Cancer molecular biology GBM molecular biology

dr.beaumontphysician.com

Cancer molecular biology GBM molecular biology

9/7/2012

Glioblastoma Cancer Stem Cells:

Translational Studies in

Therapeutic Resistance and Biomarker Discovery

John S. Kuo, MD PhD FAANS FACS

Depts. of Neurological Surgery and Human Oncology

Director, Comprehensive Brain Tumor Program

Carbone Comprehensive Cancer Center

Center for Stem Cell and Regenerative Medicine

School of Medicine and Public Health

University of Wisconsin-Madison

No financial disclosures or conflicts

for all work in this presentation

21 st Beaumont Symposium, Troy, Michigan - September 19, 2012

Objective:

Learn about the clinical challenges and

opportunities for developing more effective

GBM therapies through cancer stem cell

studies.

Question:

Describe one or more novel findings in GBM

cancer stem cell research that explain clinical

observations in GBM patients.

Cancer is a disease of mutations”

Epigenetic

regulation

Transcripts

Proteins

Networks

Cancer molecular biology

• Cell cycle regulation (p53, Rb, MDM2, CDK)

• Cell growth signaling (EGF-R, AKT, PTEN,

PI3K)

• Angiogenesis (VEGF, PDGF, FGF)

• Epigenetic regulation (MGMT promoter

methylation, differential microRNAs, histone

coding)

GBM molecular biology

• Cell cycle regulation (p53, Rb, MDM2, CDK)

• Cell growth signaling (EGF-R, AKT, PTEN,

PI3K)

• Angiogenesis (VEGF, PDGF, FGF)

• Epigenetic regulation (MGMT promoter

methylation, differential microRNAs, histone

coding)

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9/7/2012

Gliomas:

Astrocytoma

Oligodendroglioma

Ependymoma

Glioblastoma

multiforme (GBM)

Courtesy of S. Salamat

Grading Gliomas:

Nuclear atypia

Mitosis

Vascular endothelial

proliferation

Necrosis

Rapid recurrence despite aggressive

surgery, radiation and chemotherapy

Different Patients, Different GBMs

Preop Postop 6 months f/u

Preop 3 months 20 months

What’s different Molecular and Cell biology

The current standard of care

%

100

90

80

70

60

50

40

30

20

10

0

0 6 12 18 24 30 36 42

months

RT

TMZ/RT

Median OS, mo: 12.1 14.6 p


9/7/2012

Median survival after maximal

surgery + radiotherapy + temozolomide

MGMT + correlation with survival

14.6 mo (26%)

21.7 mo (46%)*

Stupp et al, NEJM 2005

*Hegi et al., NEJM 2005

Median 2 yr survival is

46% for MGMT- vs.

14% for MGMT+ GBMs

from Hegi et al, NEJM (2005)

The Cancer Genome Atlas Project

Cataloging the GBM Genome

• Prelim NCI Cancer Genome Atlas

• Analysis of 601 selected genes in 206

GBMs for DNA copy number,

transcriptome, and DNA methylation

• 91 GBM completely sequenced

• Prelim NCI Cancer Genome Atlas

• Analysis of 601 selected genes in 206

GBMs for DNA copy number,

transcriptome, and DNA methylation

• 91 GBM completely sequenced

The Cancer Genome Atlas Research Network, Nature (2008)

Cataloging the GBM Genome

Pathways frequently mutated in GBM

• sequenced and analyzed 20,661 genes

in 22 GBMs

• sequenced 689,071,123 nucleotides

• detected 2,325 somatic mutations

Kinzler group, Science, 2008 The Cancer Genome Atlas Research Network, Nature (2008)

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9/7/2012

Features of the Cancer Genome

• Systematic GBM genome analysis

on large sample sets

• under-analyze subset of biologically

and clinically significant cells

(i.e. cancer stem cells, Rx

resistant cells)

• Discovery of freq mutations (75%

GBMs contain mutations in

certain signaling pathways)

