October 11-14, 2007 Dublin, Ireland 6th International Symposium on Translational Research in...

October 11-14, 2007Dublin, Ireland

6th International Symposium on Translational Research in Oncology

This program is supported by educational grants from

Dennis J. Slamon, MD, PhDChief, Division of Hematology/OncologyDavid Geffen School of Medicine at UCLALos Angeles, California

John Crown, MD, MPHHead, Medical Oncology ResearchSt Vincent’s HospitalElm ParkDublin, Ireland


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clinicaloptions.com/oncology


Now in its sixth year, this annual symposium has a firmly established reputation as a premier meeting at which the world’s leading researchers gather to present and discuss new directions in oncology research with a focus on translating the most recent laboratory developments into improved clinical outcomes for cancer patients. Under the direction of John Crown, MD, MPH, and Dennis J. Slamon, MD, PhD, the program includes didactic presentations and interactive discussions. Faculty are carefully selected from among the researchers at the forefront of the translational work in the topic, whether from academia, government, or industry. The program encourages networking and interaction between the attendees and the renowned faculty members.

Program Overview



About These Slides

DisclaimerThe materials published on the Clinical Care Options Web site reflect the views of the authors, not those of Clinical Care Options, LLC, the CME providers, or the companies providing educational grants. The materials may discuss uses and dosages for therapeutic products that have not been approved by the United States Food and Drug Administration. A qualified healthcare professional should be consulted before using any therapeutic product discussed. Readers should verify all information and data before treating patients or using any therapies described in these materials.

Users are encouraged to include these slides in their own presentations, but we ask that content and attribution not be changed. Users are asked to honor this intent.

These slides may not be published or posted online or used for any other commercial purpose without written permission from Clinical Care Options.

We are grateful to Hasan Korkaya, DVM, PhD, the chair of the session, who aided in the preparation of this slideset.

We are also grateful to the speakers in the session who gave us permission to use a select group of their slides from the meeting to make this slideset possible: Michael Lahn, MD; James Carmichael, MD; Marian L. Waterman, PhD; and Hasan Korkaya, DVM, PhD.

Session VII: Malignant Stem Cells as Targets in Oncology

Hasan Korkaya, DVM, PhDResearch FellowInternal MedicineHematology/OncologyUniversity of MichiganAnn Arbor, Michigan

The Role of TGF-β in Translational Medicine



Cancer Stem Cell Concept

In 1867, Cohnheim proposed that cancer originates from stem cells because of similarities between fetal development and certain types of tumors such as teratocarcinomas[1]

Although the heterogeneity of tumor cells was known, Cohnheim’s hypothesis was not confirmed until 1994 when Lapidot and colleagues reported that acute myeloid leukemia is maintained by a rare population of stem cells[2]

1. Cohnheim J. Path Anat Physiol Klin Med. 1867;40:1-79.2. Lapidot T, et al. Nature. 1994;367:645-648.



The cancer stem cell concept may explain why conventional therapies fail and provides molecular targets for the effective treatment of advanced tumors

Researchers are actively studying how to target cellular self-renewal and differentiation pathways[1,2]

1. Shugar RC, et al. Gene Ther. 2007;Nov 8:[Epub ahead of print].2. Korkaya H, et al. BioDrugs. 2007;21:299-310.

Cancer Stem Cell Concept: Tumor Resistance



1. Takahashi K, et al. Biochem Soc Trans. 2005;33:1522-1525.2. Welham MJ, et al. Biochem Soc Trans. 2007;35(pt 2):225-228.3. Tannishtha R, et al. Nature. 2005;434:843-850.4. Ruscetti FW, et al. Oncogene. 2005;24:5751-5763.5. Fortunel NO, et al. Blood. 2000;96:2022-2036.6. Korkaya H, et al. BioDrugs. 2007;21:299-310.

