1 Name : Dr. Joerg Hoheisel Born : In Bad Nanheim, Germany Studies : At University of Konstanz, Germany PhD : In 1984 – 1988 P td At I i lC R hF di Postdoc : At Imperial Cancer Research Fund in London, UK 1988 – 1993 Since 1993 Group Leader at German Cancer Research Center (DKFZ) Since 2008 Chairman of Scientific Council of DKFZ Since 2008 Chairman of Scientific Council of DKFZ 2.5 μm Stockholm Stockholm Stockholm Moscow Moscow Moscow Oslo Oslo Oslo Dublin Dublin Dublin Riga Riga Riga St. Petersburg St. Petersburg St. Petersburg Helsinki Helsinki Helsinki Heidelberg Heidelberg Heidelberg Berlin Berlin Berlin Paris Paris Paris London London London Vienna Vienna Vienna Hamburg Hamburg Hamburg Warsaw Warsaw Warsaw Frankfurt Frankfurt Frankfurt Zürich Zürich Zürich Kiew Kiew Kiew Odessa Odessa Odessa Brussels Brussels Brussels Rome Rome Rome Madrid Madrid Madrid Athens Athens Athens Istanbul Istanbul Istanbul Lisbon Lisbon Lisbon Belgrade Belgrade Belgrade Toulouse Toulouse Toulouse
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Name : Dr. Joerg HoheiselBorn : In Bad Nanheim, GermanyStudies : At University of Konstanz, GermanyPhD : In 1984 – 1988P td At I i l C R h F d iPostdoc : At Imperial Cancer Research Fund in
London, UK 1988 – 1993
Since 1993 Group Leader at German Cancer ResearchCenter (DKFZ)
Since 2008 Chairman of Scientific Council of DKFZSince 2008 Chairman of Scientific Council of DKFZ
2.5 µm
StockholmStockholmStockholm
MoscowMoscowMoscow
OsloOsloOslo
DublinDublinDublin
RigaRigaRiga
St. PetersburgSt. PetersburgSt. PetersburgHelsinkiHelsinkiHelsinki
HeidelbergHeidelbergHeidelberg
BerlinBerlinBerlin
ParisParisParis
LondonLondonLondon
ViennaViennaVienna
HamburgHamburgHamburgWarsawWarsawWarsaw
FrankfurtFrankfurtFrankfurt
ZürichZürichZürich
KiewKiewKiew
OdessaOdessaOdessa
BrusselsBrusselsBrussels
RomeRomeRomeMadridMadridMadrid
AthensAthensAthens
IstanbulIstanbulIstanbul
LisbonLisbonLisbon
BelgradeBelgradeBelgradeToulouseToulouseToulouse
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Genomics ActivitiesNational Center forTumour Diseases (NCT)
• DNA-microarrays> 550 mRNA analyses > 250 miR analyses
• Antibody-microarrays• Protein-microarrays
> 350 blood & urine samples
160 genomic sequences
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MicroRNA (miR) ProfilingmiR
94 blood samples 45 cancer samples38 chronic pancreatitis11 normal donors
185 tissue samples 137 cancer samples137 cancer samples22 chronic pancreatitis26 normal donors
Chip designfeaturing 863 miRs RT-PCR confirmationcommon to miRBase 12.0 to 14.0 960 reactions
Individual miR ProfilesmiR
Bauer et al. (2012) PLoS ONE 7, e34151.
MicroRNA Diagnosis from BloodmiR
Bauer et al. (2012) PLoS ONE 7, e34151.
MicroRNA Diagnosis from BloodmiR
Healthy Multiple Sclerosis
0 0.2 0.4 0.6 0.8 1
Probability
0 0.2 0.4 0.6 0.8 1
Keller et al. (2011) Nature Meth. 8, 841-843.
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MicroRNA Diagnosis from Blood
Tumours
BrainOvary Pelvis
Skin
Stomach
Colon
Breast
Wilms
Lung
Pancreas
Prostate
Keller et al. (2013) Genome Biol., submitted.
Stable RNAi by Lentiviral Transduction
5‘ LTR shRNA Barcode 3‘ sinLTR
P1 P2
Puromycin resistance
Processed into siRNA
Amplification for construct identification
Essential target
NON-essential target
shRNA barcode
shRNA barcode
Barcode pool tzero Barcode pool tend
P1 P2
P1 P2
Böttcher et al. (2010) Curr. Genomics 11, 162-167.