Challenges in Cancer Therapy

1. What causes rapid cancer

recurrence (failure of current therapies)

2. What are keys to understanding

and overcoming therapeutic

resistance (improve current therapies)

3. How to design effective

‘personalized’ cancer therapy

(individualize therapy to max efficacy, reduce toxicity)

• p53, Rb, PI3K (RTK, ras, NF-1)

Opportunities for Better Therapies

Molecular biology of cancer stem cells

Translation into better cancer therapies

Cancer Stem Cell Hypothesis

Cancer stem cells / tumor stem-like or progenitor cells

• Small subpopulation of cells within tumors

highly efficient in tumor initiation

• Display stem cell-like characteristics of self-renewal,

enhanced proliferation, multipotent differentiation

GBM Cancer Stem Cells (GSCs)

• Originally identified in Dirks lab (Singh et al., 2003 and

2004)

• Enrichment for cell surface marker expression (CD133) or

functional isolation as neurospheres in stem cell media

• Display enhanced therapeutic resistance to radiation and

chemotherapy

Cancer Stem Cell hypothesis

Cancer Stem Cells have been successfully

isolated from many types of solid tumors

(breast, brain, lung, melanoma,

colorectal, head/neck, prostate ca…etc)

Ebben et al 2010

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9/7/2012

CSCs must also be targeted to improve outcomes

A

Human GBM CSC isolation via sphere culture

Post-chop 250 µm

B

C

1 Week

250 µm

Ebben et al 2010

4-6 weeks

100 µm

GBM specimens obtained fresh from OR

• Chopped into 100 μm cubes

• Placed into suspension culture and neurospheres develop in 4-6 weeks

• Passaged by chopping into 100 µm cubes to preserve stem cell niche

• Successfully isolated independent GBM CSC lines (12.1, 22, 33, 44, 99)

• Culture conditions similar to normal human NSC

• Serum-free culture in ‘Passaging Medium’: DMEM (70%) and Ham’s F12 (30%)

• Additional growth factors: EGF (50 ng/ml); bFGF (50 ng/ml); Insulin (4 μg/ml) in B27

supplement

Identifying human GBM CSC lines

GBM CSC initiate tumors in SCID mouse

44 CSC

33 CSC

22 CSC

• Highly efficient tumor initiation (as few as 100 - 1000 cells)

• Retain tumor-initiating potential (serial transplantation to form

secondary and tertiary tumor xenografts)

12 CSC

Clark et al., Neoplasia 2012

GBM CSC are more similar to primary

tumors than cultured cell lines

GBM CSCs are more similar in gene

expression profile to primary tumors

GBM

(NBE/SCM)

CSC culture

(SC/10%

FBS)

GBM (non-CSC)

culture

Lee et al., Cancer Cell (2006)

Lee et al., Cancer Cell (2006)

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9/7/2012

Cancer stem cells are genetically more

similar to patient GBMs, differentially

resistant to current therapies and likely

drive rapid tumor recurrence.

Therefore, a better understanding of

CSC biology is crucial for improving

clinical outcomes.

What does CSC biology reveal

about GBM therapeutic

resistance

(Differential therapeutic response

in cancer clinical trials)

Kozak & Kuo labs, unpublished

Beier et al., J Neurooncol (2012)

MGMT

ß-Actin

Different Patterns of MGMT & PTEN

Expression in CSC vs. Non-CSC

u87

u251

NHA

22 T

33 T

hNSC

12.1 CSC

22 CSC

33 CSC

Does heterogeneity of

MGMT expression

Determine TMZ sensitivity

MGMT status correlates with TMZ response

Proliferation Assays on T=non-CSC GBM cells:

‣ 22T (MGMT -) 33T (MGMT +)

GBM Tu Line Tx with TMZ

GBM Tu Line Tx with TMZ

100

120

80

100

u87

u251

NHA

22 T

33 T

hNSC

12.1 CSC

22 CSC

33 CSC

% cell growth

60

40

20

22T

33T

% cell growth

80

60

40

20

22T

33T

PTEN

ß-Actin

PTEN expression observed

in all CSC lines.

