Internship Quantification of T-cell mediated tumor regression

Supervisors Lorenzo Fanchi (NKI Amsterdam) amp Maarten Slagter (LACDR Leiden) Internship available from April May 2015 T cells have attracted widespread clinical interest due to their specific and potent tumor killing capacity Infiltration of effector T cells into tumor lesions has been associated with a favorable clinical prognosis in melanoma

1 and glioblastoma

2 Furthermore adoptive cell transfer of autologous T-cells as applied at the

NKI-AvL and in other cancer centers has shown a response rate of approximately 50 in patients with metastatic melanoma

3-

4 Predictive biomarkers of treatment success are still lacking however The

majority of research efforts are focused on qualitative aspects of the tumor infiltrating T-cells (eg TCR-repertoire activation and exhaustion) or the tumor and surrounding cells (eg antigen presentation expression of immunosuppressive molecules tumor vascularization collagen and cellular adhesion molecule density) We approach the balance between tumor growth and destruction by T-cells quantitatively can tumor size dynamics be explained by the intratumoral frequencies of T-cells that vary over time How fast do T-cells kill tumor cells and is this rate increased by inhibition of PD-L1 a molecule that is often produced by tumor cells in order to evade immune attack Does the killing rate increase linearly with T-cell levels or does it start to level off at a physiologically relevant frequency of T-cells

At the Schumacher lab time course experiments are being performed to follow the anti-tumor capabilities of various T-cell populations in an immunodeficient mouse model We have shown that observed tumor dynamics can be adequately described using a relatively simple mathematical model incorporating growth and T-cell mediated regression However due to the absence of information on T-cell infiltration within the tumors a robust parameterization of the model was not yet possible With additional measurements it will be possible to determine the typical per capita killing rate of T-cells in a tumor context as has been extensively done for T-cells targeted at virally infected cells

5 You will play a large role in generating the data required

to come to such a quantitative description of T-cell-tumor cell interactions Experimentation will consist of inducing tumor growth in mice and monitoring the anti-tumor efficacy of various T-cell populations over time Importantly we will also measure the amount of intratumoral T-cells at multiple time points and the relation with their blood levels You will work with a variety of laboratory techniques including (multimer) combi-coding flow cytometry and retroviral transduction and take part in the highly dynamic lab of Ton Schumacher that focuses on Immunotherapy and fundamental Immunology

Applicant profile

Highly motivated MSc student in Biomedical sciences Biological sciences or similar with an interest in (tumor) immunology

Preferably in the possession of Artikel 9 Proefdieronderzoek and experienced in animal experimentation Alternatively willing to take the required course

Able to work accurately

Able to write and communicate clearly in English

Available for at least six months Contact

Please send an email including a brief CV to mslagterlacdrleidenunivnl References 1 Wu R et al Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic

melanoma current status and future outlook Cancer J 18 160ndash75 (2012)

2 Lohr J et al Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired

by tumor-derived TGF-β Clin Cancer Res 17 4296ndash308 (2011)

3 Dudley M Wunderlich J amp Robbins P Cancer regression and autoimmunity in patients after clonal

repopulation with antitumor lymphocytes Science (80- ) 298 850ndash855 (2002)

4 Rosenberg S a Restifo N P Yang J C Morgan R a amp Dudley M E Adoptive cell transfer a clinical

path to effective cancer immunotherapy Nat Rev Cancer 8 299ndash308 (2008)

5 Elemans M Florins A Willems L amp Asquith B Rates of CTL killing in persistent viral infection in vivo

PLoS Comput Biol 10 e1003534 (2014)

Not available anymore

Title Identification of proteins interacting with the hepatic efflux transporter ABCC6 Location Division of Molecular Oncology Netherlands Cancer Institute Amsterdam Project coordinator Koen van de Wetering E-mail kvdweteringnkinl Duration of Internship 6-8 months Background Pseudoxanthoma elasticum (PXE) is the prototype of a heritable connective tissue disorder with clinical manifestations in skin eyes and the wall of arterial blood vessels(12) At the affected sites histology shows ectopic mineral deposits(3) The precise prevalence of PXE is unknown but estimated to range between 125000 and 1100000(34) PXE is inherited in an autosomal recessive manner without ethnic or racial predilection but women often display more serious symptoms than men(3) PXE symptoms vary widely even in one family but the morbidity is considerable(35) In fact PXE causes blindness(6) and early death from cardiovascular conditions(37) PXE is a slow progressive disease and the first signs generally appear at puberty In the majority of cases the first organ system affected is the skin showing yellowish papules in the neck and at other flexural surfaces These lesions have the tendency to coalesce into large plaques and eventually the skin loses its elasticity and becomes wrinkled and redundant(7) Clinical signs related to cardiovascular problems include intermittent claudication and gastrointestinal haemorrhages However the most serious cardiovascular complications of PXE are probably the early onset of atherosclerosis and myocardial infarctions(7) Eye pathology is also prominent in PXE which eventually leads to vision loss(6) The genetic basis of PXE has long been thought to reside in genes involved in extracellular matrix formation(89) It therefore came as a surprise when three groups reported in 2000 that PXE is caused by inactivating mutations in the gene encoding the transmembrane efflux transporter ABCC6 also known as Multidrug Resistance-associated Protein 6 MRP6(10-12) ABCC6 belongs to the C-branch of the ATP-binding cassette (ABC) family of transmembrane proteins(2) The other transporters of this subfamily are all typical organic anion efflux transporters and translocate their substrates against steep concentration gradients at the cost of ATP hydrolysis(13) In vitro ABCC6 behaves as a typical ABCC transporter as well it transports some organic anions and is inhibited by typical ABCC inhibitors like benzbromarone and indomethacin(1415) ABCC6 is predominantly expressed in the liver where it localizes to the basolateral (sinusoidal) membrane(1617) Importantly ABCC6 is only present at low levels in the tissues affected by PXE(1618) Clearly ABCC6 in the liver secretes a factor into the circulation that prevents ectopic mineralization in the periphery(119) The nature of this factor has for long remained a mystery Very recently our group has solved this mystery by showing that ABCC6 mediates the release of nucleoside-triphosphates predominantly ATP from the liver(2021) Outside the hepatocytes but still within the liver vasculature released ATP is converted into AMP and pyrophosphate (PPi) (2021) The latter is a strong inhibitor of mineralization(22) Importantly absence of functional ABCC6 in PXE patients results in reduced PPi levels in the blood circulation(21) which explains their ectopic mineralization phenotype How ABCC6 mediates ATP release is still a mystery We have been unable to confirm that ATP and other nucleotides are transported by ABCC6 in vesicular transport assays the gold standard to determine whether or not a compound is transported This does not exclude the possibility that ABCC6 directly transports ATP Maybe additional proteins or metabolites are crucial to drive transport and these could be missing in our in vitro vesicular transport experiments Alternatively ABCC6 might regulate the activity of another channel or transporter In this respect it is important to mention that ABCC6 belongs to the C-branch of the ABC superfamily of transmembrane proteins which also harbours ABCC8 and ABCC9 both regulators of complex K+-channels(2) It is even feasible that ABCC6 functions as a NTP channel similar to the chloride channel ABCC7 (also known as Cystic Fibrosis Transmembrane conductance Regulator CFTR)(2) Irrespective of the exact manner by which ABCC6 mediates ATP release this process must be tightly regulated In the present project we

want to search for proteins that interact with ABCC6 in the plasma membrane which might regulate ABCC6 activity Aim of the project Identify proteins that interact with ABCC6 using proteomics approaches Experimental approach We want to explore with which proteins ABCC6 interacts using a relatively new technique called proximity-dependent biotin identification (BioID)(2324) BioID exploits a promiscuous biotin protein ligase (BirA) of Escherichia coli to label proteins that are in close proximity (10 nm(25)) Under standard cell culture conditions biotinylating activity of BirA is low but can be induced by the addition of 50 microM biotin to the cell culture medium Proteins labeled with biotin can subsequently be purified on streptavidin-coated beads for identification by proteomics(23) BirA can be fused either to the C- or N-terminus of ABCC6 and both fusion constructs will be tested in HEK293 cells a cell line proven to be very suitable for following ABCC6 expression and functionality(2021) The N-terminus of ABCC6 sticks into the extracellular space whereas the C-terminus is present intracellularly(7) BirA fused to the N-terminus of ABCC6 will label ABCC6-interacting proteins that fully span the plasma membrane or are present in the outer leaflet of the plasma membrane If ABCC6 interacts with a channel we expect to label it using the N-terminal BirA fusion constructs BirA fused to the C-terminus of ABCC6 will label a wider range of interacting proteins Those that are present in the cytosol and in the inner leaflet of the plasma membrane C-terminal BirA ABCC6 fusion proteins have the potential to next to proteins that fully span the plasma membrane also label cytosolic regulatory proteins Biotinylated proteins can be collected on streptavidin beads and analysed by mass spectrometry (proteomics) Constructs encoding ABCC6 tagged at its N or C terminus have already been generated and are ready for use Selection of techniques used in this project

Culturing of mammalian cells

Proximity-dependent biotin identification (BioID)

DNA isolation

Introduction of cDNAs in mammalian cells

Bioluminescence-based assay to determine pyrophosphate concentrations

Bioluminescence-based assay to determine ATP concentrations

SDS-PAGE Western blotting

Confocal microscopy

Mass spectrometry based proteomics

Interested Send an e-mail to kvdweteringnkinl It would be appreciated if you would include a brief CV

