Progressione tumorale: nuovi target

terapeuticiterapeutici

Alessandra Gentile

Institute for Cancer Research and Treatment - IRCC,

University of Turin School of Medicine

Outline

• About Invasive Growth• About the MET gene and Cancer• A new gene involved in cancer

About Invasive Growth

• Invasive growth is a physiological process that occurs during embryonic development and post-natal tissue regeneration.

• The genetic program includes control of proliferation, cell-scattering, migration and proliferation, cell-scattering, migration and protection from apoptosis.

• The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth

About Invasive Growth

• Invasive growth is a physiological process that occurs during embryonic development and post-natal tissue regeneration.

• The genetic program includes control of proliferation, cell-scattering, migration and proliferation, cell-scattering, migration and protection from apoptosis.

• The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth

About Invasive Growth

• Invasive growth is a physiological process that occurs during embryonic development and post-natal tissue regeneration.

• The genetic program includes control of proliferation, cell-scattering, migration and proliferation, cell-scattering, migration and protection from apoptosis.

• The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth.

Sema domain

G-P richIg like domains

500 AAMRS (PSI)

HGF (Scatter Factor)

The MET oncogene, encodes the HGF receptor

Kringles Protease-like

Ig like domains400 AA

Tyrosine Kinase

Met(HGF Receptor)

PP

PP

Invasive growth is controlled by specific factors

EGF

MLP-29 (Liver stem/progenitor cells)

EGF

HGF

Invasive growth is controlled by specific factors

HGF

MLP-29 (Liver stem/progenitor cells)

Met Receptor(tyrosine kinase)

HGF

Signal transduction

Expression ofa selective set of Genes

(The Met Signature)

Invasion & Growth

MET

P

CD44v6

Plexin B1

Integrin α6β 4

Link to cytoskeleton

P

P

Link to cytoskeleton

GRB2RAS

Transient MAP kinaseactivation (proliferation)

PI3Kp85

PP

GRB2

PI3Kp85

GAB1

P

SHP2

SRC

Sustained MAP and PI3 kinase activation (invasion / apoptosis protection)

PLCγ

CRKL

C.Boccaccio and P.M.Comoglio. Nature Rev. Cancer.2006, 6: 637-645.

STAT

Cell-Polarity and Morphogenesis

Met Receptor(tyrosine kinase)

HGF

Signal transduction

Expression ofa selective set of Genes

(The Met Signature)

Invasion & Growth

About MET

• The MET oncogene, controls the selective expression of a functional cluster of genes (The “Invasive Growth Signature”)“Invasive Growth Signature”)

• The “Signature” can be exploited for the prognosis of some invasive-metastatic cancers

MET regulated genes( The Invasive Growth Signature )

HGF (hours)

1 6 24

Delayed Early

Delayed Early Late

Immediate Early

Biphasic

Immediate Early

Tonic

Biphasic

Late

Biphasic

Tonic

Total = 250 genes

2x induced

2x suppressed

Tumor samples, ordered by survival time

ClusterA

Avg.Cluster A- Avg. Cluster B

The “metagene”

ClusterB

5 years

2x suppressed

2x induced

E.Medico et al.(unpublished)

About the MET geneand Cancer

• It is over-expressed in response to unfavourable micro-environmental conditions, such as hypoxia : “Oncogene Expedience”.

• It is constitutively activated in some cancers, by amplification, mutations or autocrine loops:“Oncogene Addiction”.

• It is good candidate for targeted therapies

Proteasomedegradation

Cellular [O2]

α

Nucleus

HIF-1α(regulated)α -OH

pVHL

Cytoplasm

HIF Proline Hydroxylase

Hypoxia promotes invasive growthby transcriptional activation of the MET Oncogene

HIF-1β(constitutive)

Nucleus

βHIF-1αβ

The MET promoter

P (-2619) S (-345) A (+353)S (+89)

HRE-1 HRE-2 asHRE-1

AP-1

HRE-4 HRE-5

STARTA (-253)

asHRE-2/HRE-3

S (-411) A (-32)

S (-295)

Pennacchietti et al. , Cancer Cell 3: 347 (2003)

About the MET geneand Cancer

• It is over-expressed in response to unfavourable micro-environmental conditions, such as hypoxia or ionizing radiations: “Oncogene Expedience”.

• It is constitutively activated in some cancers, by • It is constitutively activated in some cancers, by amplification, mutations or autocrine loops:“Oncogene Addiction”.

• It is good candidate for targeted therapies

Juxtamembrane

Sema

IPT

PSI

P S985

MET oncogene mutations in Human Ca.

KD ActivationLoop

ATP-bindingregion

C-terminalloop

Y1349

Y1356

Juxtamembranedomain

P

P

PP

Y1234

Y1235

Docking site

PP S

Y1003

G.Stella, S.Benvenuti et al, submitted

MET gene amplification

C GTL16

MET oncogene amplification in Human Ca.

