“RECETTORI PER CHEMOCHINE COME MARCATORI BIOLOGICI E

MOLECOLARI DI RISPOSTA CLINICA E TARGET TERAPEUTICO”

Stefania ScalaIstituto Tumori di Napoli - “Fondazione G. Pascale”

Istituto Tumori di Napoli- Fondazione G. Pascale

Responsabile Scientifico UO 1 Stefania Scala1. Radiodiagnostica Alfredo Siani2. Immunologia Clinica Stefania Scala3. Oncologia Medica B Vincenzo Rosario Iaffaioli4. Chirurgia Oncologica C Paolo del Rio5. Unità di sperimentazione su modello animale Claudio Arra

Gruppi di ricerca afferenti ad altre Istituzioni di Ricerca:1. Istituto di Chimica Biomolecolare (ICB) CNR PietroAmodeo2. Istituto di Biostrutture e Bioimmagini (IBB) CNR Stefania de Luca

Istituto Superiore di Sanità

Responsabile Scientifico UO 2 Ruggero Marchiano De Maria

1. Dipartimento di Ematologia Oncologia e MedicinaMolecolare. Reparto di Oncologia Molecolare. Alessandra Carè

2. Dipartimento di Ematologia, Oncologia e MedicinaMolecolare. Reparto di Oncologia Medica. Ugo Testa

3. Dipartimento di Biologia Cellulare e Neuroscienze.Repartodi Imaging Molecolare e Cellulare. Franca Podo

4. Dipartimento di Tecnologie e Salute. Metodi Ultrastrutturaliper Terapie Innovative Antitumorali. Giuseppe Arancia

Fondazione Centro San Raffaele del Monte Tabor

Responsabile Scientifico UO 3 Matteo Bellone1.Unità di Immunologia Cellulare Matteo Bellone2. Cancer Stem Cell Unit Rossella Galli3. Unità operativa Anatomia Patologica Claudio Doglioni

CXCR4 antagonists

Burger JA, Peled A. CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers.Leukemia. 2009 Jan;23(1):43-52. Epub 2008 Nov 6.

Rationale for CXCR4 antagonists

Affecting stromal interactions with cancer cellsMobilizing tumor cells from tissue sites, suchas the marrow, improving access toconventional therapy;Blocking of migration and dissemination of tumor cellsBlocking of paracrine growth and survivalsignals through activation of the CXCR4-CXCL12 axis

5. blocking pro-angiogenesis effects of CXCL12.

CXCR4 Inhibitors in Clinical Trials

Golay J, Introna M Chemokines and antagonists in non-Hodgkin's lymphoma. Expert Opin Ther Targets. 2008 May;12(5):621-35

There is a need for developing new potent CXCR4 antagonists with a safety profile suitable for human

clinical use

Objective

Rationale Drug DesignvMIP-II (vCCL2), “chemokine-like” (CK-like) synthesized by Herpes virus-Kaposi associated. 40% identity with human CK

vMIP-II (vCCL2) binds and inhibits CXC, CC e XC such as CCR1, CCR2, CCR5, CCR8, CXCR4 e XCR

CXCL12

SDF-1α Crystallographic structure

CXCR4 Binding and transduction involves residues in N-Terminal Domain

N-terminal Domain

Rationale Drug Design

CXCR4 ligand design focused on the comparative studies of N-terminal SDF-1αagonist and vMIP-II antagonist CXCR4 ligand design considered a conserved structural motif associated to an homolog sequence, although in reverse orientation, between SDF-1α e vMIP-II

SDFSDF--1, (11, (1--17)17)

vMIP-II, (1-10) LGAS PDK

KPVSLSYR CPC ESHKPVSLSYR CPC ESHRFFRFF

WHR

1 10 15

1 10

Tridimensional structure of the conserved amminoacidic motif shared by SDF-1α (green) e

vMIP-II (purple).

Design Phase I

The motif was the core of cyclic peptides aimed to preserve the structure.

The peptides differ for terminal sequences and for the cyclization

A group of peptides were synthesized in both orientation for the homologue sequence

Also protection at the extremities of the peptides were evaluated

Peptides

vMIP-II-mimetic (X-X-Arg)‏

Ac-Ciclo-Hys-OHAc-Ciclo-Hys-NH2Ac-Ciclo-Phe-OHAc-Ciclo-Phe-NH2Ac-Ciclo-Tyr-OHAc-Ciclo-Tyr-NH2Ciclo-Hys-OHCiclo-Hys-NH2 Ciclo-Phe-OHCiclo-Phe-NH2 Ciclo-Tyr-OH Ciclo-Tyr-NH2 Ciclo-Trp-OHCiclo-Hys-7 Ciclo-Phe-7 Ciclo-Tyr-7Ciclo-Hys-b-ALA.