OCTOBER 2014

References 1 Jiang Q Endo M Dibra F Wang K Uitto J Pseudoxanthoma elasticum is a metabolic disease

J Investig Dermatol 2009129348ndash54

2 Borst P Oude Elferink R Mammalian ABC transporters in health and disease Annu Rev Biochem 200271537ndash92

3 Uitto J Bercovitch L Terry SF Terry PF Pseudoxanthoma elasticum Progress in diagnostics and research towards treatment Am J Med Genet 20111551517ndash26

4 Plomp AS Hu X de Jong PTVM Bergen AAB Does autosomal dominant pseudoxanthoma elasticum exist Am J Med Genet 2004126A403ndash12

5 Li Q Jiang Q Pfendner E Vaacuteradi A Uitto J Pseudoxanthoma elasticum clinical phenotypes molecular genetics and putative pathomechanisms Exp Dermatol 2009181ndash11

6 Georgalas I Tservakis I Papaconstaninou D Kardara M Koutsandrea C Ladas I Pseudoxanthoma elasticum ocular manifestations complications and treatment Clinical and Experimental Optometry 201094169ndash80

7 Vaacuteradi A Szaboacute Z Pomozi V de Boussac H Fuumlloumlp K Araacutenyi T ABCC6 as a target in pseudoxanthoma elasticum Curr Drug Targets 201112671ndash82

8 Sherer DW Sapadin AN Lebwohl MG Pseudoxanthoma elasticum an update Dermatology (Basel) 19991993ndash7

9 Smith JG Davidson EA CLARK RL Dermal elastin in actinic elastosis pseudoxanthoma elasticum Nature 1962195716ndash7

10 Bergen AA Plomp AS Schuurman EJ Terry S Breuning M Dauwerse H et al Mutations in ABCC6 cause pseudoxanthoma elasticum Nat Genet 200025228ndash31

11 Le Saux O Urban Z Tschuch C Csiszar K Bacchelli B Quaglino D et al Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum Nat Genet 200025223ndash7

12 Ringpfeil F Lebwohl MG Christiano AM Uitto J Pseudoxanthoma elasticum mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter Proc Natl Acad Sci USA 2000976001ndash6

13 Borst P Evers R Kool M Wijnholds J The multidrug resistance protein family Biochim Biophys Acta 19991461347ndash57

14 Madon J Hagenbuch B Landmann L Meier PJ Stieger B Transport function and hepatocellular localization of mrp6 in rat liver Mol Pharmacol 200057634ndash41

15 Ilias A Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6) J Biol Chem 200227716860ndash7

16 Scheffer GLG Kool MM de Haas MM de Vree JMLJ Pijnenborg ACLMA Bosman DKD et al Tissue distribution and induction of human multidrug resistant protein 3 Lab Invest 200282193ndash201

17 Pomozi V Le Saux O Brampton C Apana A Ilias A Szeri F et al ABCC6 Is a Basolateral Plasma Membrane Protein Circ Res 2013112e148ndash51

18 Gorgels TGMF Teeling P Meeldijk JD Nillesen STM van der Wal AC van Kuppevelt TH et

al Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruchs membrane Exp Eye Res Elsevier Ltd 201210459ndash64

19 Jiang Q Oldenburg R Otsuru S Grand-Pierre AE Horwitz EM Uitto J Parabiotic Heterogenetic Pairing of Abcc6minusminusRag1minusminus Mice and Their Wild-Type Counterparts Halts Ectopic Mineralization in a Murine Model of Pseudoxanthoma Elasticum The American Journal of Pathology 20101761855ndash62

20 Jansen RS Kuumlccediluumlkosmanoglu A de Haas M Sapthu S Otero JA Hegman IEM et al ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release Proc Natl Acad Sci USA 201311020206ndash11

21 Jansen RS Duijst S Mahakena S Sommer D Szeri F Varadi A et al ABCC6-Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation--Brief Report Arterioscler Thromb Vasc Biol 2014341985ndash9

22 Nitschke Y Rutsch F Genetics in Arterial Calcification Lessons Learned From Rare Diseases Trends Cardiovas Med Elsevier Inc 201222145ndash9

23 Roux KJ Kim DI Raida M Burke B A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells J Cell Biol 2012196801ndash10

24 Firat-Karalar EN Rauniyar N Yates JR III Stearns T Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Curr Biol Elsevier Ltd 201424664ndash70

25 Kim DI KC B Zhu W Motamedchaboki K Doye V Roux KJ Probing nuclear pore complex architecture with proximity-dependent biotinylation Proc Natl Acad Sci USA 2014111E2453ndash61

Genome-nuclear lamina interactions and resistance to targeted therapy in melanoma Background Melanoma is one the most aggressive human malignancies Novel targeted therapies (vemurafenib and dabrafenib which target BRAF

E600 and trametinib which targets MEK) have been developed and

showed dramatic clinical responses However resistances invariably occur and the prognosis of melanoma patients still remains poor Extensive research efforts to search for resistance mechanisms identified secondary mutations in the components of the MAPK pathway upstream or downstream of BRAF or in parallel pathways such as the PI3K pathway

1 These alterations occur late and are the

result of a massive selection pressure imposed by prolonged treatment However when performing short term response curves with vemurafenib in cultured melanoma cells we and others found that an appreciable of proportion cells survive the treatment at a dose that otherwise kills the majority of the population arguing for the existence of an early nonmutational mechanism of resistance A cell population of drug-tolerant cells has also been described for lung cancer lines treated with EGFR inhibitors and has been termed drug tolerant persisters (DTPs)

2 Parallel observations were done in

the clinic where the majority of responses to targeted therapies are only partial which suggest that survival of DTPs also occurs in patients As relapses arise frequently at site of residual tumors DTPs are likely to be responsible for most of therapy failures Therefore it is important to understand the mechanism underlying the drug tolerant state This phenotype is reversible as DTPs cells regain sensitivity to the drug when cultured in drug-free media

3 This suggests the involvement of epigenetic

mechanisms and raises the hypothesis that a particular chromatin configuration may underlie the drug tolerant state One important aspect of chromatin organization is its interaction with the nuclear lamina (NL) Indeed the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs)

4

Goals of the project In this project we will set out to investigate a role for genome-NL interactions in the establishment of the drug tolerant state in the context of BRAF mutant melanoma treated with vemurafenib For this we will make use of DamID a technique developed by the laboratory of Bas Van Steensel that allows for genome-wide study of genome-protein interactions

5 After having selected a vemurafenib-sensitive

melanoma cell line and optimized the drug concentration that allows obtaining the tolerant state we will perform DamID with lamin B1 a component of the nuclear lamina in parental cells and DTPs to search for LADs changes during the establishment of the drug tolerant state RNA sequencing will be performed in parallel in order to investigate a relationship between changes in LADs and changes in gene expression that may occur during this transition Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture (lenti)viral infections cell proliferation and viability assays immunofluorescence and Western blot analysis In addition you will be introduced to genomic approaches such as DamID and RNA sequencing which involve processing of genomic DNA or RNA and next generation sequencing Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl References 1 Sullivan R J amp Flaherty K T Resistance to BRAF-targeted therapy in melanoma Eur J

Cancer 49 1297-1304 doi101016jejca201211019 (2013) 2 Sharma S V et al A chromatin-mediated reversible drug-tolerant state in cancer cell

subpopulations Cell 141 69-80

want to search for proteins that interact with ABCC6 in the plasma membrane which might regulate ABCC6 activity Aim of the project Identify proteins that interact with ABCC6 using proteomics approaches Experimental approach We want to explore with which proteins ABCC6 interacts using a relatively new technique called proximity-dependent biotin identification (BioID)(2324) BioID exploits a promiscuous biotin protein ligase (BirA) of Escherichia coli to label proteins that are in close proximity (10 nm(25)) Under standard cell culture conditions biotinylating activity of BirA is low but can be induced by the addition of 50 microM biotin to the cell culture medium Proteins labeled with biotin can subsequently be purified on streptavidin-coated beads for identification by proteomics(23) BirA can be fused either to the C- or N-terminus of ABCC6 and both fusion constructs will be tested in HEK293 cells a cell line proven to be very suitable for following ABCC6 expression and functionality(2021) The N-terminus of ABCC6 sticks into the extracellular space whereas the C-terminus is present intracellularly(7) BirA fused to the N-terminus of ABCC6 will label ABCC6-interacting proteins that fully span the plasma membrane or are present in the outer leaflet of the plasma membrane If ABCC6 interacts with a channel we expect to label it using the N-terminal BirA fusion constructs BirA fused to the C-terminus of ABCC6 will label a wider range of interacting proteins Those that are present in the cytosol and in the inner leaflet of the plasma membrane C-terminal BirA ABCC6 fusion proteins have the potential to next to proteins that fully span the plasma membrane also label cytosolic regulatory proteins Biotinylated proteins can be collected on streptavidin beads and analysed by mass spectrometry (proteomics) Constructs encoding ABCC6 tagged at its N or C terminus have already been generated and are ready for use Selection of techniques used in this project

Culturing of mammalian cells

Proximity-dependent biotin identification (BioID)

