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Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsWebinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScience
18 May, 201118 May, 2011
Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsReal-time, Label-free Cell Monitoring in RNAi ProfilingReal-time, Label-free Cell Monitoring in RNAi Profiling
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Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceId tif i N l C Th ti T tId tif i N l C Th ti T t
Brought to you by the Science/AAAS Business Office18 May, 201118 May, 2011
Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsReal-time, Label-free Cell Monitoring in RNAi ProfilingReal-time, Label-free Cell Monitoring in RNAi Profiling
Participating Experts:
Brought to you by the Science/AAAS Business Office
Kristina Cole, M.D.-Ph.D.Children’s Hospital of PhiladelphiaPhiladelphia, PA
David Azorsa, Ph.D.Translational Genomics Research Institute (TGen)Scottsdale, Arizona
Sponsored by:
An RNAi screen of the protein kinome identifies CHK1 as a therapeutic target in neuroblastoma
Kristina A. Cole, MD/PhD
The Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine
Neuroblastoma (NB)
Common pediatric cancerN bl t i i t t li i l bl Neuroblastoma remains an important clinical problemDespite intense therapy over 50% of HR pts will suffer a relapse that is invariably fatal
There is a need for targeted therapies
Low Risk100%
80%
60%
High Risk
40%
20%
Park et al,JCO 2010
10%
An RNAi screen of the kinome (using an impedance based assay)(using an impedance based assay)
S tti thSetting up the screen:1. choice of platform2. choice of cell lines3 i l di i3. experimental conditions4. validation
An RNAi screen of the kinome identifies a candidate list of targetsidentifies a candidate list of targets
Cole et al, PNAS 2011
CHK1 depletion was broadly potent in NB
120
% Growth Inhibition after CHK1 depletion
60
80
100
120
0
20
40
RPE‐1
SKNAS
SKNDZ
IMR‐5
BE2
BE2C
EBC1
KELLY
NLF
NGP
SKNSH
CHK1
• Central mediator of the DDRhalts cell cycle progression- halts cell cycle progression
- replication fork progression
• maps to 11q24 maps to 11q24
• CHK1 inhibitors are in clinical trials- chemo-radiation sensitizer- normal tissues spared
• Single agent activity in AMLCavelier et al Cancer Res, 2009
- not in other cancer types
Dai and Grant: Clin Ca Res, 16(2), 376-83, 2010
CHK1 is over‐expressed in MYCNA tumors p
CHK1 is constitutively phosphorylatedin NB cell lines and tumorsin NB cell lines and tumors
Blot 1
Blot 2
Sensitivity Correlates with CHK1 PhosphorylationPhosphorylation
Spearman’s Correlation = - 0.85 (p=.0004)
CHK1 inhibition causes apoptosis during replicationapoptosis during replication
MYCN induction results in CHK1 phosphorylationphosphorylation
CHK1 inhibition is potent in vivop
NB-1643
P=0.001
As a single agent and in combination with chemotherapy
Conclusions/Future Directions
An RNAi screen of the kinome identified novel candidatesAn RNAi screen of the kinome identified novel candidates
CHK1 is constitutively phosphorylated in NeuroblastomaCHK1 is constitutively phosphorylated in Neuroblastoma- biomarker of inhibitor sensitivity and activity- MYC(N) induced replication stress
evaluation of mechanism underway- evaluation of mechanism underway
CHK1 is a rational target for clinical development in NBCHK1 is a rational target for clinical development in NB- small molecule inhibitors are in trials for adult cancers
Acknowledgements
The Children’s Hospital of Philadelphia
Cole LabCole LabMike RussellMike RussellJonathan HugginsJonathan Huggins
Maris LabMaris LabPatrick MayesPatrick MayesJosh CourtrightJosh Courtright
Mössé LabMössé LabM. Laudenslager
PathologyPathologyBruce Pawal
Jonathan HugginsJonathan HugginsChase Chase HuldermanHuldermanMichael Michael LaQuagliaLaQuaglia
Josh CourtrightJosh CourtrightCindy WinterCindy WinterSharon DiskinSharon Diskin
AttiyehAttiyeh LabLabGeoffrey Norris
StatisticsStatisticsYimie LiYimie Li
PfizerKenneth HookJames Christiansen