Cancer Stem Cells: Malignant Transformation Malignant transformation of tissue stem cells is believed to result from

dysregulation of self-renewal pathways including

– PI3K-Akt[1,2]

– Wnt/-catenin[3]

– TGF-[4,5]

– Tumor suppressor proteins: p53 and PTEN[6]

The deregulation of such pathways has been reported in a number of malignancies including breast, colon, and prostate cancer



Signal Transduction of TGF-ßTGF-ß ligands

Receptor

type I

PPReceptor

type II

Gene transcription or repression

PSmad 2,3

PSmad

4

P



Many advanced tumors have abrogated the TGF- growth inhibitory pathway

Overexpression of TGF- has been observed in

– Breast cancer

– Prostate cancer

– Colon cancer

– Lung cancer

TGF- overexpression correlates with poor prognosis in many tumor types

Preclinical antitumor efficacy has been observed in mouse models with TGF-–neutralizing antibodies, soluble receptors, and small-molecule kinase inhibitors targeting the TGF-RI kinase

TGF- in Cancer: Introduction



Normalepithelium

Carcinomain situ

Invasivecarcinoma

Metastasis

T cells

Immune suppression

IL-11

PTHrP

VEGFCTGF

Smooth muscle

TGF-

FibrosisGrowthfactors

EMT

Boneosteolysis

Vesselsangiogenesis

Tumor cells

TGF-TGF-

TGF- in Cancer: Tumorigenesis

TF

TGF- mRNA

TGF-

TGF- TGF-

SMAD2/3

SMAD2/3

JNK

II I PPP

SMAD4

TFTarget gene expression

RasTAK1

RhoA

p38

ERK1,2

PKB/Akt

Nucleus

Cytoplasm

AP-12009AP-11014

TGF- DNA

Vaccine NovaRx

LY580276 SB-505124SD-208

SR2F

LerdelimumabMetelimumabGC-1008

PP

SMAD4

SMAD2/3

Co-TFs

PI3

Lahn M, et al. Expert Opin Investig Drugs. 2005;14:629-643.

TGF-–Associated Therapeutic Targets



Compound Company/Sponsor

Preclinical Antitumor Activity

Clinical Studies

Antisense Oligonucleotide

AP 12009 Antisense Pharma

GlioblastomaPancreatic cancer

Phase I/II in glioblastoma

AP 11014 Antisense Pharma

NSCLCProstate cancer

Colon cancer

N/A

NovaRx NovaRx Glioblastoma Phase I/II in glioblastoma

Phase I/II in NSCLC


TGF- Inhibitors: Clinical Investigation Overview



Compound Company/Sponsor Preclinical Antitumor Activity

Clinical Studies

Large-Molecule Inhibitors

Lerdelimumab CAT N/A N/A

Metelimumab CAT/Genzyme N/A N/A

GC-1008 CAT/Genzyme N/A Phase I

SR-2F NCI/NIH N/A N/A

Small-Molecule Inhibitors

LY2157299 Eli Lilly & Co Breast cancerNSCLC

Ongoing phase I study

SB-505124 GlaxoSmithKline N/A N/A

SM16 Biogen Mesothelioma N/A

SD-208 Scios GlioblastomaMultiple myeloma

N/A


TGF- Inhibitors: Clinical Investigation Overview (cont’d)




Patients with activated pSMAD in circulation or high levels of TGF-1

– Study JBAG: nondrug interventional trial to determine pharmacodynamic markers for future application in drug trials of LY2157299

– Patients with skeletal metastasis

– Evaluation of pSMAD expression in PBMCs

– Evaluation of TGF-1 levels

Patients with a specific gene expression profile based on their original tumor biopsy (data not shown)

Patient Selection Strategies



TGF- inhibitors may be appropriately used in patients with advanced metastatic malignancies

Nonclinical data can be applied to establish PK/PD models and reduce the uncertainty in phase I studies

Baseline patient selection methods may be used to further optimize the role of TGF- inhibitors either as single agents or in combination with other anticancer drugs

TGF-β in Cancer: Conclusions

PARP Inhibition



DNA repair defects lead to increased cancer susceptibility and increased sensitivity to DNA-damaging agents

Novel targeted therapeutic approach

Normal cells have multiple DNA repairpathways but some are lost in cancer cells

DNA damage frequently occurs in all cells

Inhibiting DNA repair in cancer cells that have impaired repair pathways leads to selective cell killing and an increased therapeutic ratio

Why is DNA repair a good

target?