DNA isolation

Introduction of cDNAs in mammalian cells

Bioluminescence-based assay to determine pyrophosphate concentrations

Bioluminescence-based assay to determine ATP concentrations

SDS-PAGE Western blotting

Confocal microscopy

Mass spectrometry based proteomics

Interested Send an e-mail to kvdweteringnkinl It would be appreciated if you would include a brief CV

OCTOBER 2014

References 1 Jiang Q Endo M Dibra F Wang K Uitto J Pseudoxanthoma elasticum is a metabolic disease

J Investig Dermatol 2009129348ndash54

2 Borst P Oude Elferink R Mammalian ABC transporters in health and disease Annu Rev Biochem 200271537ndash92

3 Uitto J Bercovitch L Terry SF Terry PF Pseudoxanthoma elasticum Progress in diagnostics and research towards treatment Am J Med Genet 20111551517ndash26

4 Plomp AS Hu X de Jong PTVM Bergen AAB Does autosomal dominant pseudoxanthoma elasticum exist Am J Med Genet 2004126A403ndash12

5 Li Q Jiang Q Pfendner E Vaacuteradi A Uitto J Pseudoxanthoma elasticum clinical phenotypes molecular genetics and putative pathomechanisms Exp Dermatol 2009181ndash11

6 Georgalas I Tservakis I Papaconstaninou D Kardara M Koutsandrea C Ladas I Pseudoxanthoma elasticum ocular manifestations complications and treatment Clinical and Experimental Optometry 201094169ndash80

7 Vaacuteradi A Szaboacute Z Pomozi V de Boussac H Fuumlloumlp K Araacutenyi T ABCC6 as a target in pseudoxanthoma elasticum Curr Drug Targets 201112671ndash82

8 Sherer DW Sapadin AN Lebwohl MG Pseudoxanthoma elasticum an update Dermatology (Basel) 19991993ndash7

9 Smith JG Davidson EA CLARK RL Dermal elastin in actinic elastosis pseudoxanthoma elasticum Nature 1962195716ndash7

10 Bergen AA Plomp AS Schuurman EJ Terry S Breuning M Dauwerse H et al Mutations in ABCC6 cause pseudoxanthoma elasticum Nat Genet 200025228ndash31

11 Le Saux O Urban Z Tschuch C Csiszar K Bacchelli B Quaglino D et al Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum Nat Genet 200025223ndash7

12 Ringpfeil F Lebwohl MG Christiano AM Uitto J Pseudoxanthoma elasticum mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter Proc Natl Acad Sci USA 2000976001ndash6

13 Borst P Evers R Kool M Wijnholds J The multidrug resistance protein family Biochim Biophys Acta 19991461347ndash57

14 Madon J Hagenbuch B Landmann L Meier PJ Stieger B Transport function and hepatocellular localization of mrp6 in rat liver Mol Pharmacol 200057634ndash41

15 Ilias A Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6) J Biol Chem 200227716860ndash7

16 Scheffer GLG Kool MM de Haas MM de Vree JMLJ Pijnenborg ACLMA Bosman DKD et al Tissue distribution and induction of human multidrug resistant protein 3 Lab Invest 200282193ndash201

17 Pomozi V Le Saux O Brampton C Apana A Ilias A Szeri F et al ABCC6 Is a Basolateral Plasma Membrane Protein Circ Res 2013112e148ndash51

18 Gorgels TGMF Teeling P Meeldijk JD Nillesen STM van der Wal AC van Kuppevelt TH et

al Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruchs membrane Exp Eye Res Elsevier Ltd 201210459ndash64

19 Jiang Q Oldenburg R Otsuru S Grand-Pierre AE Horwitz EM Uitto J Parabiotic Heterogenetic Pairing of Abcc6minusminusRag1minusminus Mice and Their Wild-Type Counterparts Halts Ectopic Mineralization in a Murine Model of Pseudoxanthoma Elasticum The American Journal of Pathology 20101761855ndash62

20 Jansen RS Kuumlccediluumlkosmanoglu A de Haas M Sapthu S Otero JA Hegman IEM et al ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release Proc Natl Acad Sci USA 201311020206ndash11

21 Jansen RS Duijst S Mahakena S Sommer D Szeri F Varadi A et al ABCC6-Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation--Brief Report Arterioscler Thromb Vasc Biol 2014341985ndash9

22 Nitschke Y Rutsch F Genetics in Arterial Calcification Lessons Learned From Rare Diseases Trends Cardiovas Med Elsevier Inc 201222145ndash9

23 Roux KJ Kim DI Raida M Burke B A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells J Cell Biol 2012196801ndash10

24 Firat-Karalar EN Rauniyar N Yates JR III Stearns T Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Curr Biol Elsevier Ltd 201424664ndash70

25 Kim DI KC B Zhu W Motamedchaboki K Doye V Roux KJ Probing nuclear pore complex architecture with proximity-dependent biotinylation Proc Natl Acad Sci USA 2014111E2453ndash61

Genome-nuclear lamina interactions and resistance to targeted therapy in melanoma Background Melanoma is one the most aggressive human malignancies Novel targeted therapies (vemurafenib and dabrafenib which target BRAF

E600 and trametinib which targets MEK) have been developed and

showed dramatic clinical responses However resistances invariably occur and the prognosis of melanoma patients still remains poor Extensive research efforts to search for resistance mechanisms identified secondary mutations in the components of the MAPK pathway upstream or downstream of BRAF or in parallel pathways such as the PI3K pathway

1 These alterations occur late and are the

result of a massive selection pressure imposed by prolonged treatment However when performing short term response curves with vemurafenib in cultured melanoma cells we and others found that an appreciable of proportion cells survive the treatment at a dose that otherwise kills the majority of the population arguing for the existence of an early nonmutational mechanism of resistance A cell population of drug-tolerant cells has also been described for lung cancer lines treated with EGFR inhibitors and has been termed drug tolerant persisters (DTPs)

2 Parallel observations were done in

the clinic where the majority of responses to targeted therapies are only partial which suggest that survival of DTPs also occurs in patients As relapses arise frequently at site of residual tumors DTPs are likely to be responsible for most of therapy failures Therefore it is important to understand the mechanism underlying the drug tolerant state This phenotype is reversible as DTPs cells regain sensitivity to the drug when cultured in drug-free media

3 This suggests the involvement of epigenetic

mechanisms and raises the hypothesis that a particular chromatin configuration may underlie the drug tolerant state One important aspect of chromatin organization is its interaction with the nuclear lamina (NL) Indeed the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs)

4

Goals of the project In this project we will set out to investigate a role for genome-NL interactions in the establishment of the drug tolerant state in the context of BRAF mutant melanoma treated with vemurafenib For this we will make use of DamID a technique developed by the laboratory of Bas Van Steensel that allows for genome-wide study of genome-protein interactions

5 After having selected a vemurafenib-sensitive

melanoma cell line and optimized the drug concentration that allows obtaining the tolerant state we will perform DamID with lamin B1 a component of the nuclear lamina in parental cells and DTPs to search for LADs changes during the establishment of the drug tolerant state RNA sequencing will be performed in parallel in order to investigate a relationship between changes in LADs and changes in gene expression that may occur during this transition Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture (lenti)viral infections cell proliferation and viability assays immunofluorescence and Western blot analysis In addition you will be introduced to genomic approaches such as DamID and RNA sequencing which involve processing of genomic DNA or RNA and next generation sequencing Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl References 1 Sullivan R J amp Flaherty K T Resistance to BRAF-targeted therapy in melanoma Eur J

Cancer 49 1297-1304 doi101016jejca201211019 (2013) 2 Sharma S V et al A chromatin-mediated reversible drug-tolerant state in cancer cell

subpopulations Cell 141 69-80

References 1 Jiang Q Endo M Dibra F Wang K Uitto J Pseudoxanthoma elasticum is a metabolic disease

J Investig Dermatol 2009129348ndash54

2 Borst P Oude Elferink R Mammalian ABC transporters in health and disease Annu Rev Biochem 200271537ndash92

3 Uitto J Bercovitch L Terry SF Terry PF Pseudoxanthoma elasticum Progress in diagnostics and research towards treatment Am J Med Genet 20111551517ndash26

4 Plomp AS Hu X de Jong PTVM Bergen AAB Does autosomal dominant pseudoxanthoma elasticum exist Am J Med Genet 2004126A403ndash12

5 Li Q Jiang Q Pfendner E Vaacuteradi A Uitto J Pseudoxanthoma elasticum clinical phenotypes molecular genetics and putative pathomechanisms Exp Dermatol 2009181ndash11

6 Georgalas I Tservakis I Papaconstaninou D Kardara M Koutsandrea C Ladas I Pseudoxanthoma elasticum ocular manifestations complications and treatment Clinical and Experimental Optometry 201094169ndash80

7 Vaacuteradi A Szaboacute Z Pomozi V de Boussac H Fuumlloumlp K Araacutenyi T ABCC6 as a target in pseudoxanthoma elasticum Curr Drug Targets 201112671ndash82

8 Sherer DW Sapadin AN Lebwohl MG Pseudoxanthoma elasticum an update Dermatology (Basel) 19991993ndash7

9 Smith JG Davidson EA CLARK RL Dermal elastin in actinic elastosis pseudoxanthoma elasticum Nature 1962195716ndash7

10 Bergen AA Plomp AS Schuurman EJ Terry S Breuning M Dauwerse H et al Mutations in ABCC6 cause pseudoxanthoma elasticum Nat Genet 200025228ndash31

