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Integrating Immunoregulatory and Vascular
Signaling Programs through Lectin-Glycan
Interactions:
A sweet liaison
Gabriel Rabinovich
Presenter Discloser Information
Gabriel Rabinovich
The following relationships exist to this
presentation
No Relationships to Disclose
GENOMICS
PROTEOMICS
GLYCOMICS CO
MP
LE
XIT
Y
KN
OW
LE
DG
E
4 different nucleotides
1 type of bond
20 amino acids
1 type of bond
Diverse types of bonds
and multiple branching
Complexity of the Glycome
The glycome has the potential to display over
100 million more information than the genome…
Rabinovich & Croci, Immunity 2012
Galectins are a family of
animal lectins that have
affinity for multiple N-acetyl-
lactosamine units (Gal1-4
NAcGlc) present in N- and O-
glycans
Galectins are highly
conserved throughout animal
evolution
Galectins are secreted
through a non-classical
pathway
Galectins bind to cell
surface glycoconjugates and
control receptor segregation,
retention and turnover
Galectins Deciphering the biological information encoded by the
glycome: a major role for lectins including galectins
Galectins: structure and classification
Rabinovich & Toscano, Nat Rev Immunol 2009
Galectin-1 expression correlates with the
aggressiveness of human tumors and the
acquisition of metastatic phenotypes
• Gal-1 substantially contributes to the immunosuppressive
activity of melanoma cells
• Silencing of Gal1 gene expression induces markedly
increased Th1-type responses in vivo
Rubinstein et al, Cancer Cell (2004)
• Blockade of Gal-1 within the tumor
microenvironment results in CD4+ and CD8+ T cell-
mediated tumor rejection
Galectin-1 promotes tumor cell evasion of T cell
responses
Juszczynski et al, PNAS (2007)
Rodig et al, Clinical Cancer Research (2008)
Ouyang et al, Blood (2011)
Ouyang et al, Blood (2013)
Gal-1 is overexpressed in
classical Hodgkin lymphoma
compared to diffuse large B
cell lymphoma (DLBCL) and
mediastinal large B cell
lymphoma (MLBCL)
An AP-1-dependent
enhancer regulated by EBV
(LMP1 and LMP2) controls
Gal-1 expression in Reed
Sternberg cells
Secretion of Gal-1
contributes to the Th2-
skewed immunosuppressive
microenvironment of cHL
Gal1 serum levels reflect
tumor burden and adverse
clinical outcome in cHL
Gal-1 contributes to create a Th2-skewed microenvironment
in classical Hodgkin lymphoma
Banh A et al. Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis. Cancer Res. 2011 71:4423-31.
• Tang D. et al. High expression of Galectin-1 in pancreatic stellate cells plays a role in the development and maintenance of an immunosuppressive microenvironment in pancreatic cancer. Int J Cancer. 2011; 130:2337-48 .
Martínez-Bosch, et al. Galectin-1 drives pancreatic carcinogenesis through stroma remodeling and hedgehog signaling activation. Cancer Res. 2014;press
Soldati R. et al. Neuroblastoma triggers an immunoevasive program involving galectin-1-dependent modulation of T cell and dendritic cell compartments.
Int J Cancer. 2011; 131:1131-41.
HODGKIN LYMPHOMA
LUNG ADENOCARCINOMA
PANCREATIC ADENOCARCINOMA
Gal1 activates immunoevasive programs in several
tumor types
NEUROBLASTOMA
GLIOBLASTOMA
Verschuere T et al. Glioma-derived galectin-1 regulates innate and adaptiveantitumor immunity. Int J Cancer. 2014; 134:873-84 Baker et al. NK cells eradicate galectin-1-deficient glioma in the absence of adaptiveimmunity. Cancer Res 2014; in press
Dalotto Moreno et al., Cancer Res, Feb 2013 Laderach et al., Cancer Res, Jan 2013
√ Blocking Gal1 expression suppresses breast cancer metastasis by targeting FoxP3+ Treg cells
√ Delineating the galectin signature of the tumor microenvironment
Mgat5
C2GnT1
Generation for Gal1 ligands
Differential glycosylation of Th1, Th2 and Th17 effector
cells selectively regulates susceptibility to cell death
Th1 and Th17 cells are
sensitive to Gal-induced cell
death
Th2 effector cells are
covered by α2,6 sialic
acid which protects
these cells from Gal-1
binding and death
Toscano et al, Nature Immunology 2007
Ilarregui et al, Nature Immunology 2009
IL-27IL-10
Tolerogenic signals delivered from dendritic cells to T cells
through an immunoregulatory circuit mediated by IL-27 and
IL-10
Do Gal-1-glycan lattices control endothelial cell biology?
Endothelial cell glycosylation controls Gal1
binding and angiogenesis
Croci et al, Cell 2014
Hypoxia reprograms the endothelial cell glycome
Hypoxia regulates Gal1 expression in tumor cells through
HIF-independent, NF- B-dependent mechanisms
Gal1 promotes angiogenesis through N-glycan–dependent mechanisms
Gal1 activates VEGF-like signaling through binding to complex N-glycans on VEGFR2
Proliferation
Migration
Tubulogenesis
Glycosylation-dependent binding of Gal1 to VEGFR2
mimics VEGF function
VEGF-like signaling
N-glycans are essential for Gal1, but
not VEGF signaling
VEGFR2, but not other glycosylated
receptors, mediates Gal1 function
Gal1 binds to VEGFR2 through N-glycan-dependent mechanisms
FRET
Gal1 binds to N-glycans on Ig-3, -4 and -7 domains
of VEGFR2
Differential sialylation of VEGF receptors
accounts for differential binding of Gal1 to
VEGFR2
Gal1-N-glycan interactions promote
segregation of VEGFR2 into
membrane microdomains
By interacting with N-glycans Gal-1 prolongs cell surface residency of VEGFR2 and slows down its internalization
Angiogenesis
OUR HYPOTHESIS
FGF2
HGF-c-Met
Bv8
MDSCs
FGF2, PIGF, Bv8, IL-17?
