This document summarizes the key steps involved in translational research for systemic sclerosis (SSc), from target identification to preclinical testing. It discusses expression profiling of SSc patient samples to identify potential drug targets. Functional characterization of targets involves in vitro testing using patient cells and in vivo testing in mouse models. Promising targets then undergo therapeutic modification and efficacy/toxicity testing. The document provides examples of STAT3 and SOCS3 as potential targets, showing how their expression and role is characterized in SSc and mouse models. Epigenetic changes like DNA methylation are also explored, with DNMT3A and SOCS3 methylation implicated in the SSc fibrotic process.

1. Translational research in SSc
Jörg Distler
Department of Internal Medicine 3 and Institute for Clinical Immunology
University of Erlangen-Nuremberg
Germany

2. Expression profiling
Overview about key steps of target development
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc

3. Expression profiling
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc 4

4. • Does the expression differ between SSc and healthy?
• Do the expression levels/pattern correlate with disease activity?
• Are the differences restricted to a subpopulation of SSc patients?
• Which cells express the molecule of interest?
Expression profiling - Confirmation of target activation

5. Healthy SSc Healthy SSc
Healthy SSc
Healthy SSc
β-actin
P-STAT3
STAT3
P-STAT3
STAT3
β-actin
Chakraborty et al., Nature Commun. 2017 Oct 24;8(1):1130.
Expression profiling - STAT3 signaling is active in SSc

6. Expression profiling
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc 7

7. Stimulus
Inter-
vention
Functional characterization – in vitro
• Characterization of the mode of action
• First confirmation of the mode of action
• IMPORTANT: Use primary cells of interest from SSc patients and matched controls!
2D cell culture of isolated
cells

8. 0
2
4
6
8
0
2
4
6
8
Acta2 mRNA
x-foldchange
x-foldchange
stressfibres
TGFβ + LacZ
control + LacZ
TGFβ + Cre
control + Cre
Ctgf mRNA
**
**
0
2
4
6
8
0
1
2
3
0
2
4
6
8
10
Col1a1 mRNA Col1a2 mRNA
x-foldchange
x-foldchange
x-foldchange
*** **
Collagen protein
**
Functional characterization – in vitro – STAT3

9. Expression profiling
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc 10

10. Functional characterization – in vivo
Which model to choose? Decision needs to be based on mode of action
myofibroblasts
macro-
phages
neutro-
philes
ECM
resident
fibroblasts
epithelial cells
pericyts /
endothial cells
fibrozytes
T and B cells

11. Functional characterization – in vivo
Which model to choose? Decision needs to be based on mode of action

12. Intratracheal injection
WNT5AAAVLacZ AAV
25μm
WNT5AAAVLacZ AAV
100μm
Subcutaneous injection
WNT5A AAV
Functional characterization – in vivo
• Activation of the pathways induces in SSc-like phenotype in healthy mice
• Pathway inhibition ameliorates experimental SSc
Wnt5afl/flCol1a2/Cre+/-
NaCl 100μm Bleo NaCl Bleo
Subcutaneous injection
Wnt5afl/flCol1a2/Cre-
Bleomycin (Bleo)

13. 0
1
2
3

0
1
2
3
4 
0
1
2
3

0.0
0.5
1.0
1.5
2.0 
0
1
2
3

0.0
0.5
1.0
1.5
2.0
2.5

0
1
2
3 
0
1
2
3
4

Fibroblast-specific knockout of STAT3 ameliorates fibrosis

14. Expression profiling
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc 15

15. Therapeutic modification – efficacy testing
Evaluation of the translation potential:
• Evaluate multiple drug candidates
• Test different doses in vitro and in vivo
• Analyse multiple models to represent the different subpopulation of SSc
• Evaluate the effects on different organs
• Test different dosing schemes: E.g. preventive dosing vs. therapeutic dosing
• Analyze the outsome of your drug candidate on other clinical outcomes, e.g.
assessment of fibrosis and vasculopathy to avoid unexpected adverse effects
• Do an in-depth toxicity screening
• Consider combination therapies