11 Le Saux O Urban Z Tschuch C Csiszar K Bacchelli B Quaglino D et al Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum Nat Genet 200025223ndash7

12 Ringpfeil F Lebwohl MG Christiano AM Uitto J Pseudoxanthoma elasticum mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter Proc Natl Acad Sci USA 2000976001ndash6

13 Borst P Evers R Kool M Wijnholds J The multidrug resistance protein family Biochim Biophys Acta 19991461347ndash57

14 Madon J Hagenbuch B Landmann L Meier PJ Stieger B Transport function and hepatocellular localization of mrp6 in rat liver Mol Pharmacol 200057634ndash41

15 Ilias A Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6) J Biol Chem 200227716860ndash7

16 Scheffer GLG Kool MM de Haas MM de Vree JMLJ Pijnenborg ACLMA Bosman DKD et al Tissue distribution and induction of human multidrug resistant protein 3 Lab Invest 200282193ndash201

17 Pomozi V Le Saux O Brampton C Apana A Ilias A Szeri F et al ABCC6 Is a Basolateral Plasma Membrane Protein Circ Res 2013112e148ndash51

18 Gorgels TGMF Teeling P Meeldijk JD Nillesen STM van der Wal AC van Kuppevelt TH et

al Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruchs membrane Exp Eye Res Elsevier Ltd 201210459ndash64

19 Jiang Q Oldenburg R Otsuru S Grand-Pierre AE Horwitz EM Uitto J Parabiotic Heterogenetic Pairing of Abcc6minusminusRag1minusminus Mice and Their Wild-Type Counterparts Halts Ectopic Mineralization in a Murine Model of Pseudoxanthoma Elasticum The American Journal of Pathology 20101761855ndash62

20 Jansen RS Kuumlccediluumlkosmanoglu A de Haas M Sapthu S Otero JA Hegman IEM et al ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release Proc Natl Acad Sci USA 201311020206ndash11

21 Jansen RS Duijst S Mahakena S Sommer D Szeri F Varadi A et al ABCC6-Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation--Brief Report Arterioscler Thromb Vasc Biol 2014341985ndash9

22 Nitschke Y Rutsch F Genetics in Arterial Calcification Lessons Learned From Rare Diseases Trends Cardiovas Med Elsevier Inc 201222145ndash9

23 Roux KJ Kim DI Raida M Burke B A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells J Cell Biol 2012196801ndash10

24 Firat-Karalar EN Rauniyar N Yates JR III Stearns T Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Curr Biol Elsevier Ltd 201424664ndash70

25 Kim DI KC B Zhu W Motamedchaboki K Doye V Roux KJ Probing nuclear pore complex architecture with proximity-dependent biotinylation Proc Natl Acad Sci USA 2014111E2453ndash61

Genome-nuclear lamina interactions and resistance to targeted therapy in melanoma Background Melanoma is one the most aggressive human malignancies Novel targeted therapies (vemurafenib and dabrafenib which target BRAF

E600 and trametinib which targets MEK) have been developed and

showed dramatic clinical responses However resistances invariably occur and the prognosis of melanoma patients still remains poor Extensive research efforts to search for resistance mechanisms identified secondary mutations in the components of the MAPK pathway upstream or downstream of BRAF or in parallel pathways such as the PI3K pathway

1 These alterations occur late and are the

result of a massive selection pressure imposed by prolonged treatment However when performing short term response curves with vemurafenib in cultured melanoma cells we and others found that an appreciable of proportion cells survive the treatment at a dose that otherwise kills the majority of the population arguing for the existence of an early nonmutational mechanism of resistance A cell population of drug-tolerant cells has also been described for lung cancer lines treated with EGFR inhibitors and has been termed drug tolerant persisters (DTPs)

2 Parallel observations were done in

the clinic where the majority of responses to targeted therapies are only partial which suggest that survival of DTPs also occurs in patients As relapses arise frequently at site of residual tumors DTPs are likely to be responsible for most of therapy failures Therefore it is important to understand the mechanism underlying the drug tolerant state This phenotype is reversible as DTPs cells regain sensitivity to the drug when cultured in drug-free media

3 This suggests the involvement of epigenetic

mechanisms and raises the hypothesis that a particular chromatin configuration may underlie the drug tolerant state One important aspect of chromatin organization is its interaction with the nuclear lamina (NL) Indeed the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs)

4

Goals of the project In this project we will set out to investigate a role for genome-NL interactions in the establishment of the drug tolerant state in the context of BRAF mutant melanoma treated with vemurafenib For this we will make use of DamID a technique developed by the laboratory of Bas Van Steensel that allows for genome-wide study of genome-protein interactions

5 After having selected a vemurafenib-sensitive

melanoma cell line and optimized the drug concentration that allows obtaining the tolerant state we will perform DamID with lamin B1 a component of the nuclear lamina in parental cells and DTPs to search for LADs changes during the establishment of the drug tolerant state RNA sequencing will be performed in parallel in order to investigate a relationship between changes in LADs and changes in gene expression that may occur during this transition Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture (lenti)viral infections cell proliferation and viability assays immunofluorescence and Western blot analysis In addition you will be introduced to genomic approaches such as DamID and RNA sequencing which involve processing of genomic DNA or RNA and next generation sequencing Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl References 1 Sullivan R J amp Flaherty K T Resistance to BRAF-targeted therapy in melanoma Eur J

Cancer 49 1297-1304 doi101016jejca201211019 (2013) 2 Sharma S V et al A chromatin-mediated reversible drug-tolerant state in cancer cell

subpopulations Cell 141 69-80

al Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruchs membrane Exp Eye Res Elsevier Ltd 201210459ndash64

19 Jiang Q Oldenburg R Otsuru S Grand-Pierre AE Horwitz EM Uitto J Parabiotic Heterogenetic Pairing of Abcc6minusminusRag1minusminus Mice and Their Wild-Type Counterparts Halts Ectopic Mineralization in a Murine Model of Pseudoxanthoma Elasticum The American Journal of Pathology 20101761855ndash62

20 Jansen RS Kuumlccediluumlkosmanoglu A de Haas M Sapthu S Otero JA Hegman IEM et al ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release Proc Natl Acad Sci USA 201311020206ndash11

21 Jansen RS Duijst S Mahakena S Sommer D Szeri F Varadi A et al ABCC6-Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation--Brief Report Arterioscler Thromb Vasc Biol 2014341985ndash9

22 Nitschke Y Rutsch F Genetics in Arterial Calcification Lessons Learned From Rare Diseases Trends Cardiovas Med Elsevier Inc 201222145ndash9

23 Roux KJ Kim DI Raida M Burke B A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells J Cell Biol 2012196801ndash10

24 Firat-Karalar EN Rauniyar N Yates JR III Stearns T Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Curr Biol Elsevier Ltd 201424664ndash70

25 Kim DI KC B Zhu W Motamedchaboki K Doye V Roux KJ Probing nuclear pore complex architecture with proximity-dependent biotinylation Proc Natl Acad Sci USA 2014111E2453ndash61

Genome-nuclear lamina interactions and resistance to targeted therapy in melanoma Background Melanoma is one the most aggressive human malignancies Novel targeted therapies (vemurafenib and dabrafenib which target BRAF

E600 and trametinib which targets MEK) have been developed and

showed dramatic clinical responses However resistances invariably occur and the prognosis of melanoma patients still remains poor Extensive research efforts to search for resistance mechanisms identified secondary mutations in the components of the MAPK pathway upstream or downstream of BRAF or in parallel pathways such as the PI3K pathway

1 These alterations occur late and are the

result of a massive selection pressure imposed by prolonged treatment However when performing short term response curves with vemurafenib in cultured melanoma cells we and others found that an appreciable of proportion cells survive the treatment at a dose that otherwise kills the majority of the population arguing for the existence of an early nonmutational mechanism of resistance A cell population of drug-tolerant cells has also been described for lung cancer lines treated with EGFR inhibitors and has been termed drug tolerant persisters (DTPs)

2 Parallel observations were done in

the clinic where the majority of responses to targeted therapies are only partial which suggest that survival of DTPs also occurs in patients As relapses arise frequently at site of residual tumors DTPs are likely to be responsible for most of therapy failures Therefore it is important to understand the mechanism underlying the drug tolerant state This phenotype is reversible as DTPs cells regain sensitivity to the drug when cultured in drug-free media

3 This suggests the involvement of epigenetic

mechanisms and raises the hypothesis that a particular chromatin configuration may underlie the drug tolerant state One important aspect of chromatin organization is its interaction with the nuclear lamina (NL) Indeed the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs)

4

Goals of the project In this project we will set out to investigate a role for genome-NL interactions in the establishment of the drug tolerant state in the context of BRAF mutant melanoma treated with vemurafenib For this we will make use of DamID a technique developed by the laboratory of Bas Van Steensel that allows for genome-wide study of genome-protein interactions