NCI / NIHNCI / NIHK08-CA136979-01 (to KAC)R01-CA87847 (to JMM)
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceId tif i N l C Th ti T tId tif i N l C Th ti T t
Brought to you by the Science/AAAS Business Office18 May, 201118 May, 2011
Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsReal-time, Label-free Cell Monitoring in RNAi ProfilingReal-time, Label-free Cell Monitoring in RNAi Profiling
Participating Experts:
Brought to you by the Science/AAAS Business Office
Kristina Cole, M.D.-Ph.D.Children’s Hospital of PhiladelphiaPhiladelphia, PA
David Azorsa, Ph.D.Translational Genomics Research Institute (TGen)Scottsdale, Arizona
Sponsored by:
Use of label free assays for targetUse of label free assays for target validation in high throughput
RNAi screeningRNAi screening
David O. Azorsa, Ph.D.Translational Genomics Research InstituteTranslational Genomics Research Institute
Scottsdale, AZ
Use of label free assays for target validation in HT RNAi iHT-RNAi screening
• Background: HT RNAi screening• Background: HT-RNAi screening
• Phenotypic profiling for the identification of “Achilles’ Heel” targets in Ewing’s Sarcoma
• Other examples of label-free impedance analysis forOther examples of label free impedance analysis for siRNA screening validation
High-Throughput RNA-interference (HT-RNAi)
• a platform for performing genome-wide functional screening using gene silencingscreening using gene silencing
• also know as loss of function (LOF) screening• also know as loss-of-function (LOF) screening
• can be performed using siRNA or shRNA libraries• can be performed using siRNA or shRNA libraries
siRNA Screening vs shRNA Screening
From the NIH Chemical Genomic Center Website: http://assay.nih.gov/index.php/section14:RNAi_Loss-of-function_Screens
Use of “Reverse” Transfection of HT-RNAi
“Reverse” TransfectionStandard Transfection
siRNA:Lipidcomplex Cells
Cell Monolayer siRNA:Lipid ComplexCell Monolayer siRNA:Lipid Complex
Steps in HT-RNAi Screening
HT RNAiAssayAssayDevelopment
DataAnalysis HitHT-RNAiy
ValidationDevelopment& Optimization
Analysis& KM Validation
Transfection Reagent Optimization Functional Assays
Hit
Small Scale Screen
Genome-wide Screen
Identification
Combination Cell Seeding Optimization
Pathway Analysis
Assays
Drug Dose Response
Growth Kinetics Analysis
Use of Label Free Impedance Assay for Cell Density Optimization
Use of Label Free Impedance for Assay Time Optimization
xC
ell I
ndex
Treatment
Time (Hours)
Use of label free assays for target validation in HT RNAi iHT-RNAi screening
• Background: HT RNAi screening• Background: HT-RNAi screening
• Phenotypic profiling for the identification of “Achilles’ Heel” targets in Ewing’s Sarcoma
• Other examples of label-free impedance analysis forOther examples of label free impedance analysis for siRNA screening validation
Ewing’s Sarcoma
• Pediatric tumor found in bones and soft tissue
• Incidence is about 3 per 1,000,000 in U.S.
• Around 90% of Ewing’s sarcoma cases result from aAround 90% of Ewing s sarcoma cases result from a chromosomal translocation t(11;22)(q24;q12) involving EWS and FLI1
• Five year survival for localized disease is about 70%; Long term survival for metastatic disease is between 10-20%
Screening for “Achilles’ Heel” Targets
+ siRNAt G Xto Gene X
Normal Cell No Effect (Normal Cell Growth)
+ siRNAto Gene X
Growth Arrest
Ewing’s Sarcoma
Cell DeathCell Death
RNAi Screen: 572 KinasesSK-ES-1TC-32
RD-ESTC-71
Phenotypic Profiling Analysis
2 a
2 b
1 a
1 b
S-1
a
S-1
b
S a
S b
5659
a
5659
b
TC-32 a
TC-32 b
TC 71 a
TC-3
2
TC-3
2
TC-7
1
TC-7
1
SK
-ES
SK
-ES
RD
-ES
RD
-ES
GM
05
GM
05
TC-32 a
GM05659 a
GM05659 b 0.920
TC-71 a
TC-71 b
SK-ES-1 a
SK-ES-1 b
RD-ES a
TC 32 a
TC-32 b
TC-71 a
TC-71 b
RD-ES a
0.5180.971
0.972 0.701
0 33RD ES a
RD-ES b
GM05659 a
GM05659 b
SK-ES-1 a
SK-ES-1 b
RD ES a
RD-ES b 0.959
0.975
0.826
0.733
RNAi Screen of Ewing’s sarcomaTC-32 SK-ES-1
ore re
4
0
2
4
2
Z-sc
o
Z-sc
or
STK10
TNK2STK10
TNK2-2
-4
0
-2
-4
TC 71 RD ES
-6
-8
4
4
-6
4TC-71 RD-ES