0

0

0 30 60 90

0 30 90

uM TMZ

uM TMZ

‣Data from three representative experiments

Kozak & Kuo labs, unpublished

Kozak & Kuo labs, unpublished

MGMT status correlates with TMZ response

Proliferation Assays Cancer Stem Cells (CSC)

vs. Tumor (non-CSC) from the same patient:

‣ 33CSC (MGMT -) 33T (MGMT +)

Aberrant EGF signaling

in GBM and other cancers

% Cell Growth

120.0

100.0

33CSC vs 33T TMZ TX

80.0

60.0

40.0

20.0

0.0

0 20 40 60 80 100

uM TMZ

‣3d TMZ Tx

33 CSC

33T

Kozak & Kuo labs, unpublished

• Constitutively active EGFRvIII mutation (loss

of ligand binding domain) freq found in GBM

• EGFR-targeting therapies successful for lung

cancer but fail against GBM (Lassman et al., Clin Cancer Res 2005)

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9/7/2012

Multiple EGFR mutations in lung cancer are

associated with therapeutic response to TKI

EGFR mutations frequently found in

lung cancer are not present in GBM CSC

G719(S/C) mutation

hNSC

A:0.0%

T:0.0%

G:100.0%

Mutation locus

200

• Exon 19 deletion: 46.2% of patients

• Exon 21 L858R: 43.0%

• Exon 18 G719S or G719C: 4.8%

• Exon 20 E709K: 2.8%

• Exon 21 non-L858R (i.e. L861Q): 2.8%

Pan Q et al., J Mol Diagn. 2005

100

22 GBM CSC

300

200

100

Cell line

E S C A T G A C T C G T

5 10

A:0.0%

T:1.0%

G:99.0%

E S C A T G A C T C G T

5 10

E709K

Exon 19 deletion

(Δnt2235‐2249)

L858R L861Q G719(S/C)

hNSC (control) N/A N/A N/A N/A N/A

12.1 CSC Not present Not present Not present Not present Not present

12.1 NGF Not present Not present Not present Not present Not present

22 CSC Not present Not present Not present Not present Not present

22 NGF Not present Not present Not present Not present Not present

33 CSC Not present Not present Not present Not present Not present

33 NGF Not present Not present Not present Not present Not present

44 CSC Not present Not present Not present Not present Not present

Kuo lab, unpublished

GBM CSCs do not need EGF to survive

GBM CSCs do not need EGF to survive

But EGFR signaling is

essential for CSC proliferation

• Normal NSC and GBM fail to propagate without EGF

GBM CSCs in ‘no growth factor’ (NGF) conditions

survive, proliferate, and initiate tumors

Clark et al., Neoplasia 2012

What explains these

contradictory observations

Kelly et al., 2009

Soeda et al, 2008

Clark et al., 2012

Inhibited EGFR

EGFR

CETUXIMAB

EGFR

PIP2

PIP3

Constitutive EGFRvIII

mutant signaling

PIP2

EGFR

vIII

GBM CSC do not express

constitutively active EGFRvIII

PI3K

PI3K

PTEN

PTEN

Cell Death

AKT

mTOR

Proliferation

Clark et al., Neoplasia 2012

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9/7/2012

GBM CSC survive and proliferate long term

without exogenous EGF and bFGF

Observation:

Compensatory Her 2/3 activation

in GBM CSC preserves

downstream signaling –

therefore prediction is that multi-

HER family targeting is required

for effective anti-CSC therapy.