5 After having selected a vemurafenib-sensitive

melanoma cell line and optimized the drug concentration that allows obtaining the tolerant state we will perform DamID with lamin B1 a component of the nuclear lamina in parental cells and DTPs to search for LADs changes during the establishment of the drug tolerant state RNA sequencing will be performed in parallel in order to investigate a relationship between changes in LADs and changes in gene expression that may occur during this transition Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture (lenti)viral infections cell proliferation and viability assays immunofluorescence and Western blot analysis In addition you will be introduced to genomic approaches such as DamID and RNA sequencing which involve processing of genomic DNA or RNA and next generation sequencing Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl References 1 Sullivan R J amp Flaherty K T Resistance to BRAF-targeted therapy in melanoma Eur J

Cancer 49 1297-1304 doi101016jejca201211019 (2013) 2 Sharma S V et al A chromatin-mediated reversible drug-tolerant state in cancer cell

subpopulations Cell 141 69-80

Genome-nuclear lamina interactions and resistance to targeted therapy in melanoma Background Melanoma is one the most aggressive human malignancies Novel targeted therapies (vemurafenib and dabrafenib which target BRAF

E600 and trametinib which targets MEK) have been developed and

showed dramatic clinical responses However resistances invariably occur and the prognosis of melanoma patients still remains poor Extensive research efforts to search for resistance mechanisms identified secondary mutations in the components of the MAPK pathway upstream or downstream of BRAF or in parallel pathways such as the PI3K pathway

1 These alterations occur late and are the

result of a massive selection pressure imposed by prolonged treatment However when performing short term response curves with vemurafenib in cultured melanoma cells we and others found that an appreciable of proportion cells survive the treatment at a dose that otherwise kills the majority of the population arguing for the existence of an early nonmutational mechanism of resistance A cell population of drug-tolerant cells has also been described for lung cancer lines treated with EGFR inhibitors and has been termed drug tolerant persisters (DTPs)

2 Parallel observations were done in

the clinic where the majority of responses to targeted therapies are only partial which suggest that survival of DTPs also occurs in patients As relapses arise frequently at site of residual tumors DTPs are likely to be responsible for most of therapy failures Therefore it is important to understand the mechanism underlying the drug tolerant state This phenotype is reversible as DTPs cells regain sensitivity to the drug when cultured in drug-free media

3 This suggests the involvement of epigenetic

mechanisms and raises the hypothesis that a particular chromatin configuration may underlie the drug tolerant state One important aspect of chromatin organization is its interaction with the nuclear lamina (NL) Indeed the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs)

4

Goals of the project In this project we will set out to investigate a role for genome-NL interactions in the establishment of the drug tolerant state in the context of BRAF mutant melanoma treated with vemurafenib For this we will make use of DamID a technique developed by the laboratory of Bas Van Steensel that allows for genome-wide study of genome-protein interactions

5 After having selected a vemurafenib-sensitive

melanoma cell line and optimized the drug concentration that allows obtaining the tolerant state we will perform DamID with lamin B1 a component of the nuclear lamina in parental cells and DTPs to search for LADs changes during the establishment of the drug tolerant state RNA sequencing will be performed in parallel in order to investigate a relationship between changes in LADs and changes in gene expression that may occur during this transition Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture (lenti)viral infections cell proliferation and viability assays immunofluorescence and Western blot analysis In addition you will be introduced to genomic approaches such as DamID and RNA sequencing which involve processing of genomic DNA or RNA and next generation sequencing Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl References 1 Sullivan R J amp Flaherty K T Resistance to BRAF-targeted therapy in melanoma Eur J

Cancer 49 1297-1304 doi101016jejca201211019 (2013) 2 Sharma S V et al A chromatin-mediated reversible drug-tolerant state in cancer cell

subpopulations Cell 141 69-80

3 Sharma S V Haber D A amp Settleman J Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents Nature reviews Cancer 10 241-253 doi101038nrc2820 (2010)

4 Guelen L et al Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions Nature 453 948-951 (2008)

5 Vogel M J Peric-Hupkes D amp van Steensel B Detection of in vivo protein-DNA interactions using DamID in mammalian cells Nat Protoc 2 1467-1478 (2007)

OCTOBER 2013

Dissecting DC antigen acquisition and licencing mechanisms in DNA vaccination Supervisors Dr Yanling Xiao and Dr Adriaan Bins Division Immunology Research group Prof J Borst NKIAvL Background The recent successes of immune therapy in clinical oncology have confirmed the notion that manipulation of the immune system can cure cancer patients However the immune interventions that are currently employed in clinical trials are not targeting tumor-specific T cells directly (eg ipilumimab) or are very complex (cellular therapies) An immune intervention that purposely elicits potent antigen-specific T cell immunity in patients does not exist In mice however DNA vaccines can do that DNA vaccination employs gene therapy for vaccination purposes By transient transfection of tissue in a vaccinee the immune system is exposed to this antigen and mounts an immune response The method is clinically attractive due to its simplicity and robustness At the same time it offers a unique flexibility that in laboratory animals can help in answering complex immunological questions Question In this project we dissect the mode of antigen transfer en presentation of dermal DNA vaccines We want to know what the relevant antigen presenting cells are how they aquire the dermally expressed antigen and what the additional signals are that result in optimal lsquolicencingrsquo of these APCrsquos for T cel priming Furthering the insight in the mechanisms involved in antigen presentation is crucial for successful translation of DNA vaccines from mice to patients On the long run this may lead to a safe and efficient tumor specific vaccine that can be used in the early stages of melanoma as an adjuvant treatment modality Approach In order to visualize antigen transfer and presentation we employ a DNA vaccine encoding a model antigen This antigen is a green fluorescent protein fused to T and B cell epitopes Employing the principles of biolistic transfection the pDNA is coated on gold bullets that are lsquoshotrsquo in the skin of mice using a gene gun The brightly green protein produced by transfected skin cells can be traced through the organism due to its extraordinary fluorescence and APC containing the model antigen can be easily identified in the draining lymph node This way we will identify the DC subset that cross-presents the DNA vaccine-encoded antigen The dermis containes 5 DC subsets discernible by CD207 CD11b and CD103 expression (among CD11c MHC Class II positive cells) Recently the DC subset that cross-presents antigen expressed in keratinocytes in non-inflamed lsquosteady statersquo skin has been identified (Henri et al) CD207+CD103+ dermal DC appeared to be the cross-presenting subset in steady-state However this may not apply to antigens expressed by keratinocytes upon dermal DNA vaccination We will sort the antigen presenting DC subsets from DLN of vaccinated mice By in vitro co-culture with resting CFSE-labeled antigen specific CD8 T cells we will assess which DC subset cross-presents the antigen by reading out CFSE dilution To dissect the impact of CD4 help we have created additional model antigens that either do or do not contain CD4 epitopes We will pinpoint the molecular substrate of DC licencing by deep sequencing the relevant APC and comparing the gene signature of DC that present antigen in either the lsquohelpedrsquo or lsquohelplessrsquo setting Techniques The work will entail standard molecular cloning techniques flow-cytometry cell-sorting intravital confocal microscopy various in vitro immunological essays and (supervised) work with laboratory animals Depending on you skills it may also involve RNA isolation and computer-based analysis of deep sequencing data The project allows for a high level of independency on your part and the lab of Prof Borst provides a very stimulating environment to learn basic immunology

Background reading Nat Immunol 2010 Nov11(11)989-96 doi 101038ni1946 Epub 2010 Oct 19 B cell follicles and antigen encounters of the third kind Cyster JG J Invest Dermatol 2011 Nov131(11)2178-85 doi 101038jid2011198 Epub 2011 Jul 7 Skin as a peripheral lymphoid organ revisiting the concept of skin-associated lymphoid tissues Egawa G Kabashima K J Exp Med 207 (2010) 189-206 Henri S Poulin LF Tamoutounour S Ardouin L Guilliams M de Bovis B Devilard E Viret C

Azukizawa H Kissenpfennig A Malissen B CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-