SDF-1α mimetic (Arg-X-X)‏

Ciclo-Hys-7 invCiclo-Phe-7 inv Ciclo-Tyr-7 inv

Peptides functional evaluation

Indirect binding evaluation to CXCR4

Calcium efflux evaluation MigrationP-Erk Induction

20) Trp-OH

19) Hys-b-ALA

18) Tyr-7 inv

17) Hys-7 inv

16) Phe-7 inv

15) Tyr-7

14) Hys-7

13) Phe-7

12) Tyr-NH2

11) Tyr-OH

10) Hys-NH2

9) Hys-OH

8) Phe-NH2

7) Phe-OH

6) Ac Tyr-NH2

5) Ac.Tyr-OH

4) Ac Hys-NH2

3) Ac Hys-OH

2) Ac Phe-NH2

1) Ac.Phe-OH

AMD

P.ERKMigrationIndirect binding Calcium efflux

ANCI MI

Inhibitor peptides

Ciclo-Hys-7 inv

Ciclo-Phe-7 inv

Ciclo-Tyr-7 inv

Ciclo-HYs-OH

Ac-Hys-NH2

Ciclo-Hys-7 inv inhibits CXCR4 receptors in 4 assays: migration, Ca2+efflux, p-ERK induction and indirect binding.

Competitive binding curves of Ciclo Phe-7 invfor the binding of 125ISDF-1α in CCRF-CEM cells

Serum degradation Hys-OH

1 Ora 24 Ore

Serum degradation Phe-7 Inv

1 hr 24 hrs

Serum degradation Hys-7 Inv

1 hr 24 hrs

Negative Ctr Phe-7inv

Fluorescent Phe-7 inv in human PES 43 melanoma cells

In VIVO TAG-S 750

Inhibition of lung metastases in mouse model B16-CXCR4 cells

Peptide HYS-7 Inv

Normal lung Control AMD

Peptide PHE-7 Inv Peptide HYS-OH

Not Injected PBS AMD3100

Hys-7 inv Phe-7 inv Hys-OH

Magnification 50X

0

1

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3

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5

6

7

8

9

PBS AMD Hys-7 inv Phe-7 inv Hys-OH

Num

ero

Met

asta

si

Numero medio Metastasi

Microscopic evaluation of mice metastases

Proposta di brevetto

“Peptidi, peptidomimetici e molecole organiche derivanti da un motivo strutturale comune a due proteine che legano il recettore CXCR4 e loro uso come agenti diagnostici e terapeutici in patologie tumorali che sovraesprimano tale recettore.”

Ongoing experiments

Peptides Inhibition of HIV entry Peptides treatment on PES 43 xenograftPeptides treatment of murine osteosarcoma K7M2Acute and chronic peptides toxicities in mice

Istituto Superiore di Sanità

Responsabile Scientifico UO 2 RuggeroMarchiano De Maria

1. Dipartimento di Ematologia Oncologia e Medicina Molecolare. Reparto di OncologiaMolecolare. Alessandra Carè

ISOLATION OF MELANOMA CANCER STEM CELLS

S100

Patient Xenograft

MART1

H&E

40X 40X

20X 20X

20X 20X

High tumorigenic potential of melanomaCancer stem cells (down to 5 cells are able to

Generate patient like tumors in mice)

1

100

10000

1000000

control tw-RFP-Luc

H&E

S100

KI-67H&E

control

TW-RFP-Luc

In vivo detection of melanomas generated bylentivirally transduced melanoma CSCs

Red fluorescence Luciferase activity

In vivo imaging (IVIS100, Xenogen)

0,09 1,51

IgG pe cy5 CXCR4 pe cy5

#1sphere

0,20 1,10

#3sphere

CXCR4 is expressed on a small percentageof melanoma CSC

Control CM0

200

400

600

800

1000

1200CXCR4 -

CXCR4 +

< 0.05

N.o

fmig

rate

dce

lls

CXCR4+ melanoma CSCs display higher migrationCapacity in vitro compared with CXCR4- cells

Migration assay

In vivo tumorigenic potential: similar for CXCR4+ and CXCR4- CSCsIn vivo metastatic potential: ongoing experiments

10 minexposition

In vivo models of CSC-generated glioblastomasfor drug testing

GBM I-sc GBM Q- sc

CXCR4 is expressed at high level in glioblastoma CSCs

Istituto Superiore di Sanità

Responsabile Scientifico UO 2 Ruggero Marchiano De Maria

1. Dipartimento di Ematologia, Oncologia e MedicinaMolecolare. Reparto di Oncologia Medica. Ugo Testa

The pathway comprising PLZF/miR-146a and its target CXCR4 operates in leukemic cells.

The regulatory pathway involving PLZF control on miR-146a expression and miR-146a control on CXCR4 translation is found in AMLs

Istituto Superiore di Sanità

Responsabile Scientifico UO 2 RuggeroMarchiano De Maria

3. Dipartimento di Biologia Cellulare e Neuroscienze.Reparto di Imaging Molecolare e Cellulare. Franca Podo

Recettori per Chemochine come Marcatori Biologici e Molecolari di Risposta Clinica e Target Terapeutico

Gruppo di Ricerca 3

Franca PodoGiulia CarpinelliAlessandro RicciSerena CecchettiMaria Elena PisanuEgidio IorioMassimo Giannini

The Phosphocholine (PCho) level as indicator of multiple alterations in the PC-cycle

The intracellular PCho pool in cancer cells reflects genomic expression and activation state of multiple PC cycle enzymes, notably choline kinase (chok) in the biosynthetic Kennedy pathway and PC-specific phospholipases (PC-plc, PC-pld) in the catabolic pathways, in response to agonist-mediated cell receptor stimulation.

