2011 AACR OncoPanel Poster

2011 AACR 102nd Annual Meeting

Title: Tumor cell line profiling of eighteen cancer

therapeutics to evaluate relationships between

tumor genotypes and cancer cell sensitivities

Authors: Yulia Y. Ovechkina, Christine O'Day, Karen Marcoe, Robert Keyser, Karen

Bernards, Jessica Chesnut-Speelman, Phuong T.B. Nguyen, Jenny Mulligan, Teddy

Lin, Rodney Shively, Jim Hnilo, Brian Nelson. Ricerca Biosciences, LLC, Bothell,

WA

Introduction

In vitro cellular response profiling of tumor human cell lines has become a

widely used approach for the targeted cancer therapeutics development. Correlation

of the drug sensitivity and resistance cellular response with genomic data offers a

robust and sensitive system for predicting clinical efficacy and identifying more

efficacious patient populations.

We have developed a high throughput cellular approach to evaluate the

relationship between tumor genotypes and drug sensitivity over 240 human tumor

cell lines. A panel of eighteen cancer therapeutic agents was tested for proliferative,

apoptotic and cell cycle arrest responses using multiplexed high content screening

with automated fluorescence microscopy and image analysis based technology (GE

Healthcare INCell Analyzer 1000). Growth index was measured using nuclear dye.

Activated caspase 3 antibodies were used for the apoptosis induction detection.

Phospho-histone H3 antibodies were used to measure the cell cycle block. We

generated cell line profiles to reveal drug sensitivity and resistance patterns and

identified examples of the genomic markers associated with a specific response. This

approach could provide insight into the mechanisms of enhanced susceptibility or

resistance which in turn could be used for the optimization of the targeted cancer

therapeutics.

• 240 human tumor-derived cell

line panel from different origins

with broad genetic

heterogeneity

• Cell lines are quality controlled

(procured from ATCC and

other banks)

• Sensitive High Content cellular

image analysis

• High throughput (384 well plates)

• Multiplexed data output

• 10 concentrations (in triplicates)

240_OncoPanel™ - Human Tumor Cell Line Profiling

1

6

6

7

7

10

11

12

19

19

20

20

20

20

29

33

Other

Endocrine

Head and Neck

Liver

Prostate

Kidney

Bladder

Pancreas

Lung

Skin

Breast

CNS

Female GU

Soft Tissue

Haematopoietic

Colon/GI

240_OncoPanel™ - Cancer Types

Available genetic information

mRNA expression data

http://www.ebi.ac.uk/microarray-as/ae/files/E-MTAB-37

Gene copy number and mRNA expression data

https://cabig.nci.nih.gov/caArray_GSKdata/

Sanger gene copy number data

http://www.sanger.ac.uk/genetics/CGP/translation/data/

http://www.broadinstitute.org/cgi-bin/cancer/datasets.cgi

Sanger gene mutation data

http://www.sanger.ac.uk/genetics/CGP/Celllines

The mutation data was obtained from the Sanger Institute Catalogue

Of Somatic Mutations In Cancer web site,

http://www.sanger.ac.uk/cosmic

Bamford et al (2004) The COSMIC (Catalogue of Somatic Mutations

in Cancer) database and website. Br J Cancer, 91,355-358.

50+ Gene Mutation data

APC BRAF BRCA1 BRCA2

CDH1 CDKN2A CTNNB1 CYLD

ERBB2 FBXW7 FGFR3 FLCN

GNAS HRAS JAK2 KIT

MAP2K4 MLH1 MSH2 MSH6

NF1 NF2 NOTCH1 NPM1

NTRK3 PALB2 PDGFRA PIK3C2A

PTCH1 PTEN RB1 RET

SMAD4 SMO SOCS1 STK11

TP53 TSC1 TSC2 VHL

CBFB EGFR FLT3 KRAS

NRAS PIK3CA RUNX1 MYC








https://cabig.nci.nih.gov/caArray_GSKdata/

http://www.sanger.ac.uk/genetics/CGP/translation/data/




http://www.sanger.ac.uk/genetics/CGP/Celllines

High Throughput Screening

Cell proliferation

Cell cycle

Apoptosis

High Content Analysis

Compound

C

N

N

O

N C

O

OCH3

H

NCH2CH2CH2CH3

H

240 human tumor cell line panel

Sensitive cells Resistant cells

Genotype correlation analysis

240_OncoPanel™ - Assay Workflow

72 hrs

-11 -10 -9 -8 -7 -60

50

100

log [Vinblastine], M

PO

C

-11 -10 -9 -8 -7 -60

10

20

30

40


Fo

ld In

du

cti

on

-12 -11 -10 -9 -8 -70

1

2

3

4


Fo

ld I

nd

ucti

on

Criteria for Positive Responses

• Cell proliferation measured by relative cell counts

» EC50 is a concentration at the curve inflection point (parameter C)

» IC50 is a concentration at 50% of maximal possible response

» GI50 is a concentration needed to reduce the growth of treated cells to half that of untreated cells.