derived antigens irrespective of the presence of Langerhans cells

Contact yxiaonkinl abinsnkinl

AUGUST 2013

Dissecting DC antigen acquisition and licencing mechanisms in DNA vaccination Supervisors Dr Yanling Xiao and Dr Adriaan Bins Division Immunology Research group Prof J Borst NKIAvL Background The recent successes of immune therapy in clinical oncology have confirmed the notion that manipulation of the immune system can cure cancer patients However the immune interventions that are currently employed in clinical trials are not targeting tumor-specific T cells directly (eg ipilumimab) or are very complex (cellular therapies) An immune intervention that purposely elicits potent antigen-specific T cell immunity in patients does not exist In mice however DNA vaccines can do that DNA vaccination employs gene therapy for vaccination purposes By transient transfection of tissue in a vaccinee the immune system is exposed to this antigen and mounts an immune response The method is clinically attractive due to its simplicity and robustness At the same time it offers a unique flexibility that in laboratory animals can help in answering complex immunological questions Question In this project we dissect the mode of antigen transfer en presentation of dermal DNA vaccines We want to know what the relevant antigen presenting cells are how they aquire the dermally expressed antigen and what the additional signals are that result in optimal lsquolicencingrsquo of these APCrsquos for T cel priming Furthering the insight in the mechanisms involved in antigen presentation is crucial for successful translation of DNA vaccines from mice to patients On the long run this may lead to a safe and efficient tumor specific vaccine that can be used in the early stages of melanoma as an adjuvant treatment modality Approach In order to visualize antigen transfer and presentation we employ a DNA vaccine encoding a model antigen This antigen is a green fluorescent protein fused to T and B cell epitopes Employing the principles of biolistic transfection the pDNA is coated on gold bullets that are lsquoshotrsquo in the skin of mice using a gene gun The brightly green protein produced by transfected skin cells can be traced through the organism due to its extraordinary fluorescence and APC containing the model antigen can be easily identified in the draining lymph node This way we will identify the DC subset that cross-presents the DNA vaccine-encoded antigen The dermis containes 5 DC subsets discernible by CD207 CD11b and CD103 expression (among CD11c MHC Class II positive cells) Recently the DC subset that cross-presents antigen expressed in keratinocytes in non-inflamed lsquosteady statersquo skin has been identified (Henri et al) CD207+CD103+ dermal DC appeared to be the cross-presenting subset in steady-state However this may not apply to antigens expressed by keratinocytes upon dermal DNA vaccination We will sort the antigen presenting DC subsets from DLN of vaccinated mice By in vitro co-culture with resting CFSE-labeled antigen specific CD8 T cells we will assess which DC subset cross-presents the antigen by reading out CFSE dilution To dissect the impact of CD4 help we have created additional model antigens that either do or do not contain CD4 epitopes We will pinpoint the molecular substrate of DC licencing by deep sequencing the relevant APC and comparing the gene signature of DC that present antigen in either the lsquohelpedrsquo or lsquohelplessrsquo setting Techniques The work will entail standard molecular cloning techniques flow-cytometry cell-sorting intravital confocal microscopy various in vitro immunological essays and (supervised) work with laboratory animals Depending on you skills it may also involve RNA isolation and computer-based analysis of deep sequencing data The project allows for a high level of independency on your part and the lab of Prof Borst provides a very stimulating environment to learn basic immunology

Background reading Nat Immunol 2010 Nov11(11)989-96 doi 101038ni1946 Epub 2010 Oct 19 B cell follicles and antigen encounters of the third kind Cyster JG J Invest Dermatol 2011 Nov131(11)2178-85 doi 101038jid2011198 Epub 2011 Jul 7 Skin as a peripheral lymphoid organ revisiting the concept of skin-associated lymphoid tissues Egawa G Kabashima K J Exp Med 207 (2010) 189-206 Henri S Poulin LF Tamoutounour S Ardouin L Guilliams M de Bovis B Devilard E Viret C

Azukizawa H Kissenpfennig A Malissen B CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-

derived antigens irrespective of the presence of Langerhans cells

Contact yxiaonkinl abinsnkinl

AUGUST 2013

Background reading Nat Immunol 2010 Nov11(11)989-96 doi 101038ni1946 Epub 2010 Oct 19 B cell follicles and antigen encounters of the third kind Cyster JG J Invest Dermatol 2011 Nov131(11)2178-85 doi 101038jid2011198 Epub 2011 Jul 7 Skin as a peripheral lymphoid organ revisiting the concept of skin-associated lymphoid tissues Egawa G Kabashima K J Exp Med 207 (2010) 189-206 Henri S Poulin LF Tamoutounour S Ardouin L Guilliams M de Bovis B Devilard E Viret C

Azukizawa H Kissenpfennig A Malissen B CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-

derived antigens irrespective of the presence of Langerhans cells

Contact yxiaonkinl abinsnkinl

AUGUST 2013

Pattern of physical mental and psychosocial recovery following curative lung resection in lung cancer patients diagnosed with NSCLC

Lung resection still provides the best long-term outcome for lung cancer but causes a considerable decay of physical and psychosocial health status even years following surgery Pulmonary rehabilitation which includes exercise training education and psychosocial and behavioral interventions is shown to improve recovery following lung resection The increase in physical activity is considered the most beneficial aspect of pulmonary rehabilitation in lung cancer patients Surprisingly no studies so far have reported the use of objective measures to assess daily physical activity levels for longer periods of time in this population Next to that psychological symptoms seem to be important for successful rehabilitation in terms of adherence and effects on self-reported outcomes So far no studies could be found describing the role of psychological symptoms in recovery following lung resection or the effect of rehabilitation on psychological symptoms Objectives In this study we aim to explore

- the recovery of physical and psychological health status physical activity patterns and HRQoL in

cancer patients treated with lung resection from pre-surgery until 6 months post-surgery

- the relationship between daily physical activity and recovery after lung resection

- the role of psychological symptoms in recovery of (self-reported) physical health and HRQoL

To reach the objectives of the study a prospective cohort study started at the NKI-AVL hospital in Amsterdam in which lung cancer patients treated with lung resection (with curative intention) are monitored from 1 month pre-surgery until 6 months post-surgery Outcome measures include but are not limited to lung function physical fitness physical functioning and symptoms psychological symptoms and daily activity levels Candidate profile We are looking for a motivated student who has a background in physiotherapy human movement sciences health sciences medicine or comparable field who is interested in clinical research and is willing to spend a period of 3-6 months working on this project You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and letter of motivation by email to jtimmermannkinl

JULY 2013

Sister chromatid co-entrapment and release by the cohesin complex During each cell division the entire genome must be duplicated and separated in such a manner that each of the daughter cells inherits a complete copy of the genome without any alterations Sister chromatid cohesion mediated by the cohesin complex is crucial to maintain such genomic integrity Cohesin holds the sisters together by co-entrapping sister DNA fibres inside its ring-shaped structure from S phase until mitosis Cohesin is of vital importance to resist the pulling forces of microtubules up to the satisfaction of the spindle assembly checkpoint when the abrupt cleavage of cohesin rings triggers the segregation of sister chromatids to the opposite poles of the cell in anaphase Cohesin is also essential for high fidelity DNA repair How cohesin manages to specifically co-entrap the sister DNAs of each individual chromosome is largely a mystery We find that this process is dependent on the acetylation of cohesinrsquos Smc3 subunit by the Eco1 acetyltransferase This acetylation stabilizes cohesinrsquos association with chromatin and makes it resistant to the cohesin removal factor Wapl Our current understanding is that cohesin has a continuous cycle of DNA entrapment and release and that Smc3 acetylation in S phase acts as a lock to stabilize specifically the cohesin rings that hold the sister DNAs together Many unanswered questions remain regarding the cohesin complex For example What are the upstream regulators that determine which cohesin rings are acetylated What are the mechanistic consequences of Smc3 acetylation How does Wapl remove cohesin from chromatin And what are the cellular pathways that drive cohesin from chromosome arms in early mitosis These are the kind of questions that keep us awake at night and drive our research We are addressing such questions using a combination of genetics biochemistry and microscopy in both budding yeast and mammalian cells Are you equally fascinated by this topic and would you like to join our team for a Masters internship of at least 6 months Feel free to send an enquiring email including your CV and motivation to Dr Benjamin D Rowland The Netherlands Cancer Institute Division of Cell Biology I Plesmanlaan 121 1066 CX Amsterdam browlandnkinl 020-512 1932

JUNE 2013

Dynamics and role of genome-nuclear lamina interactions in oncogene-induced senescence Backgound Cellular senescence is a virtually irreversible form of cell cycle arrest that occurs in response to diverse stress signals including oncogene activation (Oncogene-Induced Senescence or OIS) We and others have provided evidences that OIS acts as a robust tumor suppressor mechanism in vivo that prevents the progression of many types of pre-malignant lesions to full-blown cancers Given this pivotal role of senescence in the pathogenesis of human cancer there is a crucial need to dissect the mechanisms underlying this failsafe program We are investigating whether and how the nuclear envelope (NE) and its interaction with the genome change during OIS We discovered that in primary human cells undergoing OIS display expression of all lamins and of several nuclear envelope proteins is strongly suppressed by a mechanism involving protein degradation by the lysosomal machinery Immunofluorescence study showed that the residual NE proteins display an aberrant pattern of localization while electron microscopy analysis revealed invagination of the NE indicating that OIS cells undergo a global disruption of the NE structure Objectives The goal of this project is to investigate whether the above-described alterations in NE structure and composition affect genome-nuclear lamina interactions during OIS Using DamID a technique that allows for genome-wide mapping of protein-genome interactions the laboratory of Bas van Steensel has previously identified in normal human fibroblasts large chromosomal domains interacting with the nuclear lamina called Lamina-Associated Domains (LADs) In collaboration with the laboratory of Bas van Steensel (NKI) we are optimizing the DamID technique with lamin B1 a component of the nuclear lamina for OIS cells Once the technique optimized in this setting we will generate LAD profile in cells induced to senesce by the expression of the BRAF

E600 oncogene as well as in normal proliferating

quiescent cells and in cells that have bypassed BRAFE600

- induced senescence to identify LADs changes that are specific for OIS Should we identify such changes we will determine whether they contribute to the transcriptional alterations previously reported in OIS cells and identify the pathways that are regulated by this type of chromatin rearrangement Techniques In this project a range cellular biology techniques will be used including but not limited to cell culture cell proliferation assays and Western blot analysis In addition you will be introduced to the DamID technique which involves isolation and processing of genomic DNA for deep sequencing and bioinformatic analysis of deep sequencing data Candidate profile We are looking for a motivated student (HLOWO) who has a background in cellular and molecular biology and biochemistry who is highly interested in basic research and is willing to spend a period of 6-8 months working on this project Previous research experience is preferred but not mandatory You should be able to work with others but also independently Contact If you are interested andor you need more information please send an email with a CV and a letter of motivation by email to clenainnkinl

