“RECETTORI PER CHEMOCHINE COME MARCATORI BIOLOGICI E...
Transcript of “RECETTORI PER CHEMOCHINE COME MARCATORI BIOLOGICI E...
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“RECETTORI PER CHEMOCHINE COME MARCATORI BIOLOGICI E
MOLECOLARI DI RISPOSTA CLINICA E TARGET TERAPEUTICO”
Stefania ScalaIstituto Tumori di Napoli - “Fondazione G. Pascale”
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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
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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
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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
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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.
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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.
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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
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There is a need for developing new potent CXCR4 antagonists with a safety profile suitable for human
clinical use
Objective
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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
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CXCL12
SDF-1α Crystallographic structure
CXCR4 Binding and transduction involves residues in N-Terminal Domain
N-terminal Domain
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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
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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).
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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
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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
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Peptides functional evaluation
Indirect binding evaluation to CXCR4
Calcium efflux evaluation MigrationP-Erk Induction
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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
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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.
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Competitive binding curves of Ciclo Phe-7 invfor the binding of 125ISDF-1α in CCRF-CEM cells
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Serum degradation Hys-OH
1 Ora 24 Ore
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Serum degradation Phe-7 Inv
1 hr 24 hrs
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Serum degradation Hys-7 Inv
1 hr 24 hrs
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Negative Ctr Phe-7inv
Fluorescent Phe-7 inv in human PES 43 melanoma cells
In VIVO TAG-S 750
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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
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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
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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.”
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Ongoing experiments
Peptides Inhibition of HIV entry Peptides treatment on PES 43 xenograftPeptides treatment of murine osteosarcoma K7M2Acute and chronic peptides toxicities in mice
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Istituto Superiore di Sanità
Responsabile Scientifico UO 2 RuggeroMarchiano De Maria
1. Dipartimento di Ematologia Oncologia e Medicina Molecolare. Reparto di OncologiaMolecolare. Alessandra Carè
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ISOLATION OF MELANOMA CANCER STEM CELLS
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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)
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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)
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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
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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
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10 minexposition
In vivo models of CSC-generated glioblastomasfor drug testing
GBM I-sc GBM Q- sc
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CXCR4 is expressed at high level in glioblastoma CSCs
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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
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The pathway comprising PLZF/miR-146a and its target CXCR4 operates in leukemic cells.
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The regulatory pathway involving PLZF control on miR-146a expression and miR-146a control on CXCR4 translation is found in AMLs
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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
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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
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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
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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.
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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.
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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
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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
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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.
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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
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M14 WT M14 ADR
CXCR4 CXCR4
M14 WT
P-gpP-gp
M14 ADR
CXCR4 Expression in M14 human melanoma cell lines
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Tyr 7 inv is not toxic to M14 human melanoma cells
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M14 WT
CTR
CXCL12
Tyr 7 inv + CXCL12
24 h
24 h
24 h
Migration of M14 human melanoma cells
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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
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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
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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
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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
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Thanks