PCho

PC-cycle

The PCho level is therefore proposed as a possible fingerprintof tumor progression and pharmacodynamic endpoint

of conventional and targeted therapies

Is there any relation between CXCR4/CXCL12 axis and PC-cycle?

PI3K

MEKERK

Raf

PCho

PC

Signal transduction pathways triggered bytyrosine kinase receptors and/or controlledby oncogenes or intracellular proteinkinases (e.g. Ras, Raf-1, MEK and ERK1/2), have been reported to be related to PC metabolism at the level of either chokand/or PC specific phospholipases.

Are there relationships between the CXCR4/CXCL12 axis and the PC cycle?

1) To investigate the role of non invasively MRS-detected PCho signal as a possible indicator of CXCR4/CXCl12 activity;

2) To evaluate the effects of activation/inhibition of PC-cycle enzymes on the biological role of this receptor-chemokineaxis in cancer cells.

Relation between CXCR4/CXCL12 axis and intracellularPCho content in CEM cells

The cells were grown overnight in serum-free media and then treated with or without SDF-1α (100 ng/ml), AMD3100 (10µM) and serum (10%) for 24 h. Under these experimental conditions, FCS deprivation resulted ina significant increase of PCho (P < 0.05) compared with cells grown in complete medium (control). When the cells were re-stimulated with SDF-1α (100 ng/ml) , the average intracellular PCho content returned to basal levels of control cells.Furthermore, preliminary results indicated that cells treated with AMD3100 practically reverted the effect of SDF-1α.

PCho

PCho

control

deprived of FCS

0

2

4

6

8

10

control deprived of FCS deprived of FCS+ SDF-1a

deprived of FCS + SDF-1a +AMD3100

deprived of FCS+ AMD3100

nmol

es P

Cho/

106

cells

PCho

Expression of PC-plc enzyme and D609-induced down-modulation of CXCR4 receptor on

membrane surface of CEM cells

CXCR4 membrane expression

0

20

40

60

80

100

CTR 15 30 60 180 300

time (min)

Mea

n Fl

uore

scen

ce

Inte

nsity

D609

Cell

coun

ts

PC-plc expression

97% positive cells118 mean fluorescence intensity

Further clarification of these mechanisms may contribute to the development of non invasive MRS approaches to monitor the effects of activation or inhibition of the CXCR4-CXCL12 axis in tumor cells in vivo.

Istituto Superiore di Sanità

Responsabile Scientifico UO 2 Ruggero Marchiano De Maria

4. Dipartimento di Tecnologie e Salute. MetodiUltrastrutturali per Terapie Innovative Antitumorali. Giuseppe Arancia

M14 WT M14 ADR

CXCR4 CXCR4

M14 WT

P-gpP-gp

M14 ADR

CXCR4 Expression in M14 human melanoma cell lines

Tyr 7 inv is not toxic to M14 human melanoma cells

M14 WT

CTR

CXCL12

Tyr 7 inv + CXCL12

24 h

24 h

24 h

Migration of M14 human melanoma cells

Fondazione Centro San Raffaele del Monte Tabor

Responsabile Scientifico UO 3 Matteo Bellone1.Unità di Immunologia Cellulare Matteo Bellone2. Cancer Stem Cell Unit Rossella Galli3. Unità operativa Anatomia Patologica Claudio Doglioni

Synapthopysin

20X20X

Stem Cell Research Institute and CIGTP - San Raffale - Milano

Characterization of Prostate Cancer Stem/Initiating Cells

TSV 070116TNE 070116 TAD 071122

20X80X 20X

Androgen Receptor Androgen Receptor

20X 10X

CXCR4 expression on Prostate Cancer Stem/Initiating cells

CXCR4

TSV 070116

FSC

SSC

Stem Cell Research Institute and CIGTP - San Raffale - Milano

0 200 400 600 800 1000

0

200

400

600

800

1000

68

0 200 400 600 800 1000

0

200

400

600

800

1000

73.7

0 200 400 600 800 1000

0

200

400

600

800

1000

49.3

TNE 070116 TAD 071122

100

101

102

103

104

FL4-H: CXCR4 APC

0

20

40

60

80

100

% o

f Max

100

101

102

103

104

FL4-H: CXCR4 APC

0

20

40

60

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% o

f Max

100

101

102

103

104

FL4-H: CXCR4 APC

0

20

40

60

80

100

% o

f Max

Acknowledgments

Cellular Immunology UnitCIGTP

Elena JachettiMatteo GrioniMatteo Bellone

Stem Cells Research Institute

Stefania MazzoleniSara MorosiniRossella Galli

Unità Funzionale Anatomia Patologica

Massimo FreschiClaudio Doglioni

Istituto Scientifico Universitario San Raffaele - Milano

Thanks