16. Pharmacologic inhibition of STAT3 ameliorates experimental
fibrosis
Myofibroblast
count
Hydroxyproline
content
Dermal
thickness
x-foldchangex-foldchange
NaCl Bleo Bleo+S3l-201
AdLacZ AdTBR AdTBR+S3l-201
0.0
0.5
1.0
1.5
2.0
2.5******
0
1
2
3
4
5 ******
0
2
4
6
8
******
0
5
10
15
*** **
0
1
2
3
4
5
*****
0
5
10
15
20 *****
X-foldchangesin
P-STAT3/STAT3
3.0
2.5
2.0
1.5
1.0
0.5
* *
0.5
1.0
1.5
2.0  
100µm
100µm
Chakraborty et al., Nature Commun. 2017 Oct 24;8(1):11

17. Myofibroblast differentiation
JAK1/2
STAT3
STAT3
TGFß
Summary
JNK SRC cABL
Inflammation
JAK2
STAT3
STAT3
IL6
STAT3
inhibitor

18. Role of patients in preclinical drug development
Expression profiling
Functional
characterization
In vitro testing
(cell culture)
In vivo testing
(mouse models)
Therapeutic
modification
Efficacy testing Toxicity testing
Proof-of-Concept
studies in SSc
Requires
patient
samples
Requires
patient
samples
Depends on
participation
of patients
Partially
dependent on
samples

19. SOCS3
STAT3
ATP
+ ADP
InhibitionActivation
JAK2 / STAT3 signaling and its regulation by SOCS3
Babon J. et al., Immunity. 2012
Babon J. and Nicola NA. et al., Growth Factors. 2012
Yoshimura A. and Yasukawa H., Immunity. 2012
TGFß
/IL6
SOCS3

20. SOCS3 is downregulated in SSc fibroblasts
600x200x
healthy SSc
SOCS3+P4Hb+
P4Hb+
A
+
I
P
D
healthy SSc
SOCS3P4Hboverlay

21. Epigenetics
healthy SSc
Fibroblasts after 4 passages in vitro
without exogenous stimulation

22. DNA methylation
N
N
O
NH 2
OP
N
N
O
NH2
OP
H3C
OH H
5-Methylcytosine
+ CH3
OH H
Cytosine
Methyltransferases
Dnmt-1
Dnmt-3a
Dnmt-3b
Inhibition of transcription
→ several inhibitors (MTIs) in clinical use:
5-aza-2’-deoxycytidine, 5-azacytidine, Procainamide, Hydralazine

23. Fibroblast-specific knockout of SOCS3 exacerbates
experimental fibrosis
0.0
0.5
1.0
1.5
2.0
2.5
3.0
**
**
***
**
foldchangesof
dermalthickness
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
**
***
Socs3fl/fl NaCl
Socs3fl/fl Bleomycin
Socs3fl/fl;Col6Cre NaCl
Socs3fl/fl;Col6Cre Bleomycin
foldchangesof
myofibroblastcounts
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
* *
*
Socs3fl/fl Bleomycin + 5-aza Socs3fl/fl;Col6Cre Bleomycin + 5-aza
** * n.s.
n.s.
foldchangesof
hydroxyprolinecontent
0.0
0.5
1.0
1.5
2.0
2.5
3.0
** *
**
*
foldchangesof
dermalthickness
0.0
0.5
1.0
1.5
2.0
2.5
3.0
* *
*
*
foldchangesof
hydroxyprolinecontent
5.0
4.5
4.0
3.5
3.0 *
2.5
2.0
1.5
1.0
0.5
0.0
*
*
Socs3fl/fl LacZ-AAV5
Socs3fl/fl TGFβRIact-AAV5
Socs3fl/fl;Col6Cre LacZ-AAV5
Socs3fl/fl;Col6Cre TGFβRIact-AAV5
Socs3fl/fl TGFβRIact-AAV5+ 5-aza Socs3fl/fl;Col6Cre TGFβRIact-AAV5 + 5-aza
*
foldchangesof
myofibroblastcounts
Socs3fl/fl
Socs3fl/fl;
Col6Cre
NaCl Bleomycin
Socs3fl/fl
Socs3fl/fl;
Col6Cre
LacZ-AAV5 TGFβRIact-AAV5