MET Protein overexpression

C GTL16

MET gene amplification

About the MET geneand Cancer

• The oncogene encodes a tyrosine-kinase receptor for HGF (“Scatter factor”)

• It is over-expressed in response to unfavourable micro-

environmental conditions, such as hypoxia or ionizing environmental conditions, such as hypoxia or ionizing

radiations: “Oncogene Expedience”.

• It is constitutively activated in some cancers, by

amplification, mutations or autocrine loops:

“Oncogene Addiction”.

• It is good candidate for targeted therapies

Therapeutic inactivation of the MET oncogenecan be achieved by:

1. Small molecules inhibiting the Tyrosine Kinase1. Small molecules inhibiting the Tyrosine Kinase

2. Monoclonal Antibodies inducing Met“Shedding”

In vitro therapy of Met–addicted human gastric Cato a specific kinase inhibitor (small molecule)

A. Bertotti, L.Trusolino et al., Science Signaling, 2, issue 100, Dec. 2009

MET Dependency correlates with gene amplification

6MKN-45

5.8EBC-1

MET copy N°Cell Line< 3 %

ADDICTION

97 %EXPEDIENCE

6.3HS746T

6.1GTL-16

6MKN-45

P.M.Comoglio and L.Trusolino, Nature Rev. Cancer, In preparation

Gene Therapy with MET antibody

Gene transfer of Lentiviral vector carrying the cDNA for DN30into the tumor: Cancer cells produce the Monoclonal Antibody

Gene transfer of DN30 RF anti-Met antibodiesInhibits growth of U87-MG Glioblastoma

xenotransplants

60

70

80

90

100

free

ani

mal

s

.

0

10

20

30

40

50

60

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58

Time (days)

% tu

mor

-fre

e an

imal

s

.

ctrl

DN30 RF MAb cDNA

E.Vigna et al.,Cancer Res. 2008;68:9176

What next ?

• Oa-5d5 antibody, Genetech, preclinical

• DN30 antibody, Metheresis, preclinical

• PF2341066, small molecule, Pfizer, phase I/II clinical

• XL880, small molecule, Exelesis, phase I clinical• XL880, small molecule, Exelesis, phase I clinical

• ARQ197, small molecule, ArQule, phase I/II clinical

• MK2461, small molecule, Merck, phase I/II clinical

• SGX523, small molecule, SGX pharma., Phase I clinical

• JNJ38877605, small molecule, J&J, preclinical

From P. Comoglio et al, Nature Rev. Drug Discovery, 7, 504

Receptor Tyrosine Kinase (RTKs) superfamily

from Blume-Jensen and Hunter, 2001

Orphan Receptor tyrosine kinase family

Immunoglobulin-like motif

Cysteine rich domain

(Frizzled-like domain)

1 2

Kringle domain

Serine/Threonine rich domain 1

Proline rich domain

Tyrosine kinase domain

Serine/Threonine rich domain 2937 aa 943 aa

Orphan Receptor protein expression

in cancer cell lines

Screened cancer cells

85

98%

2

2%

human cancer cell panel

phospho-Ror1 positive cells

NCI-H1993

HS746T

Orphan Receptor knock-down impairs cell

proliferation

Orphan Recptor* Orphan Recptor*

Control shRNAshRNA(A)_Orphan Receptor 1shRNA(B)_Orphan Receptor 1

Orphan Receptor knock-down impairs

anchorage independent cellular growth

Control shRNA

NCI-H1993 HS746T PC3Orphan Recptor* Orphan Recptor* Orphan Recptor

shRNA(A)_Orphan Receptor 1

shRNA(B)_Orphan Receptor 1

80,0

100,0

120,0

% o

f tum

or fr

ee a

nim

als

Orphan Receptor knock-down impairs tumor growth

HS746THS746THS746THS746TOrphan Recptor*

0,0

20,0

40,0

60,0

0 10 20 30 40 50 60 70

Time (day)

% o

f tum

or fr

ee a

nim

als

Control shRNA

shRNA(A)_Orphan Receptor 1

shRNA(B)_Orphan Receptor 1

Orphan Recptor*

Met & HypoxiaP.MichieliS.Pennacchietti M.GalluzzoC.Basilico

Met and miRNAS. GiordanoS. CorsoA. PetrelliC. Migliore

IRCC – Candiolo (Italy)

Director: Paolo M. Comoglio MD

Met gene therapyE.VignaG.PacchianaR.Albano

Plexins L.TamagnoneA.Casazza

Met AddictionL.TrusolinoA.BertottiD.TortiF.Galimi

Met MutationsS. BenvenutiM.F. Di Renzo

Met- signatureE.MedicoL.Lazzari

Met & Stem cellsC.BoccaccioF.De BaccoP.LuraghiS. GastaldiG.Reato

“Today Science, Tomorrow Medicine”

IRCC: Institute for Cancer Research and Treatment