“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
2
3
4
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
80
100
% 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