Targeting DNA Repair in Oncology: Rationale



Base excision

repair

Type of damage:

Bulky adducts

Insertionsand deletions

O6-alkylguanine

Repairpathway:

Nucleotide-excision

repair

Mismatch repair

Directreversal

Repairenzymes:

Double-strandbreaks

Single-strand breaks

PARP

Recombinationalrepair

ATM DNA-PK

HR NHEJ

XP, poly-

merasesMSH2,MLH1

AGT

Types of DNA Damage and Repair



PARP recruitmentPARP

DNA damage

PARP activation and assembly of repair factors

NAD+

poly(ADP-ribose)PARP

PAR degradation via PARG

PARGPARP

End processing,gap filling, and ligation

PNK 1pol β

XRCC1 LigIII

pol β

XRCC1 LigIII

PNK 1

PARP and Base Excision Repair



Targeted Killing of Cancer Cells With Defective DNA-Repair Mechanisms

Survival

Normal cell

Repair by HR pathway

Exploits inherent weakness of cancer cells that have defective DNA repair

Double-stranded break

BRCA deficient ordeficiency of other

HR proteins

No repair(No HR pathway)

Cell death

Cancer cell with defective repair



Genomic instability

B AB

Pathway B inhibitor

Death Survival

Tumor cell Healthy cell

AB

ABB

Loss of repair pathway

Inhibition of DNA Repair in Cancer Cells



DNA Repair Inhibitors in Cancer Cells: 2 Modes of Action Potentiation

– Inhibition of DNA repair following DNA-damaging agents

– Original hypothesis

Synthetic lethality

– Selected cancer cells lose DNA repair pathways, whereas normal cells remain unaffected

– Targeting these defective cells may cause selective cell kill with an increased therapeutic ratio

– May allow for a novel targeted approach to cancer treatment



23 mm

Strong family history

Ovarian BRCA1-/-

Unpublished data.



Days

CA

-125

(U

/mL

)

*GCIG CA-125 response. (Rustin G, et al. J Clin Oncol. 2004;22:4035-4036) †Ongoing response.

Unpublished data.

50

150

250

350

-350 -250 -150 -50 0 50 150

Family history

93% decline*

BRCA1 185delAG mutation

50

150

250

-20 0 20 40 60 80 100 120

BRCA1 4184delTCAA mutation

4

8

12

16

-20 -10 0 10 20 30 40 50 60

BRCA1 4693delAA mutation

Nonsecretor†

100

200

300

400

-100 -50 0 50 100 150 200


76% decline*†

200

600

1000

1400

-100 -50 0 50 100 150 200


98% decline*†

79% decline*†

10002000300040005000

-40 -20 0 20 40 60 80 100

36% decline

PR+ SD+

PR PR+

PR PR+

Hereditary Ovarian Cancer: Responses and CA-125 Levels

The Role of HER2 in Regulating Cancer Stem Cell Self-Renewal



HER2 is amplified in 20% to 30% of human breast cancers and is associated with a poor clinical outcome[1]

Although trastuzumab produces significant clinical benefit in the treatment of HER2-amplified breast tumors, one third of patients do not respond to trastuzumab and a majority of initial responders demonstrate disease progression within 1 year of treatment[2,3]

HER2 in Malignant Transformation of Mammary Epithelium

1. Slamon DJ, et al. Science. 1989;244:707-712.2. Miller KD. Oncologist. 2004;9(suppl 3):16-19.3. Seidman AD, et al. J Clin Oncol. 2001;19:2587-2595.



Potential Mechanisms of Trastuzumab Resistance Cell signaling pathways, including PTEN, PI3K/Akt, and

IGF-I, have been implicated in the resistance of breast tumors to trastuzumab therapy

The mechanism of resistance is not well understood

Nagata Y, et al. Cancer Cell. 2004;6:117-127.Chan CT, et al. Breast Cancer Res Treat. 2005;91:187-201.Grothey A, et al. J Cancer Res Clin Oncol. 1999;125:166-173.



SCSelf renewal

Progenitor cellsER+

Myoepithelial cells

Ductal epithelial cells

Alveolar epithelial cells

Differentiation

SCSCSC

Early progenitor cells

Cancer stem cell

Cancer stem cell

Mutations,deregulation of pathways

PTEN

HER2PI3-K/Akt

p53Notch, Hedgehog Bmi-1

Wnt/-catenin

Malignant Transformation of Mammary Stem Cells



Korkaya H, et al. BioDrugs. 2007;21:299-310.