Clark et al., Neoplasia 2012

Clark et al., Neoplasia 2012

Overcoming anti-EGFR therapy resistance

Blockade of multiple EGFR family receptors

• Lapatinib (dual EGFR/HER2 inhibitor) inhibits colony formation via

pAKT and pERK1/2 inhibition

Clark et al., Neoplasia 2012

GBM CSC Model of Patient Clinical Course

GBM CSC heterogeneity

Surgical

Removal

Months

Anti-EGFR treatment

Recurrent: Anti-EGFR

(+radiation/temozolomide)

therapy resistant

-EGF

NGF CSC: EGF

GBM CSC

-bFGF

independent

4-6 wks

Passage

Passage every

+EGF

every week

2 weeks

+bFGF

Re-sensitize GBM to

anti-EGFR therapy

Uncover mechanisms of

EGF independence

Clinicians need rapid isolation and analysis of patient-specific

CSC in order to individualize therapeutic strategies

Zorniak et al., Clin Canc Res 2012

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9/7/2012

GBM CSC lines show differential treatment response

Vehicle CTX CTX+HER LAP

99 CSC 33 CSC 15 CSC 12.1 CSC 22 CSC

• 2000 CSC/well onto

laminin-coated 6 wells

• 4 weeks after seeding

(stain colonies with

crystal violet)

• Timing varies by

cell line (33 CSC r

≈ 2 wks ; 12.1

CSC r ≈ 5 wks )

• LAP: lapatinib (5 µM),

HER: herceptin (20

µg/ml), CTX:

cetuximab (100 nM)

Clark et al, unpublished

Variations in clinical response likely

due to biological differences between

the cancers and cancer stem cells of

different patients – we need efficient

means of rapid, patient-specific

cancer stem cell isolation and

characterization to enable

personalized cancer therapy.

Multiple CSC analysis strategies

1. Differential gene/protein expression analysis

2. Biopanning with yeast display antibody library

3. Metabolomic analysis

4. MicroRNA analysis

5. Novel CSC and GBM imaging and therapeutic agents

Goal: To identify new clinically relevant biomarkers

Multiple CSC analysis strategies

1. Differential gene/protein expression analysis

2. Biopanning with yeast display antibody library

3. Metabolomic analysis

4. MicroRNA analysis

5. Novel CSC and GBM imaging and therapeutic agents

Goal: To identify new clinically relevant biomarkers

Differential Gene Expression Profiling to

Identify CSC‐specific Markers

Upregulated

CSC Transcripts

GBM Primary Brain Tumor

n=21

Zorniak et al, unpublished

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9/7/2012

Downregulated

CSC Transcripts

qPCR Validation of

Upregulated CSC Transcripts

Zorniak et al, unpublished

Zorniak et al, unpublished

Multiple CSC analysis strategies

Yeast Antibody Display Technology

1. Differential gene/protein expression analysis

2. Biopanning with yeast display antibody library

Single‐chain Fv

(scFv)

HA

tag

Aga2

scFv

c‐myc

tag

3. Metabolomic analysis

4. MicroRNA analysis

5. Novel CSC and GBM imaging and therapeutic agents

Goal: To identify new clinically relevant biomarkers

IgG

Aga2

scFv

yeast

•Yeast cell express specific scFv‐encoded antibody

•scFvsare presented on the yeast cell surface for binding

•Multivalent ‐ 50,000 scFvs per yeast cell

Boder E.T., Wittrup, K.D. Nat. Biotechnol. 15, 553‐557 (1997)

Pepper, L., Cho, Y.K., Boder E.T., Shusta E. V., Comb. Chem. High Throughput Screen., 11, 127‐34 (2008)

Aga1

Cell wall

Cell membrane

Screening for CSC markers via yeast

display human scFv antibody library

Selection Procedure with the

scFv Antibody Library

Roughly 500,000,000 Unique Human scFv Antibodies

Marasco WA, Sui J. The growth and potential of human antiviral monoclonal antibody therapeutics. Nat Biotechnol. 2007 Dec;25(12):1421-34.

Marasco WA, Sui J. The growth and potential of human antiviral monoclonal antibody therapeutics. Nat Biotechnol. 2007 Dec;25(12):1421-34.