Characterization of anti-VEGF sensitive and resistant tumors
Differential EC glycosylation induced by anti-VEGF refractory
versus sensitive tumors: an in vitro approach
Differential glycosylation of vessels associated to anti-VEGF
sensitive or resistant tumors in vivo
sensitive
resistant
Anti-VEGF treatment selectively increases Gal1 secretion by
tumor cells
Anti-VEGF treatment increases Gal1 expression in a Kras-
driven genetically engineered mouse model (GEMM) of
pancreatic ductal adenocarcinoma (resistant to anti-VEGF)
Silencing Gal1 eliminated refractoriness to anti-VEGF
Disruption of complex N-glycans abrogates resistance to
anti-VEGF therapy
Elimination of 2,6 sialic acid converts sensitive into anti-
VEGF refractory tumors
Elimination of 2,6 sialic acid converts sensitive into anti-
VEGF refractory tumors by facilitating Gal1 interactions with
VEGFR2 N-glycans
Croci et al, Cell 2014
Can we capitalize on Gal-1-glycan interactions for
therapeutic purposes?
Glycoamines (lactulosamines) (SLA) Rabinovich et al, Glycobiology 2006; 16:210-220.
Anti-Gal-1 mAb .
Anti-Gal1 mAb blocks Gal1 binding to ECs, suppresses angiogenesis and inhibits VEGFR2 phosphorylation
Therapeutic administration of the F8.G7 mAb promotes tumor regression in established KS
The anti-Gal1 mAb eliminates refractoriness to anti-VEGF in LLC1 and R1.1 tumors
The anti-Gal1 mAb promotes vessel normalization early after treatment
The anti-Gal1 mAb promotes pericyte coverage and maturation and alleviates tumor hypoxia
Croci et al, 2014
The anti-Gal1 mAb modulates immune responses in both sensitive and refractory tumors
N-glycan deficiency phenocopies the immunostimulatory effect of anti-Gal1 mAb
Treatment with Gal1-specific neutralizing mAb increases influx of immune cells to tumor parenchyma
T cell purification
1.5 x105
B16F0 cells
Day 0
Inoculation of 1 x 107
CFSE+ cells
24h# of splenic and
intratumoral CFSE+
T cells
CFSE labeling
1.5 x105
B16 or LLC1
F8.G7 or isotype
Cell, 2014
Rutowski et al, Cancer Cell, In press
Gal1 activates immunoevasive programs in several tumor types
Rubinstein et al, Cancer Cell 2004; Juszczynski et al, PNAS 2007; Rutowski et al, Cancer Cell 2014
Effector T cells: Differential glycosylation of Th1, Th2 and Th17 cells selectively
regulates susceptibility to Gal1-induced cell death. Toscano et al, Nature Immunol 2007
Dendritic cells: Gal1-glycan interactions activate a tolerogenic circuit mediated
by IL-27 and IL-10 which amplifies tumor immunosuppression Ilarregui et al, Nature Immunol 2009
Vascular compartment: Gal1-N-glycan interactions can link tumor hypoxia to
angiogenesis through HIF-independent, NF-B-dependent mechanismsCroci et al., J Exp Med 2012
Regulatory T cells: Gal1 promotes the differentiation of FoxP3+ Treg cells which
contribute to the metastatic potential of breast cancer cells
Blois et al., Nat Med 2007; Dalotto-Moreno et al, Cancer Res 2013.
Vascular compartment: Glycosylation-dependent Gal1-VEGFR2 interactions
preserve angiogenesis in anti-VEGF refractory tumorsCroci et al., Cell 2014
Lessons and Take Home Messages
THERAPEUTIC: Ab-mediated Gal1-blockade overcomes tumor-induced
immunosuppression and promotes vascular remodelingCroci et al, JEM 2012; Croci et al, Cell 2014
DIAGNOSTIC: A ‘galectin-specific signature’ may serve as biomarker to
delineate tumor progression
Laderach et al, Cancer Res 2013
Implications and Potential Impact on the Field
DIEGO CROCI
MARTA TOSCANO
JUAN PABLO CERLIANI
MARIANA SALATINO
JUAN M ILARREGUI
SANTIAGO DI LELLA
SANTIAGO MENDEZ HUERGO
SEBASTIAN DERGAN DYLON
TOMAS DALOTTO MORENO
VERONICA MARTINEZ ALLO
ADA BLIDNER
KARINA MARIÑO
LUCIANO MOROSI
ROBERTO DAVICINO
JAVIER ELICABE
LUCAS GENTILINI
ANA CUTINE
VICKY SUNDBLAD
CHARLY GUARDIA
ROSA MORALES
JUANK STUPIRSKI
NICOLAS PINTO
SEBASTIAN MALLER
DIEGO LADERACH
ALEJANDRO CAGNONI
COLLABORATORS
MARGARET SHIPP
CARLOS BAIS
ADRIANA ALBINI
JOSE CONEJO
GARCIA
SUPPORT: AGENCIES AND
FOUNDATIONS