-sco
re
scor
e
TNK2
0
2
2
0
-2
2
Z- Z-
STK10
TNK2
STK10
TNK2-2
-4
-6
-4
-6
-8
-8 -10
Phenotypic Profiling
32 a
32 b
71 a
71 b
ES
-1 a
ES
-1 b
ES
a
ES
b
0565
9 a
0565
9 b
93
TC-71TC-32
98 43
AKAP3_BAKT1_ACDK5R2_AGRK4_BMAST1_BMK-STYX_ANEK3 A
TC-3
TC-3
TC-7
TC-7
SK
-E
SK
-E
RD
-E
RD
-E
GM
0
GM
0
ES GM05659
6998 51
25NEK3_ANEK9_ANTRK3_BPIK3AP1_APRKCA_BPXK_ASTK10_ASTK10_BSTK11 B
5725 8Cell lines
17specific
SK-ES-1 RD-ESSTK11_B
PLK1_A
TAOK3_BTNK2_B
Silencing of TNK2 and STK10 inhibits growth of E i ’ llEwing’s sarcoma cells
TC-32 SK-ES-1
l)
iabi
lity
(% o
f con
trol)
Via
bilit
y (%
of c
ontro
STK10_5 STK10_6 TNK2_5 TNK2_6 PLK1_6 PLK1_7ControlSTK10_5 STK10_6 TNK2_5 TNK2_6 PLK1_6 PLK1_7Control
Vi
TC-71 RD-ES
ntro
l)
cont
rol)
Via
bilit
y (%
of c
on
Via
bilit
y (%
of
STK10_5 STK10_6 TNK2_5 TNK2_6 PLK1_6 PLK1_7ControlSTK10_5 STK10_6 TNK2_5 TNK2_6 PLK1_6 PLK1_7Control
Kinetic analysis of growth inhibition by TNK2 and STK10 il i i E i ’ llSTK10 silencing in Ewing’s sarcoma cells
Silencing of TNK2 and STK10 induces apoptosis E i ’ llEwing’s sarcoma cells
80
100
*
**
**
**80
100
tive
116/144
62/94
20
40
60
Hoechst 33258
40
60
Anne
xin
V Po
si
195/35779/155
0Control STK10_5 STK10_6 TNK2_5 TNK2_6STK10_5 STK10_6 TNK2_5 TNK2_6Control
Annexin V0
20
Control STK10_5 STK10_6 TNK2_5 TNK2_6
% A
18/548
Use of label free assays for target validation in HT RNAi iHT-RNAi screening
• Background: HT RNAi screening• Background: HT-RNAi screening
• Phenotypic profiling for the identification of “Achilles’ Heel” targets in Ewing’s Sarcoma
• Other examples of label-free impedance analysis forOther examples of label free impedance analysis for siRNA screening validation
Label Free Impedance Assay: Effect of knock-in of growth promoting genes
HT-RNAi Screen for Gemcitabine Sensitizing Targets
tabi
ne
abin
e 5 nM Gemcitabine 10 nM Gemcitabine
nM G
emci
M G
emci
ta
Viab
ility)
10
Viab
ility)
5 n
CHK1_BCHK1_A
CHK1_A
Log
(V
Log
(V CHK1_B
Log (Viability) Vehicle
Log (Viability) Vehicle
Validation of CHK1 as a Gemcitabine Sensitizing Target
MIA PaCa-2
% C
ontr
ol%
Con
trol
rol
BxPC3
rol
% C
ontr
% C
ont
Label Free Impedance Analysis: Growth Inhibition by CHK1 Silencing and Gemcitabineby CHK1 Silencing and Gemcitabine
73 hrs+ Vehicle109 hrs
Non-Sil siRNA
Addition
51 hrs
CHK1-A siRNA
CHK1-B siRNA+ 10 nM Gem
35 hrs 39 hrsAddition
Summary
• High Throughput RNAi screening is a useful l tf f th id tifi ti f d l t fplatform for the identification of modulators of
biological functions
• Label Free Impedance assays are instrumental in both assay development of HT-RNAi and validation of functional hitsof functional hits.
AcknowledgmentsgTGenSpyro Mousses
National Cancer Institute Javed Khan
Shilpi AroraTanner HagelstromMeredith HendersonIrma Gonzales
Natasha Caplen
Very Special Thanks toIrma GonzalesKristen BisanzTanya LittleChristian Beaudry
Robert & Allyson KavnerReed KavnerEwing’s Research FoundationV F d tiHolly Yin
Jeff KieferDaniel Von HoffJeffrey Trent
V Foundation
Jeffrey Trent
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceId tif i N l C Th ti T tId tif i N l C Th ti T t
Brought to you by the Science/AAAS Business Office18 May, 201118 May, 2011
Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsReal-time, Label-free Cell Monitoring in RNAi ProfilingReal-time, Label-free Cell Monitoring in RNAi Profiling
Participating Experts:
Brought to you by the Science/AAAS Business Office
Kristina Cole, M.D.-Ph.D.Children’s Hospital of PhiladelphiaPhiladelphia, PA
David Azorsa, Ph.D.Translational Genomics Research Institute (TGen)Scottsdale, Arizona
Sponsored by:
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceId tif i N l C Th ti T tId tif i N l C Th ti T t
Brought to you by the Science/AAAS Business Office18 May, 201118 May, 2011
Identifying Novel Cancer Therapeutic TargetsIdentifying Novel Cancer Therapeutic TargetsReal-time, Label-free Cell Monitoring in RNAi ProfilingReal-time, Label-free Cell Monitoring in RNAi Profiling
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