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9/7/2012

Screening enriched clones &

making complete antibodies

Screening protocol

scFv Library

Washed Away

Amplified

Washed Away

Recovered Library

Recovered Library

1 st Round

2 nd Round

Enrichment protocol is repeated until a high recovery percentage is achieved

Marasco WA, Sui J. The growth and potential of human antiviral monoclonal antibody therapeutics. Nat Biotechnol. 2007 Dec;25(12):1421-34.

Wang XX, Cho YK, Shusta EV. Mining a yeast library for brain endothelial cell-binding antibodies. Nat Methods. 2007 Feb;4(2):143-5.

Round Two

Round Six

40X

40X

Round Eight

Antibodies Enriched for GBM CSC binding

Round 2 Round 6 Round 9

Round 1 2 3 4 5 6 7 8 9

Total Yeast 5x10 9 2x10 8 5x10 7 5x10 7 5x10 7 5x10 7 5x10 7 5x10 7 5x10 7

No.

Recovered

Recovery

%

ND 2.58x10 6 4.28x10 5 9.05x10 5 ND 2.584x10 6 7.8x10 6 5.86x10 6 6.26x10 6

ND 1.29 0.856 1.808 ND 5.168 15.6 11.72 12.52

40X

Zorniak et al., unpublished

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9/7/2012

scFv expression is required for yeast

binding to cancer stem cells

DNA Fingerprinting

to Identify Unique Clones

scFv OFF

scFv ON

Prelim PCR

screening showing

repertoire of unique

scFV clones

20X

Zorniak et al., unpublished

Zorniak et al., unpublished

Predicted classes of binding antibodies

Clone 1.7 shows ‘Stem Cell’‐specific binding

Class

I

II

hNSC BT22 NHA U87


+ + ‐ ‐ ‐


Zorniak et al., unpublished

CSC Antigen Identification Strategy

Multiple CSC analysis strategies

Antigen separated

in a gel

Tandem Mass

Spectrometer

1. Differential gene/protein expression analysis

2. Biopanning with yeast display antibody library

3. Metabolomic analysis

•At least 25 – 100 ng of antigen needed

•YDIP elution products can be combined and concentrated (>20‐fold)

Low abundance membrane proteins can be purified and

concentrated

4. MicroRNA analysis

5. Novel CSC and GBM imaging and therapeutic agents

Goal: To identify new clinically relevant biomarkers

Cho, Y. K., Chen, I., Wei, X., Li, L., Shusta, E. V., J. Immunol. Methods, 341, 117‐126 (2009).

12


9/7/2012

Genomics

• Genetic Maps

• Regulatory Sequences

• Gene Prediction

• Polymorphisms

Metabolomics via

Secretome analysis

Metabolomics of human embryonic stem cells and

derivatives is a source of biomarkers and mechanistic

pathways to elucidate neurodevelopmental disorders

Proteomics

Transcriptomics

• Gene Expression

• Exon-Intron model

• Sense-antisense

Metabolomics

• Metabolites

• Functional pathways

• Biochemical

Phenotype

• Subtle Phenotypes

Biomarker: biochemical feature used to predict, diagnose or

measure the progress of disease or toxic response

• Protein ID

• Isoforms

• Post-translation mods

• Interactions

GBM and GBM CSC are radioresistant

Metabolomic correlates of radiation response in

glioblastoma multiforme (GBM)

• Define the metabolic profile of tumor response to

ionization radiation (IR) gain insight into cellular

processes contributing to intrinsic radiation

resistance.

• Develop translational biomarkers to measure

progress or predict personalized response to

radiation therapy.

• Establish a tumor-specific signature which could be

monitored by a simple blood test screening tool

and marker for recurrent disease.

Kuo lab, unpublished

Metabolomic correlates of radiation response

in glioblastoma multiforme (GBM)

Statistically significant

mass features in

response to 3Gy of IR

U251

U373

16

159

142

T98G

• The small molecules

altered in response

to IR were a diverse

group of metabolites

involved in fatty acid

metabolism,

products of collagen

matrix degradation,

and other cellular

processes.

