All slides Cancer Biology FINAL 18may11.ppt...Tanner Hagelstrom Meredith Henderson Irma Gonzales Natasha Caplen Very Special Thanks to Irma Gonzales Kristen Bisanz Tanya Little Christian

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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Brought to you by the Science/AAAS Business Office18 May, 201118 May, 2011


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)








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)





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





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








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All slides Cancer Biology FINAL 18may11.ppt...Tanner Hagelstrom Meredith Henderson Irma Gonzales Natasha Caplen Very Special Thanks to Irma Gonzales Kristen Bisanz Tanya Little Christian