but protects from other defined apoptotic stimuli, indicating specificity of BH3-only function. Bmf mRNA is significantly up-regulated upon loss of matrix attachment or disruption of the actin cytoskeleton, but not in response to several other stresses. Interestingly, constitutive activation of the Mek/Erk or phosphatidylinositol 3-kinase/Akt pathways suppresses the transcriptional up-regulation of Bmf during anoikis. Thus, Bmf is a central mediator of anoikis in mammary cells and a target of oncogenes that contribute to the progression of glandular epithelial tumors. Finally, Bmf is expressed during involution of the mouse mammary gland, suggesting that Bmf may also critically contribute to developmental processes in vivo.
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http://dx.doi.org/10.1073/pnas.0700115104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820662PMC
March 2007

BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms.

Dev Cell 2007 Feb;12(2):221-34

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.
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http://dx.doi.org/10.1016/j.devcel.2006.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698712PMC
February 2007

Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast.

Mol Cell Biol 2004 Jan;24(1):338-51

Division of Biochemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

The TOR (target of rapamycin) and RAS/cyclic AMP (cAMP) signaling pathways are the two major pathways controlling cell growth in response to nutrients in yeast. In this study we examine the functional interaction between TOR and the RAS/cAMP pathway. First, activation of the RAS/cAMP signaling pathway confers pronounced resistance to rapamycin. Second, constitutive activation of the RAS/cAMP pathway prevents several rapamycin-induced responses, such as the nuclear translocation of the transcription factor MSN2 and induction of stress genes, the accumulation of glycogen, the induction of autophagy, the down-regulation of ribosome biogenesis (ribosomal protein gene transcription and RNA polymerase I and III activity), and the down-regulation of the glucose transporter HXT1. Third, many of these TOR-mediated responses are independent of the previously described TOR effectors TAP42 and the type 2A-related protein phosphatase SIT4. Conversely, TOR-controlled TAP42/SIT4-dependent events are not affected by the RAS/cAMP pathway. Finally, and importantly, TOR controls the subcellular localization of both the protein kinase A catalytic subunit TPK1 and the RAS/cAMP signaling-related kinase YAK1. Our findings suggest that TOR signals through the RAS/cAMP pathway, independently of TAP42/SIT4. Therefore, the RAS/cAMP pathway may be a novel TOR effector branch.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC303340PMC
http://dx.doi.org/10.1128/mcb.24.1.338-351.2004DOI Listing
January 2004

Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry.

Proc Natl Acad Sci U S A 2003 Feb 22;100(3):880-5. Epub 2003 Jan 22.

Department of Biochemistry, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.
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http://dx.doi.org/10.1073/pnas.232735599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC298695PMC
February 2003

The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae.

Mol Microbiol 2002 Sep;45(5):1433-41

Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase-activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7,but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1-controlled PKC1-MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1-mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.
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http://dx.doi.org/10.1046/j.1365-2958.2002.03110.xDOI Listing
September 2002

Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast.

Mol Cell Biol 2002 Mar;22(5):1329-39

Department of Biochemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

The PKC1-associated mitogen-activated protein (MAP) kinase pathway of Saccharomyces cerevisiae regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. Activation of PKC1 occurs via the GTPase RHO1 and the kinase pair PKH1 and PKH2. Here we report that YPK1 and YPK2, an essential pair of homologous kinases and proposed downstream effectors of PKH and sphingolipids, are also regulators of the PKC1-controlled MAP kinase cascade. ypk mutants display random distribution of the actin cytoskeleton and severely reduced activation of the MAP kinase MPK1. Upregulation of the RHO1 GTPase switch or the PKC1 effector MAP kinase pathway suppresses the growth and actin defects of ypk cells. ypk lethality is also suppressed by overexpression of an uncharacterized gene termed TUS1. TUS1 is a novel RHO1 exchange factor that contributes to cell wall integrity-mediated modulation of RHO1 activity. Thus, TUS1 and the YPKs add to the growing complexity of RHO1 and PKC1 regulation in the cell integrity signaling pathway. Furthermore, our findings suggest that the YPKs are a missing link between sphingolipid signaling and the cell integrity pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC134704PMC
http://dx.doi.org/10.1128/mcb.22.5.1329-1339.2002DOI Listing
March 2002