D Log GI50 is the log difference from the average GI50 value

• Apoptosis:» >5-fold increase in activated caspase-3 signal

indicates apoptosis induction

• Cell cycle block:» >2-fold increase in phospho-histone-3 indicates

G2/M cell cycle block

» <2-fold decrease in phospho-histone-3 indicates G1/S cell cycle block

High Content Multiplexed Output

Cell Nuclei – blue; Apoptotic cells-

green ; Mitotic cells - red

Cancer therapeutic agents

Midpoint of the GI50 range was used to distinguish sensitive and resistant populations. The GI50 of the sensitive and resistant populations

was then averaged to produce the “sensitive and resistant response mean GI50”.

Compound Synonym(s) Target(s) Source CAS No.Concentration

range, microM

Sensitive

response

mean GI50,

Resistant

response mean

GI50, microM

Ratio of resistant

GI50 mean to

sensitive GI50 mean

Log GI50

range

BMS-387032 SNS-032 CDK2, 7 and 9 Selleck 345627-80-7 10 - 0.0003 0.15 1.45 10 2.1

Cl 1040 PD184352 Mek1/2 Selleck 212631-79-3 10 - 0.0003 0.18 4.43 25 3.1

PD0325901 N/A Mek1/2 Calbiochem 391210-10-9 30 - 0.001 0.06 15.53 259 4.5

PD173074 N/A FGFR1, FGFR3 Calbiochem 219580-11-7 5 - 0.0002 0.24 4.63 19 2.4

API-2 Triciribine AKT Tocris 35943-35-2 20 - 0.0006 0.38 12.79 34 2.8

Lapatinib TykerbEGFR, ErbB2, Erk-1/2

and AKT

Sequoia

Research 388082-78-8 20 - 0.0006 0.28 9.26 33 3.4

BMS-536924 N/A IGF-1R Selleck 468740-43-4 10 - 0.0003 0.19 3.41 18 3

Erlotinib Tarceva EGFR, ErbB2Sequoia

Research 183321-74-6 14 - 0.0004 0.15 4.48 30 3.2

Geldanamycin N/A HSP-90Sequoia

Research 30562-34-6 5 - 0.0002 0.03 0.12 4 2.4

Sorafenib NexavarBRAF, PDGF, C-Kit,

VEGF

Sequoia

Research 284461-73-0 20 - 0.0006 0.29 4.33 15 2.6

Dasatinib SprycelBCR-ABL, SRC,

Ephrins, EGFR

Sequoia

Research 863127-77-9 5 - 0.0002 0.008 0.87 109 4.5

VX-680 TozasertibAurora-A,-B,-C

kinases

Sequoia

Research 639089-54-6 10 - 0.0003 0.06 2.23 37 3.3

Sunitinib SutentFLT3, PDGFRs,

VEGFRs, Kit

Sequoia

Research 557795-19-4 20 - 0.0006 0.21 4.37 21 3.2

Everolimus N/A mTOR Sequoia

Research 159351-69-6 10 - 0.0003 0.009 4.83 537 4.5

Tandutinib N/A FLT3 ,PDGFR, KITSequoia

Research 387867-13-2 10 - 0.0003 0.22 4.58 21 2.8

Doxorubicin N/A topoisomerase II Calbiochem 25316-40-9 5 - 0.0002 0.005 0.04 8 2.8

Paclitaxel Taxol tubulin Calbiochem 33069-62-4 0.3 - 9.55E-06 0.001 0.009 9 3.5

Staurosporine N/APKC, PKA,PKG, p60v-

src, CaM kinase IICalbiochem 62996-74-1 1 - 3.18E-05 0.003 0.025 8 2.4

Multiplexed high content approach provides insight into the drug

mechanisms of action

PD0325901 treated Colo829 cell line PD173074 treated KatoIII cell line

Cell

pro

lifera

tion

Apopto

sis

Cell

cycle

Cell

pro

lifera

tion

Apopto

sis

Cell

cyc

le

Lapatinib treated CHL-1 cell line

Cell

pro

lifera

tion

Apopto

sis

Cell

cyc

le

BMS-536924 treated HT-29 cell line

Cell

pro

lifera

tion

Apopto

sis

Cell

cycle



BMS-387032 treated MV-4-11 cell line

Cell

pro

lifera

tion

Apopto

sis

Cell

cyc

le

API-2 treated T47D cell line

Cell

pro

lifera

tion

Apopto

sis

Cell

cycle



IC50 vs GI50 profiles across 240 cell lines

0.000

0.001

0.010

0.100

1.000

10.000

100.000

0.000 0.001 0.010 0.100 1.000 10.000 100.000

GI50, microM

IC50

, mic

roM

ResistantSensitive

IC5

0

GI50

PD0325901

0.01

0.10

1.00

10.00

0.01 0.10 1.00 10.00

GI50, microM

IC50

, mic

roM

PD173074

0.01

0.10

1.00

10.00

0.01 0.10 1.00 10.00

GI50, microM

IC50

, mic

roM

BMS-536924

0.01

0.10

1.00

10.00

0.01 0.10 1.00 10.00

GI50, microM

IC50

, mic

roM

BMS-387032

0.01

0.10

1.00

10.00

100.00

0.01 0.10 1.00 10.00 100.00

GI50, microM

IC50, m

icro

M

API-2 (Triciribine)

