Transcript Exchange Protein Directly Activated by cAMP

Exchange Protein Directly Activated
By cAMP (EPAC1) Modulates TregMediated Immune Suppression
Xiaodong Cheng
Department of Integrative Biology &
Pharmacology, University of Texas Health
Science Center @ Houston
FreshAir, October 23, 2014
cAMP signaling pathways
GPCR
Gβγ
GSα
G protein
cAMP
ATP AC cAMP
cAMP
Rap1
EPAC
PKA
GSα
cAMP and major discoveries
2012
Robert Lefkowitz
Brian Kobilka
cAMP and therapeutics
• One of the most targeted signaling
pathways for therapeutic intervention
• Almost all related drugs targeting
GPCRs or PDEs.
• PKA based drug discovery has not been
successful
EPAC & PKA: major cAMP sensors
(Exchange Protein directly Activated by cAMP)
EPAC1
EPAC2
PKA
A
CBD
DEP
B
CBD
A
B
R subunit
Regulatory region
REM
RA
CDC25HD
C subunit
Catalytic region
EPAC & PKA: a tale of two receptors
EPAC & PKA exhibit distinct cellular
localizations
& may act antagonistically
Epac
or synergistically in mediating a specific
PKA
cAMP cellular function.
Gaps and unmet needs
• Physiological functions of EPAC
proteins
• Disease relevance of EPAC proteins
• Lack of isoform-specific EPAC
modulators
EPAC1 and energy homeostasis
Yan, et al. Mol. Cell. Biol. 2013.
Identification of EPAC-specific inhibitors
Tsalkova, et al. PNAS. 2012.
EPAC1 KO protects mice from
fatal rickettsioses
Gong, et al. PNAS. 2013.
ESI-09 exerts excellent bioavailablity
E S I- 0 9 C o n c e n t r a t io n ( u M )
180
160
140
120
100
80
60
40
20
0
0
5
10
15
T im e ( h o u r )
20
ESI-09 protects mice from
fatal rickettsioses
EPACs and human diseases
• Diabetes
Zhang CL, et al. Science 325:607, 2009.
Song WL, et al. Diabetes. 62:2796, 2013.
• Obesity
Fukuda M, et al. Cell Metab. 3:331, 2011.
Yan J, et al. Mol Cell Biol. 33:918, 2013.
• Cancer
Almahariq M, et al. Mol Pharmacol. 83:122, 2013.
Onodera Y, et al. J Clin Inv. 124: 367, 2014.
• Cardiovascular Diseases
Okumura S, et al. J Clin Inv. 124: 2785, 2014.
Pereira L, et al. Circulation 127:913, 2013.
• Chronic Pain
Ferrari LF, et al. Neuroscience 222:392, 2012.
Wang H, et al. J Clin Inv. 123: 5023, 2013.
cAMP and regulatory T cells
• cAMP is a potent inhibitor of T cell functions
• Role of EPAC protein in Treg is not clear
AC
miR142-3p
GJ
Foxp3
IL-2 expression
IL-4 expression
Proliferation
cAMP
PDE
Treg
Teff
EPAC1 inhibition attenuates Treg functions
140
% Teff Proliferation
120
*
100
*
80
60
40
20
0
WT
Teff
WT
Teff
+
ESI-09
KO
Teff
WT
Teff
WT
Teff
KO
Teff
KO
Teff
WT
Teff
+
+
+
+
+
WT
Treg
KO
Treg
WT
Treg
KO
Treg
WT
Treg
+
ESI-09
EPAC1 inhibition boosts the immune response
OVA Immunized
Naïve
6
*
4
*
3
2
1
Total IgG (OD) x 105
OVA-IgG (OD) x 102
5
*
5
4
3
2
1
0
0
WT
Epac1-/- WT/ESI-09
WT
Epac1-/-
Tregs suppress Teff by direct cAMP transfer
Tregs
Teff
GJ
cAMP
IL-2 expression
IL-4 expression
Proliferation
?
EPAC1
Bodor J, et al. Eur J Immunol 2012
EPAC1 does not alter T cell gap junctions
Treg
Teff
Treg + Teff
10
0%
4.5%
8
Vehicle
WT
0%
0%
4.6%
% Dye Transfer
Calcein red orange AM
0%
Vehicle
ESI-09
6
4
2
+ ESI-09
0
WT Treg
+
WT Teff
0%
0%
5.9%
Vehicle
EPAC1-/-
0%
0%
CFSE
6.0%
+ ESI-09
-/-
Epac1 Treg
+
- /Epac1 Teff
EPAC1 regulates STAT3 activation
Teff
Epac1-/-
WT
(hrs) 0
2 24 48
Treg
72
0
Epac1-/-
WT
2 24 48
72
0
2
24
0
2
24
p-STAT3
STAT3
% Phosphorylation
35
*
30
WT
Epac1
*
25
-/-
20
*
15
*
10
*
5
0
0
2
24
48
72
Teff
0
2
Treg
Hrs Post Stimulation
24
EPAC1 regulates Treg activity through STAT3
Vehicle
60
StatticV
% Proliferation of Teff
50
40
*
30
*
20
*
10
0
WT Teff
+
-
+
Epac1-/- Teff
-
+
-
WT Treg
+
-
+
Epac1-/- Treg
-
+
-
ESI-09 (5 µM)
-
-
+
Inhibition of EPAC1 desensitizes T-cells to TGF-β1
A
700
Vehicle
TGF-β1
IL-2 (ng/mL)
600
500
C
400
300
*#
200
WT
WT/ESI-09
Smad7
*#
Actin
*
100
0
WT
WT/ESI-09 Epac1-/-
B
+ TGF-β1
Vehicle
WT
P-Smad2
Smad2
WT/ESI-09
EPAC1-/-
WT
WT/ESI-09
Epac1-/-
Epac1-/-
EPAC1 as a
therapeutic target in
PDA
Inhibition in PDA
Inhibition in host
Enhance
host immune
response
Enhances
tumor
immunity
Inhibit
metastasis
Tumor
regression?
Conclusion
• EPAC proteins are important signaling
molecules and drug targets
• First-in-class small molecule EPAC
pharmacological probes as therapeutic leads
• Inhibition of EPAC1 reduces Treg potency
and Teff sensitivity to suppression through
the STAT3/TGF-β1 pathway
Acknowledgements
Lab members:
Collaborators:
Muayad Almahariq
Yaohua Hu
Fang Mei
Tamara Tsalkova
Jingbo Yan
Hui Wang
Yingmin Zhu
Ju Chen (UCSD)
Mark White (UTMB)
Jia Zhou (UTMB)
Bin Gong/David Walker (UTMB)
Massoud Motamedi (UTMB)
Jin Zhang (John Hopkins)
Don Blumenthal (Utah)
George Holz (SUNY Upstate)
Funding: ACS, AHA, GCC & NIH
EPAC1 and TGF-β1 signaling
TCR
α
CD4
β
CD3
CD3
ε δ
ε δ
TGF-β1
AC
GPC
Gs R
LCK
ζ ζ
ATP
TβR-I
P
cAMP
TβR-II
EPAC1
P
P
STAT3
STAT3
SMAD 2
SMAD 4
P
P
P
STAT3
STAT3
SMAD 2
SMAD 4
P
SMAD 7
Mechanism of EPAC activation