Global shRNA Screening
shRNA-libraryin lentiviruses
pooled infection: one virus per cell
gDNA at t gDNA at t
Barcode amplification
Hybridisation
Puromycin
Gene knockdown
gDNA at tzero gDNA at t30d
culturing for 30 days
Lethal knockdowns drop out
Proliferation Ratio
- drug
Combined growth of constructs
tzero tend-
Synthetic-Lethal Screen
tzero
Proliferation Ratio = [log2 (tend / tzero)]
+ drug
tend+
zero
tend
Böttcher et al. (2010) BMC Genomics 11, 7.
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Improving the Effect of Gemcitabine
Inhibition of replication
IC20 of Gemcitabine
Result of Synthetic-Lethal Screen Validation of Screen Result
BxPC-3 treated with 6 nM gemcitabine für 72 h
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Validation of RAD17
BxPC-3 MiaPaca-2 JoPaca-1
Elucidation of Mechanism
Fredebohm et al. (2013), J. Cell Sci., in press.
Asymetric mitosis DNA fragmentation
Elucidation of Mechanism
Fredebohm et al. (2013), J. Cell Sci., in press.
Protein Expression & Structural ProfilingAntibody Microarrays
EU
Goat anti-mouse IgGGoat anti-rabbit IgG
Dual colour incubationwith two serum samples
Alhamdani et al. (2010) J. Prot. Res. 9, 963-971.Gloriam et al. (2010) Mol. Cell. Prot. 9, 1-10.Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1280.Alhamdani et al. (2010) Proteomics, 10, 3203-3207.Schmidt et al. (2011) J. Prot. Res. 10, 1316-1322.Mustafa et al. (2011) Mol. Biosyst. 7, 1795-1801.Friedrich et al. (2011) Proteomics 11, 3757-3760.Schröder et al. (2011) Meth. Mol. Biol., 203-221.Alhamdani et al. (2012) J. Proteomics 75, 3747-3759.Hoheisel et al. (2013) Prot. Clin. Appl., in press.
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EU binder projects
Binder MoleculesAntibody Microarrays
Taussig et al. (2007) Nature Meth. 4, 13-17.Gloriam et al. (2010) Mol. Cell. Prot. 9, 1-10.
PROTEOMEBINDERS AFFINITYPROTEOME AFFINOMICS
TranscriptionalProfiling
• DNA sequences of 900 genes/ESTs exhibiting differential transcription
• Peptide selection from sequences
Expression Profiling
p qaccording to 12 selection criteria
• Peptide synthesis
• Immunisation of rabbits
• Affinity purification of polyclonal antibodies
• Characterisation by in situ analyses
Current Chip LayoutAntibody Microarrays
810 different antibodies678 selected based on significant variations in transcriptional studies
67 additional keyplayers in cancer-related pathways40 from literature research and collaboration partners25 h k i t i d t l25 housekeeping proteins and controls
Goat anti-mouse IgGGoat anti-rabbit IgG
Protein labellingwith Sypro Ruby
Positional marker proteins
Human serum samples
Human urine samples
Antibody Microarrays
Cancer Binders Profiling Pancreatic Cancer TissuesAntibody Microarrays
1031 tissues
650 t i l int
ensi
ties
[AU
]
Background
650 proteinsamples
550 intraining set
100 intest set
412 samplesit bl f 99 analyses49 samples
ith t
old batch new batchSig
na
Slides
199 intest set
suitable foranalysis
99 analysesof low qualitywithout
annotation
wererepeated
Tumour type
Celltype
Histologicalgrade Localisation Lymphnodes
affectedMetastases
mRNA vs.protein
miRNA vs.protein Survival Diabetes etc.Alcohol
added totest set
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Antibody Microarrays
Dual Colour Modus
Common ReferenceSamples A818-1A818-4A818-7AsPC-1
Proteinextractions
27 pancreatic cancer cell lines
Histochemicalanalyses
650pancreatic tissues
AsPC 1AVC-1PxPC-3Capan-1Capan-2CFPACColo-357FAMPACHPAFIMIM-PC-1IMIM-PC-2MCC-1MDA-Panc-28Mi P 2