JUNE 2013

The Genetics of Cancer and Infectious Disease ndash Brummelkamp laboratory The most powerful and direct way to gain insight into complex biological systems is to remove individual components and observe the consequences By doing so genetics has revolutionized almost all aspects of biology Although human cell cultures are often used as experimental tool their genome is largely refractory to efficient mutagenesis due to its diploid state Our group recently developed an entirely novel genetic model system based on insertional mutagenesis- in haploid and near-haploid human cells We have shown that this approach enables the generation of knockouts for most human genes and can be used to pinpoint genes that are involved in phenotypes of interest Combined with next-generation sequencing approaches this method generates high-density genetic overviews of genes required for nearly any selectable cell trait In a variety of genetic screens we have identified host factors required for infection of cells by influenza virus the first entry receptor for a Clostridium difficile toxin and we identified the cholesterol transporter NPC1 as the long-sought intracellular entry receptor for Ebola virus Cells derived from Niemann-Pick patients (harbouring mutations in NPC1) are completely resistant to infection by Ebola or Marburg virus This genetic model system is currently used to further our understanding of the inner workings of human cells to search for cancer cell vulnerabilities and to identify host factors used by pathogens Techniques involved Cell culture standard molecular biological techniques (cloning PCR amplification sequencing) retroviral transduction next-generation sequencinganalysis Duration Minimum 6 months References Carette JE Guimaraes CP Varadarajan M Park AS Wuethrich I Godarova A Kotecki M Cochran BH Spooner E Ploegh HL and Brummelkamp TR (2009) Haploid genetic screens in human cells identify host factors used by pathogens Science 326 1231-1235 Carette JE Raaben M Wong AC Herbert AS Obernosterer G Mulherkar N Kuehne AI Kranzusch PJ Griffin AM Ruthel G Dal Cin P Dye JM 1 Whelan SP Chandran K and Brummelkamp TR (2011) Ebola virus entry requires the cholesterol transporter Niemann-Pick C1 Nature 477 340-343 Carette JE Giumaraes CP Wuethrich I Blomen VA Varadarajan M Sun C Bell G Yuan B Muellner MK Nijman SM Ploegh HL and Brummelkamp TR (2011) Global gene disruption in human cells to assign genes to phenotypes by deep sequencing Nat Biotechnol 29 542-6 Reiling JH Clish CB Carette JE Varadarajan M Brummelkamp TR and Sabatini DM (2011) A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin Proc Natl Acad U S A Jun 15 Miller EM Obernosterer G Raaben M Herbert AS Deffieu MS Krishnan A Ndungo E Sandesara RG Carette JE Kuehne AI Ruthel G Pfeffer SR Dye JM Whelan SP Brummelkamp TR and Chandran K (2012) Ebola virus entry requires the host-programmed recognition of an intracellular receptor EMBO J 31 1946-60 Further reading New York Times httpwwwnytimescom20120117healthnpc1-protein-may-give-ebola-its-openinghtml Boston Globe httpwwwbostoncomBostonwhitecoatnotes201108boston-scientists-reveal-how-ebola-infects-cellsT0EPT7hpzmhAt1GkxqsT7Mindexhtml Laboratory homepage

httpresearchnkinlbrummelkamplabWelcomehtml Master students interested in working in a younginternational team are encouraged to send an e-mail to tbrummelkampnkinl It would be appreciated if you would include a brief CV and indicate why you would be interested January 2013

Student projects

Our group has a long history in developing biologically relevant FRET sensors to measure for instance Estrogen Receptor functioning phosphatidyl inositol bisphosphate (PIP2) and cyclic AdenosineMonoPhosphate (cAMP) cAMP is an important second messenger that activates Protein Kinase A (PKA) Our lab recently published a new EPAC-based sensor to measure cAMP (Klarenbeek et al PlosOne 2011) However further improvements to this sensor can still be made in both sensitivity and stability of the sensor during long measurements Another way of detecting cAMP is by using a PKA-based sensor PKA is more sensitive to cAMP than EPAC but has the disadvantage that expression of the sensor often is troublesome New molecular tools have to be developed to allow reliable expression of the PKA-based sensor All new sensors have to be tested and validated using advanced microscopic techniques In line with the cAMP sensor we are also interested in (local) induced effects of cAMP by adenylate cyclase Recently a Photoactivatable Adenylate Cylcase (PAC) is published This PAC is able to activate adenylate cyclase in distinct parts of the cell by activating it by laser light This method allows us to test the effects of cAMP on distinct structures of a cell Also other photoactivatable receptors are currently made or have been published recently for instance photoactivatable G-Coupled receptors (paGPCRrsquos) By this method we are able to activate by laser light Some paGPCRrsquos have been published but others are still under construction and have to be fully characterized These developments constitute ideal student projects In the course of ~8 months you will learn about molecular cloning fluorescent proteins FRET and the instrumentation you need for that You will create new improved version(s) and extensively test them with state-of-the-art equipment Depending on the progress made you will also be able to apply your own FRET sensor to address questions embedded in one of the research lines in our lab Because of the speed of developments it is inpossible to always present the newest ideas on this site it is best to contact us and talk one-on-one about a project that would really excite you Please contact Kees Jalink kjalinknkinl tel 0205121933

January 2013

The role of histone modifying enzymes in normal development and cancer Cancer is a disease of the DNA Accumulation of genetic alterations in time results in uncontrolled

proliferation of cells which ultimately disseminate to other organs Over the years many so‐called tumor suppressor genes and oncogenes have been identified Recently through the efforts of

full‐genome sequencing of various tumors genetic mutations in histone modifying enzymes have been identified in hematological cancers Histone modifying enzymes balance chemical modifications on histones the package material of our DNA and other cellular proteins However little is known about the mechanisms controlled by these enzymes during normal development and tumorigenesis Using conditional knockout mice which enable us to inactivate histone modifying enzymes in vivo and in vitro we explore the role of these enzymes in cellular proliferation and hematopoiesis We have currently positions available for highly motivated students who wish to participate in research aimed at identifying interacting proteins and substrates of histone deacetylases Hdac1 and Hdac2 and histone

demethylase Jmjd5 In addition fibroblasts T‐ and B‐lymphocytes and bone‐marrow cells will be

isolated from conditional knock‐out mice to analyze their potential to proliferate and differentiate Gene

expression profiles obtained by RNA‐sequencing will be performed to identify the transcriptome of these histone modifying proteins Practical work will involve a wide variety of techniques such as cloning of retroviral expression constructs cell culture of rimary and tumor cells protein purification western blot analysis qPCR and FACS nalysis If you are interested please contact Dr JH Dannenberg jdannenbergnkinl

020‐5122024 Division of Gene Regulation The Netherlands Cancer Institute Amsterdam The Netherlands Relevant literature

Wilting RH Yanover E Heideman RM Jacobs H Horner J Van der Torre J DePinho RA Dannenberg JH Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and hematopoiesis

EMBO J 2010 29 2586‐97 Guan JS Haggarty SJ Giacometti E Dannenberg JH Joseph N Gao J Nieland T Zhou Y Wang X Mazitschek R Bradner JE DePinho RA Jaenisch R Tsai LH HDAC2

negatively egulates memory formation and synaptic plasticity Nature 2009 459 55‐63

January 2013

The role of histone modifying enzymes in normal development and cancer Cancer is a disease of the DNA Accumulation of genetic alterations in time results in uncontrolled

proliferation of cells which ultimately disseminate to other organs Over the years many so‐called tumor suppressor genes and oncogenes have been identified Recently through the efforts of

full‐genome sequencing of various tumors genetic mutations in histone modifying enzymes have been identified in hematological cancers Histone modifying enzymes balance chemical modifications on histones the package material of our DNA and other cellular proteins However little is known about the mechanisms controlled by these enzymes during normal development and tumorigenesis Using conditional knockout mice which enable us to inactivate histone modifying enzymes in vivo and in vitro we explore the role of these enzymes in cellular proliferation and hematopoiesis We have currently positions available for highly motivated students who wish to participate in research aimed at identifying interacting proteins and substrates of histone deacetylases Hdac1 and Hdac2 and histone

demethylase Jmjd5 In addition fibroblasts T‐ and B‐lymphocytes and bone‐marrow cells will be

isolated from conditional knock‐out mice to analyze their potential to proliferate and differentiate Gene

expression profiles obtained by RNA‐sequencing will be performed to identify the transcriptome of these histone modifying proteins Practical work will involve a wide variety of techniques such as cloning of retroviral expression constructs cell culture of rimary and tumor cells protein purification western blot analysis qPCR and FACS nalysis If you are interested please contact Dr JH Dannenberg jdannenbergnkinl

020‐5122024 Division of Gene Regulation The Netherlands Cancer Institute Amsterdam The Netherlands Relevant literature

Wilting RH Yanover E Heideman RM Jacobs H Horner J Van der Torre J DePinho RA Dannenberg JH Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and hematopoiesis

EMBO J 2010 29 2586‐97 Guan JS Haggarty SJ Giacometti E Dannenberg JH Joseph N Gao J Nieland T Zhou Y Wang X Mazitschek R Bradner JE DePinho RA Jaenisch R Tsai LH HDAC2

negatively egulates memory formation and synaptic plasticity Nature 2009 459 55‐63