24. SOCS3 is downregulated by TGFβ-induced promoter methylation
foldchangesof
SOCS3mRNA
0.0
1.0
2.0
3.0
4.0
5.0
*********** ****
**
hours-
TGFβ
1 4 6 12 16 24 48 72120
- 1 4 6 12 16 24 48 72 120hours
SOCS3
β-actin
TGFβ
SOCS3
β-actin
hours - 12 24 72 96 120
5-aza
1.0
2.0
3.0
4.0
***
***
***
***
foldchangesof
SOCS3mRNA
0.0
hours - 12 24 72 96120
5-aza
SSc fibroblasts + 5-aza
0.0
0.5
1.0
1.5
2.0
***
n.s.
**
normal control
normal 5-aza
SSc control
SSc 5-aza
foldchangesof
SOCS3mRNA
0.1
1.0
10.0
100.0
1000.0
normal control
normal 5-aza
SSc control
SSc 5-aza
** *
foldenrichment
SOCS3
β-actin
control
5-aza
control
5-aza
normal SSc
normal vs. SSc fibroblasts MeDIP

25. TGFβ upregulates the expression of DNMT3A
***
** ***
******
***
foldchange
-
2.5
2.0
1.5
1.0
0.5
0.0
hours
TGFβ
1 4 6 12 16 24 48 72 120
DNMT3A
***
***
foldchange
-
2.5
2.0
1.5
1.0
0.5
0.0
hours
TGFβ
1 4 6 12 16 24 48 72 120
DNMT1
DNMT3A
DNMT3B
β-actin
DNMT1
- 1 4 6 12 16 24 48 72 120
hours
TGFβ

26. Fibroblast-specific deletion of Dnmt3a protects from
experimental fibrosis
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*** **
**
foldchangesof
dermalthickness
0.0
0.5
1.0
1.5
2.0
2.5
**
3.0 ** **
foldchangesof
hydroxyprolinecontent
foldchangesof
myofibroblastcounts
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
** **
Dnmt3afl/fl NaCl
Dnmt3afl/fl Bleomycin
Dnmt3afl/fl Bleomycin +5-aza
Dnmt3afl/fl;Col6Cre NaCl
Dnmt3afl/fl;Col6Cre Bleomycin
Dnmt3afl/fl;Col6Cre Bleomycin + 5-aza
foldchangesof
myofibroblastcounts
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Dnmt3afl/fl;Col6Cre LacZ-AAV5
Dnmt3afl/fl;Col6Cre TGFβRIact-AAV5
Dnmt3afl/fl;Col6Cre TGFβRIact-AAV5 + 5-aza
** **
**
foldchangesof
hydroxyprolinecontent
foldchangesof
dermalthickness
0.0
0.5
1.0
1.5
2.0
2.5
**
** **
Dnmt3afl/fl LacZ-AAV5
Dnmt3afl/fl TGFβRIact-AAV5
Dnmt3afl/fl TGFβRIact-AAV5 + 5-aza
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
** **
**
Dnmt3afl/fl
Dnmt3afl/fl;
Col6Cre
NaCl Bleomycin
Bleomycin
+ 5-aza
LacZ-AAV5
TGFβRIact-AAV5
+ 5-azaTGFβRIact-AAV5
Dnmt3afl/fl
Dnmt3afl/fl;
Col6Cre

28. Physiological response: temporary upregulation of TGFβ
JAK2JAK2
STAT
3
SOCS3
STAT
3
STAT
3
JAK2JAK2
STAT
3
SOCS3
Summary
Fibrotic tissue remodeling: persistent upregulation of TGFβ
Methylation
TGFβ
JAK2JAK2
STAT
3
STAT
3
STAT
3
+
TGFβ
SMAD
SMAD
SMAD
SMAD
DNMT3A
JAK2
SOCS3
JAK2
STAT
3
JCI, in revision

29. Acknowledgements