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9/7/2012

Predictive Efficacy Screening in Cancer

Stem Cells Using Metabolomics

• Determine:

– Unique biochemical phenotype of GBM CSC

– Unique biochemical phenotype of stemness

– Small molecule, chemical CSC-specific

biomarkers of efficacy for drug screening

– Target pathways for CSC-specific drug

discovery

Multiple CSC analysis strategies

•We have identified an

unique signature of

small molecule

biomarkers in GBM CSC

compared to GBM

tumor cells and normal

neural progenitor cells.

•Sensitive, quantitative

biomarkers to predict

CSC‐specific efficacy and

treatment responses.

1. Differential gene/protein expression analysis

2. Biopanning with yeast display antibody library

3. Metabolomic analysis

4. MicroRNA analysis

5. Novel CSC and GBM imaging and therapeutic agents

Goal: To identify new clinically relevant biomarkers

Developmental Hierarchy of Neural Lineages

AP

NPC

SOX1, SOX2, Nestin, CD15

GRP

A2B5, NG2

OPC

NP

NCAM

Classifying

GBM CSC lines

with neural

developmental

lineage markers

A2B5, CD44

A2B5, NG2, PDGFRA, OLIG2

AC

OC

NC

GFAP

OLIG2, GALC, O1, O4, MBP

NCAM, MAP2, TUJ1

Zorniak et al., Clin Canc Res 2012

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9/7/2012

Classification of GBM CSC lines

with neural lineage markers

33 CSC NPC

SOX1, SOX2, Nestin, CD15

GRP

GSC classification correlates with

tumor xenograft invasiveness

AP

A2B5, NG2

OPC

12.1 & 22

CSC

NP

NCAM

A2B5, CD44

A2B5, NG2, PDGFRA, OLIG2

44 & 99

CSC

AC

GFAP

OC

OLIG2, GALC, O1, O4, MBP

NC

NCAM, MAP2, TUJ1

Zorniak et al., Clin Canc Res 2012

CNP expression is favorable for survival

Zorniak et al.,

Clin Canc Res

2012 Zorniak et al., Clin Canc Res 2012

Summary

o GBM CSC exhibit heterogeneous, patient-specific differences in biology and

therapeutic response, and are clinically relevant disease models.

o Activation of HER2/3 receptor signaling in GBM CSC confers therapeutic

resistance against EGFR-targeted monotherapy, and explains failure of

clinical trials.

o Differential GBM CSC expression of developmental neural lineage markers

correlates with xenograft invasiveness and survival

o CNP expression is associated with GBM patient survival and easily assayed on

human GBM specimens

Zorniak et al.,

Clin Canc Res 2012

o Multiple unbiased, high-throughput strategies are being pursued to identify CSCspecific

biomarkers for possible clinical use

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9/7/2012

• Credit

• Paul Clark

• Michael Zorniak

• Bahauddeen Alrfaei

• Kyle Swanson

• Kelli Pointer

• Sathyapriya Ezhilan

• Heather Leeper

• Matthew Tipping

• David Francis

• Kevin Kozak

• M. Shahriar Salamat

• H. Ian Robins

• Eric Shusta

• Jamey Weichert

• Su-Chun Zhang

• Clive Svendsen

• Paul West, Stemina

Acknowledgments

• National Institutes of Health (NCI, NIAAA,

NINDS)

• Young Clinician Investigator – AANS

Neurosurgery Research and Education

Foundation

• HEADRUSH Brain Tumor Research

Professorship

• UW CCC Translational Research grant

• UW Institute for Clinical and Translational

Medicine

• Roger Loff Memorial GBM Research Fund

• University of Wisconsin-Madison

• Center for Stem Cell and Regenerative

Medicine

• Carbone Cancer Center

• School of Medicine and Public Health

• Graduate School of Arts and Sciences

• Dept. of Neurological Surgery

21 st Beaumont Symposium, Troy, Michigan - September 19, 2012

Thank you for your attention

16

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