0.001

0.010

0.100

1.000

10.000

100.000

0.001 0.010 0.100 1.000 10.000 100.000

GI50, microMIC

50, m

icro

M

Lapatinib

ResistantSensitive

IC5

0

GI50

Erlotinib

Dasatinib

CL 1040

VX-680

Sorafenib

Everolimus

0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100

IC50, m

icro

M

GI50, microM

0.001

0.01

0.1

1

10

0.001 0.01 0.1 1 10

IC50, m

icro

MGI50, microM

0.01

0.1

1

10

100

0.01 0.1 1 10 100

IC50, m

icro

M

GI50, microM

0.0001

0.001

0.01

0.1

1

10

0.0001 0.001 0.01 0.1 1 10

IC50, m

icro

M

GI50, microM

0.001

0.01

0.1

1

10

0.001 0.01 0.1 1 10

IC50, m

icro

M

GI50, microM

0.00001

0.0001

0.001

0.01

0.1

1

10

0.0001 0.001 0.01 0.1 1 10

IC50, m

icro

M

GI50, microM


ResistantSensitive

IC5

0

GI50

Sunitinib

Paclitaxel

Tandutinib

Staurosporine

Doxorubicin

Geldanamycin

0.01

0.1

1

10

100

0.01 0.1 1 10 100

IC50, m

icro

M

GI50, microM

0.1

1

10

0.01 0.1 1 10

IC50, m

icro

MGI50, microM

0.0001

0.001

0.01

0.1

1

0.0001 0.001 0.01 0.1 1

IC50, m

icro

M

GI50, microM

0.00001

0.0001

0.001

0.01

0.1

1

1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

IC50, m

icro

M

GI50, microM

0.0001

0.001

0.01

0.1

1

0.0001 0.001 0.01 0.1 1

IC50, m

icro

M

GI50, microM

0.001

0.01

0.1

1

10

0.001 0.01 0.1 1 10

IC50, m

icro

M

GI50, microM


0.001

0.010

0.100

1.000

10.000

100.000

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

0.01

0.10

1.00

10.00

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

0.01

0.10

1.00

10.00

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

0.001

0.010

0.100

1.000

10.000

100.000

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

GI50 values across 240 cell line panel

ResistantSensitive 240 cell lines

GI5

0, m

icro

M

PD0325901

PD173074

Lapatinib

BMS-536924

BMS-387032

0.01

0.10

1.00

10.00

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

0.01

0.10

1.00

10.00

100.00

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0,

mic

roM

API-2 (Triciribine)

Erlotinib

Dasatinib

CL 1040

VX-680

Sorafenib

Everolimus


GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M



GI5

0, m

icro

M

Sunitinib

Paclitaxel

Tandutinib

Staurosporine

Doxorubicin

Geldanamycin

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

1.00E-05

1.00E-04

1.00E-03

1.00E-02

1.00E-01

1.00E+00

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M

0.0001

0.001

0.01

0.1

1

10

100

0 20 40 60 80 100 120 140 160 180 200 220 240

GI5

0, m

icro

M


Chemosensitivity profiles ranked by the D GI50 value across 240 cell lines


DL

og

GI5

0, m

icro

M

PD0325901

PD173074

Lapatinib

BMS-536924

BMS-387032

API-2 (Triciribine)

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

DL

og

GI5

0,

mic

roM

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

DL

og

GI5

0,

mic

roM

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

DL

og

GI5

0,

mic

roM

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

DL

og

GI5

0,

mic

roM


DL

og

GI5

0, m

icro

M

Erlotinib

Dasatinib

CL 1040

VX-680

Sorafenib

Everolimus

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0, m

icro

M

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

DL

og

GI5

0, m

icro

M

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

DL

og

GI5

0,

mic

roM

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM



DL

og

GI5

0, m

icro

M

Sunitinib

Paclitaxel

Tandutinib

Staurosporine

Doxorubicin

Geldanamycin

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

DL

og

GI5

0,

mic

roM

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

DL

og

GI5

0, m

icro

M

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

DL

og

GI5

0,

mic

roM

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

DL

og

GI5

0,

mic

roM


Raf/Ras mutations are associated with sensitivity to Mek inhibitor, PD0325901, while