January 2013

Preclinical investigations on drug delivery and anti-tumor efficacy against high-grade glioma by combination therapy using small molecular inhibitors targeting PI3K MAPK and Ink4a-RB pathways Project Supervisor Olaf van Tellingen PhD Stage Master Internship 2013 Project description Despite of the recent success in development of targeted therapies with small molecular inhibitors (imatinib erlotinib sorafinib) so far no targeted therapeutic drug has shown any meaningful effect in the treatment of high-grade glioma glioblastoma the most common primary brain tumor in adults The major reasons include poor drug delivery due to the blood-brain barrier and insufficient considerations of the multiple and parallel oncogenic cell signaling pathways driving this disease

With the much better view on the signaling networks involved in high-grade glioma as obtained from the cancer genome atlas (TCGA) project these issues can now be appropriately addressed by well-designed preclinical studies For this purpose we have established a unique series of in vitro in vivo models to investigate the brain penetration and antitumor efficacy of candidate drugs In particular we have established a series of spontaneous high-grade glioma models that carry the specific genetic alterations underlying the human disease such as deletion of the CDKN2 locus loss of Pten andor P53 We have also established glioma lsquostemrsquo cell lines from these tumors as well as naiumlve precursor neurosphere stem cell cultures With these powerful tools we are able to interrogate the importance of these pathways for treatment and to optimize the combination and timing of interventions This work will provide crucial information with respect to selecting those drugs that can efficiently cross the blood brain barrier and enter the brain at pharmacologically relevant levels and to select combinations of drugs that together may halt the progression of this disease Moreover by this work we may identify those patients that -based on the genetic profiles of their tumors- may benefit most of specific drug combinations Our work is supported by the presence of a high-end cone-beam irradiator for small animals that will allow us to mimic the standard chemo-radiation therapy given to patients This is important since any novel therapy will always be given in adjunct to the standard chemo-radiation therapy This project will focus on small molecular inhibitors targeting the p16Ink4ab-CDK46-Rb Ras-Raf-Mek-Erk and PI3k-Akt-mTOR pathways

This project is open for highly motivated students doing their Masters in Biomedical Sciences Biology or Pharmacology Candidates should be willing to spend a period of at least 8 months as this work involves lab-animal research Within this project you will be using various techniques covering different disciplines namely Animal experiments plasma pharmacokinetics tissue distribution drug efficacy toxicity studies Cell and molecular biology experiments glioma stem cells isolation cellstem cell culturing transwell assays cell proliferation assays PCR West blotting etc HPLC bio-analysis Furthermore the Netherlands Cancer Institute also provides the opportunity to attend the weekly seminars where speakers from international top institutions in the Cancer Research field will give a presentation of their work Contact Olaf van Tellingen (Division of Diagnostic Oncology Preclinical Pharmacology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam) Email ovtellingennkinl Tel 020-512-2792 January 2013

Project title The influence of methylating agents and adipocytokines on intestinal crypt bases isolated from a newly established Msh2-Lynch syndrome mouse model Project description Lynch syndrome (LS) also named non-polyposis colon cancer (HNPCC) is caused by an inherited defect in genes encoding the DNA mismatch repair (MMR) system (for example Msh2) Patients with LS are characterised by the early development of colorectal cancer (CRC) and tumors in other internal organs (stomach small intestine) or the skin (Muir-Torre syndrome) LS patients are heterozygous for a MMR gene and they gradually accumulate MMR-defective cells by loss of the wild-type allele To mimic this situation we established a novel Msh2-Lynch mouse model in which a MMR-defective intestinal compartment can be obtained by injections of tamoxifen These mice carry two modified Msh2 alleles one is constitutive knockout the other behaves as wild-type but exons 12 and 13 are flanked by LoxP sites Recombination between the loxP sites and hence inactivation of the Msh2 allele occurs by Cre recombinase whose activity requires tamoxifen In this way Msh2-- cells arise amidst MMR proficient Msh2+- cells Importantly this modification takes part in the intestinal stem cells since expression of Cre-recombinase relies on the promoter of Lgr5 a well-known marker of these stem cells Because of the strong mutator phenotype of MMR-deficient cells we expect development of intestinal tumors Using this model we are investigating whether food constituents in particular methylating agents can accelerate tumorigenesis In addition a correlation between obesity and cancer development has been recently suggested although conflicting results have been reported on the role of adipocytokines (produced by adipose tissue) in promotion of tumorigenesis in the intestinal tract The aim of this student project is to establish an efficient and quick in vitro experimental model system that will mimic the situation occurring in the Msh2-Lynch mouse intestine where MSH2-proficient cells are surrounded by MMR-deficient cells One of the techniques we are currently testing is the culturing of organoids as described by Sato et al (2009) who showed that intestinal crypt bases isolated from mice can successfully be grown and expanded outside the animal body This model will be used for the analysis of factors that may modulate the behavior of MMR-deficient crypt cells and their propensity to tumorigenesis Both factors with an inhibitory and a stimlulatory potential towards tumorigensis will be investigated Candidate profile We are looking for a highly motivated master student that is willing to spend a period of 6 ndash 9 months in our research group The student is familiar with at least some of the following techniques cell culture transformation and transfection PCR and Western blotting Art 9 or 12 registration allowing to work with animals is not mandatory but provides extra opportunities Both domestic and international students are welcome to apply for this project After a short period of introduction to the project the student will have the possibility to work independently References Lynch HT Lynch J (2000) Lynch syndrome genetics natural history genetic counselling and prevention J Clin Oncol 18 19S-31S Donohoe CL Doyle SL Reynolds JV (2011) Visceral adiposity insulin resistance and cancer risk Diabetol Metab Syndr 312 Vrieling A Kampman E (2010) The role of body mass index physical activity and diet in colorectal cancer recurrence and survival a review of the literature Am J Clin Nutr 92(3)471-90 Sato T Vries RG Snippert HJ van de Wetering M Barker N Stange DE van Es JH Abo A Kujala P Peters PJ Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche Nature 459(7244)262-5 Epub 2009 Mar 29 Contact The CV can be sent to Prof Hein te Riele htrielenkinl

January 2012

Project title The influence of methylating agents and adipocytokines on intestinal crypt bases isolated from a newly established Msh2-Lynch syndrome mouse model Project description Lynch syndrome (LS) also named non-polyposis colon cancer (HNPCC) is caused by an inherited defect in genes encoding the DNA mismatch repair (MMR) system (for example Msh2) Patients with LS are characterised by the early development of colorectal cancer (CRC) and tumors in other internal organs (stomach small intestine) or the skin (Muir-Torre syndrome) LS patients are heterozygous for a MMR gene and they gradually accumulate MMR-defective cells by loss of the wild-type allele To mimic this situation we established a novel Msh2-Lynch mouse model in which a MMR-defective intestinal compartment can be obtained by injections of tamoxifen These mice carry two modified Msh2 alleles one is constitutive knockout the other behaves as wild-type but exons 12 and 13 are flanked by LoxP sites Recombination between the loxP sites and hence inactivation of the Msh2 allele occurs by Cre recombinase whose activity requires tamoxifen In this way Msh2-- cells arise amidst MMR proficient Msh2+- cells Importantly this modification takes part in the intestinal stem cells since expression of Cre-recombinase relies on the promoter of Lgr5 a well-known marker of these stem cells Because of the strong mutator phenotype of MMR-deficient cells we expect development of intestinal tumors Using this model we are investigating whether food constituents in particular methylating agents can accelerate tumorigenesis In addition a correlation between obesity and cancer development has been recently suggested although conflicting results have been reported on the role of adipocytokines (produced by adipose tissue) in promotion of tumorigenesis in the intestinal tract The aim of this student project is to establish an efficient and quick in vitro experimental model system that will mimic the situation occurring in the Msh2-Lynch mouse intestine where MSH2-proficient cells are surrounded by MMR-deficient cells One of the techniques we are currently testing is the culturing of organoids as described by Sato et al (2009) who showed that intestinal crypt bases isolated from mice can successfully be grown and expanded outside the animal body This model will be used for the analysis of factors that may modulate the behavior of MMR-deficient crypt cells and their propensity to tumorigenesis Both factors with an inhibitory and a stimlulatory potential towards tumorigensis will be investigated Candidate profile We are looking for a highly motivated master student that is willing to spend a period of 6 ndash 9 months in our research group The student is familiar with at least some of the following techniques cell culture transformation and transfection PCR and Western blotting Art 9 or 12 registration allowing to work with animals is not mandatory but provides extra opportunities Both domestic and international students are welcome to apply for this project After a short period of introduction to the project the student will have the possibility to work independently References Lynch HT Lynch J (2000) Lynch syndrome genetics natural history genetic counselling and prevention J Clin Oncol 18 19S-31S Donohoe CL Doyle SL Reynolds JV (2011) Visceral adiposity insulin resistance and cancer risk Diabetol Metab Syndr 312 Vrieling A Kampman E (2010) The role of body mass index physical activity and diet in colorectal cancer recurrence and survival a review of the literature Am J Clin Nutr 92(3)471-90 Sato T Vries RG Snippert HJ van de Wetering M Barker N Stange DE van Es JH Abo A Kujala P Peters PJ Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche Nature 459(7244)262-5 Epub 2009 Mar 29 Contact The CV can be sent to Prof Hein te Riele htrielenkinl

January 2012