Advanced Tumors: Targeting and Elimination of Cancer Stem Cells

Normal stem cellCSCDead CSCDifferentiated cellDead cell

Conventional

therapies

Following

chemotherapy

Conventionaltherapies

Conventionaltherapies

CSC targetedtherapiesDifferentiation

induction

Tumorregrowth

Eliminationof tumor

Differentiationof CSCs

Eliminationof tumor

Eliminationof CSCs

Tumor shrinkage



Tang C, et al. FASEB J. 2007;11:[Epub ahead of print].

Tumor Type Cell Surface Markers

Acute myeloid leukemia CD34+CD38-

Breast CD44+CD24-ESA+

Brain CD133+

Colon CD133+

Head and neck CD44+

Prostate CD44+

Metastatic melanoma CD20+

Colorectal EpCAMhighCD44+CD166+

Pancreatic CD24+CD44+ESA+

Lung adenocarcinoma Scal+CD45-Pecam-CD34+

Bone sarcoma Strol+CD105+CD44+

Identifying Cancer Stem Cells in Tumors



GF

P

HE

R2

Tubulin

HER2

GFP HER2

Unpublished data.

HER2 Increases Mammosphere Formation in Normal Mammary Cells

0

50

100

150

200

250

300

350

400

1 2 3

DsRed Control

HER2

# passagesSu

spen

sio

n c

ult

ure

co

un

ts



H&E SMA Ki67H&E

10x 40x

Cell #

Constructs

Unsorted Aldefluor Positive Aldefluor Negative

10,000 5000 5000 500 250 5000 250

DsRed 8 2 4 0 0 0 0

HER2 23 11 53 5 3 0 0

HER2

DsRed

Unpublished data.

HER2 Expression in NMEC Cells Increases Outgrowths in Mice



Unpublished data.

HER2 Overexpression Expands Aldefluor-Positive Cell Populations

0

5

10

15

20

25

30

35

40

MCF7-DsRed

MCF7-HER2

Sum149-DsRed

Sum149-HER2

Sum159-DsRed

Sum159-HER2

Ald

eflu

or-

Po

siti

ve C

ells

(%

)



DEAB inhibited

DEAB inhibited

0.08% 36%

0.1% 37%

HER2

CellsAldefluor - +

Primary tumor

Secondary tumor

Sum159-HER2

Unpublished data.

Tumor Initiation by Aldefluor-Positive Breast Cancer Cells



Invasive Potential Observed With Aldefluor-Positive Breast Cancer Cells

Unpublished data.

020406080

100120140

MCF7-DsRed

MCF7-HER2

Inva

din

g C

ells

/Wel

l

ALDH+ALDH-

0100200300400500600700800

SUM159-DsRed

SUM159-HER2

ALDH+ALDH-

Inva

din

g C

ells

/Wel

l



Unpublished data.

Trastuzumab Treatment: Resistant and Sensitive Breast Cancer Cell Lines

0

5

10

15

20

25

30

35

40

Sum159-DsRed

Sum159-HER2

MDA-MB-453

JIMT-1

Ald

eflu

or-

Po

siti

ve C

ells

(%

)

Trastuzumab -Trastuzumab+



Sum159-HER2MDA-MB-453

Trastuzumab - + - +

pHER2

Tubulin

HER2

pAkt

Unpublished data.

Akt Phosphorylation of Resistant and Sensitive Breast Cancer Cell Lines



The PI3K/Akt pathway is important for the survival and maintenance of pluripotent embryonic stem cells[1]

This pathway also plays a role in adult stem cell self-renewal[2]

Increased Akt activation appears to mediate the resistance of cancer stem cells to chemotherapy[3]

1. Takahashi K, et al. Biochem Soc Trans. 2005;33(pt 6);1522-1525.2. Welham MJ, et al. Biochem Soc Trans. 2007;35(pt 2):225-228.3. Ma S, et al. Oncogene. 2007;[Epub ahead of print].

Embryonic and Adult Stem Cell Self-Renewal and Maintenance

The Wnt/Beta-Catenin Pathway



Overexpression of Wnt ligands

Colon cancer

Breast cancer

Melanoma

Head and neck cancers

Lung cancers

Gastric cancer

Mesothelioma

Barrett’s esophagus

Barker N, et al. Nat Rev Drug Discov. 2006;5:991-1014.