PIK3CA and RB mutations correlate with resistance

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

D L

og

GI5

0,

mic

roM

PD0325901

The log difference from the average GI50 value is plotted across the 240 cell lines

Sensitive Resistant

RB1 mutationsPIK3CA mutationsRaf/Ras mutations

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

DL

og

GI5

0,

mic

roM

Raf/Ras mutations are associated with sensitivity to Mek inhibitor, CL 1040, while PIK3CA

and RB mutations correlate with resistance

CL 1040


PIK3CA mutationsRaf/Ras mutations RB1 mutations

Sensitive Resistant

-4.00

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

DL

og

GI5

0,

mic

roM

PIK3CA mutations are associated with sensitivity to Everolimus, while KRAS

mutations confer resistance

Sensitive

KRAS mutationsPIK3CA and KRAS

mutations

PIK3CA mutations


Resistant

Everolimus

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

DL

og

GI5

0,

mic

roM

Sensitive Resistant

EphA7 & EphA3 mRNA

overexpression


BCR-ABL translocation

Dasatinib

Ephrin type-A7 & A3 receptor mRNA overexpression and BCR-ABL translocation

are associated with sensitivity to Dasatinib

CDKN2A mutations are associated with sensitivity to CDK inhibitor, BMS-387032,

while CCND2 amplification correlates with resistance

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

D L

og

GI5

0,

mic

roM

Sensitive Resistant

CDKN2A mutation


CCND2 (Cyclin D2) copy number

amplification

BMS-387032

AKT2 amplification and PTEN mutations are associated with resistance to AKT

inhibitor, API-2 (Triciribine)

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

D L

og

GI5

0, m

icro

M

Sensitive Resistant

PTEN mutation


AKT2 copy number amplification

API-2 (Triciribine)

FGFR protein family overexpression is associated with sensitivity to FGFR inhibitor,

PD173074, while EGFR overexpression correlates with resistance

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

D L

og

GI5

0,

mic

roM

FGFR overexpression in Ras/Raf or

PIK3CA mutation background

EGFR mRNA

overexpression

FGFR protein family mRNA

overexpression


Sensitive Resistant

PD173074

Raf/Ras, PIK3CA and PTEN mutations correlate with resistance to FGFR inhibitor, PD173074

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

D L

og

GI5

0,

mic

roM

PD173074

PTEN mutationsPIK3CA mutationsRaf/Ras mutations


Sensitive Resistant

EGFR overexpression is associated with sensitivity to EGFR inhibitor, Lapatinib, while

Raf/Ras, PIK3CA or PTEN mutations may confer resistance to the EGFR overexpressing cell

lines

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

D L

og

GI5

0,

mic

roM

Lapatinib

Sensitive Resistant

EGFR overexpression in Raf/Ras, PIK3CA or

PTEN mutation background

EGFR mRNA

overexpression


-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

D L

og

GI5

0,

mic

roM

Sensitive

Raf/Ras, PIK3CA and PTEN mutations may correlate with resistance to EGFR

inhibitor, Lapatinib

PTEN mutationsPIK3CA mutationsRaf/Ras mutations


Resistant

Lapatinib

EGFR overexpression is associated with resistance to IGF-1R inhibitor, BMS-

536924

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

D L

og

GI5

0,

mic

roM

BMS-536924

Sensitive Resistant

EGFR overexpression in

EGFR mutation background

EGFR mRNA

overexpression

The log of the difference from the average GI50 value is plotted across the 240 cell lines

K-Ras, PIK3CA and PTEN mutations may correlate with resistance to IGF-1R

inhibitor, BMS-536924

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

D L

og

GI5

0,

mic

roM

Sensitive

PTEN mutationsPIK3CA mutationsK-Ras mutations


Resistant

BMS-536924

Conclusions

We generated chemosensitivity profiles over 240 cell lines to reveal drug

sensitivity and resistance patterns and identified markers associated with a

specific response. It was found that Raf/Ras mutations confer sensitivity to MEK

inhibitors, PD0325901 and Cl 1040, while PIK3CA and RB mutations were

associated with resistance. Resistance to IGF-1R, FGFR, and EGFR inhibitors

correlated with PI3K/PTEN/Akt or Raf/Ras pathway activation. EGFR mRNA

overexpression was associated with resistance to FGFR and IGF-1R inhibitors.

Resistance to AKT inhibitor, API-2 (Triciribine), was associated with PTEN

mutations and amplification of AKT. PIK3CA mutations were associated with

sensitivity to Everolimus, while KRAS mutations confer resistance. Dasatinib

sensitivity was associated with BCR-ABL translocation and Ephrin type-A7 and

A3 receptor mRNA overexpression.

Technology

2011 AACR OncoPanel Poster