Cancers Linked to Aberrant Wnt Signaling

Overexpression of frizzled receptors

Colon cancer

Breast cancer

Head and neck cancer

Gastric cancer

Synovial sarcoma

Loss of APC function

Colon cancer

Barrett’s esophagus



-catenin gain-of-function Colon cancer Gastric cancer Hepatocellular cancer Hepatoblastoma Wilms’ tumor Endometrial ovarian cancer Adrenocortical tumors Pilomatricoma

Loss of Axin 1/2 function Colon cancer (microsatellite instability) Hepatocellular cancer Hepatoblastomas

Barker N, et al. Nat Rev Drug Discov. 2006;5:991-1014.

Cancers Linked to Aberrant Wnt Signaling (cont’d)

Other Wnt signaling components Colon cancer Mesothelioma Cervical cancer Bladder cancer Prostate cancer Breast cancer Leukemia Non-small-cell lung cancer



1. Reya T, et al. Nature. 2003;423:409-414. 2. Willert K, et al. Nature. 2003;423:448-452. 3. Korinek V, et al. Nat Genet. 1998;19:379-383. 4. Gat U, et al. Cell. 1998;95:605-614. 5. Huelsken J, et al. Cell. 2001;105:533-545. 6. Ito M, et al. Nature. 2007;447:316-320.

Wnt Signaling and Stem Cells

Hematopoietic stem cells

– Self-renewal of HSCs, HSC proliferation[1,2]

Intestinal epithelial cells

– TCF-4 necessary for stem cell compartments in mouse intestine[3]

Skin

– -catenin overexpression causes higher density of hair follicle formation[4]

– Deletion of -catenin or LEF1 eliminates hair follicles[5]

Wound repair

Follicular neogenesis in skin after wound repair is dependent on Wnt signaling[6]



WTXGSK3 CK1

AxinAPC

-catenin

E-cadherin

LEF/TCF

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.QuickTime™ and a

TIFF (LZW) decompressorare needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.QuickTime™ and a

TIFF (LZW) decompressorare needed to see this picture.

Wnt Signal Transduction



Chronic myelogenous leukemia: Wnt signaling increases self-renewal capacity in blast crisis and in imatinib-resistant cancers; increases in nuclear -catenin and LEF1 have also been detected

-catenin

CML stem cells Multipotent

progenitors

-catenin

Pro-B cells

Pro-T cells

Blast crisis granulocyte macrophage precursors

T cells

B cells

Blasts

Jamieson CH, et al. N Engl J Med. 2004;351:657-667.

Wnt Signaling and Cancer Stem Cells



Wnt Signaling: Mechanism of Action and Biological Outcome

Miyabayashi T, et al. Proc Natl Acad Sci U S A. 2007;104:5668-5673.

Treatment of mES cells (no feeder layer, no serum) with Wnt3a plus IQ-1 enabled long-term culture of embryoid bodies (48 days) with maintenance of pluripotency



-catenin

?

Barker N, et al. Nat Rev Drug Discov. 2006;997-1014.

Targeting Wnt Signal Transduction

NSAIDs reduce levels of -catenin in adenomatous polyps and colon cancer cell lines

– aspirin, indomethacin, sulindac

– rofecoxib, celecoxib, valdecoxib

– NO-ASA (NO-releasing aspirin)



Small-Molecule Inhibitors

IC50

3.2 M

4.1 M

8.7 M

PKF115-584

PKF222-815

CGP049090

LEAD

Lepourcelet M, et al. Cancer Cell. 2004;5:91-102.

-catenin/TCF interactions - HTS of natural compounds



Ki-67 positive

Wnt Notch

Stem cells

Progenitor cells

DifferentiatedPaneth cells

Differentiation:Goblet cellsEnterocytes

Enteroendocrine

Stem Cell Differentiation in Intestine



DifferentiatedPaneth cells

Stem cells

Differentiation:Goblet cellsEnterocytes

Enteroendocrine

Progenitor cells

Notch

Wnt Wnt

?

Stem Cell Differentiation Pathways in Intestine



More Hematology/Oncology Available Online! Medical Meeting Coverage: key data plus Expert

Analysis panel discussions exploring clinical implications

Treatment Updates: comprehensive programs covering the most important new concepts

Interactive Cases: test your ability to manage patients


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