1. Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis
Professor Janet M Lord
Rheumatology Research Group
MRC Centre for Immune Regulation
University of Birmingham

2. Lecture content
What is Rheumatoid Arthritis?
Neutrophils and their role in RA
Identifying novel drugs to regulate neutrophil function and survival
CDKs as regulators of neutrophil function and apoptosis

3. Inflammatory Response and Rheumatoid arthritisB B B B B B B T T T T T T T T T T T T T T T T T

4. Meet the Neutrophil

5. Phagocytosis. Degranulation, and
activation of NADPH oxidase
Destruction
of microbe
Microbe
Apoptosis
Rolling, adhesion and diapedesis.
Phagocytosis by
Tissue macrophages

6. Neutrophils and Rheumatoid Arthritis
high numbers can be found in Synovial Fluid (SF)
secretion of pro- inflammatory cytokines
loss of viscosity of SF and cartilage destruction caused by ROI and granule enzymes result in joint damage

7. The Big Question in RA is…….
Early synovitis
Resolution
Rheumatoid Arthritis

8. Chronic inflammation in Rheumatoid Arthritis
Division X
Emigration
Recruitment
TNF-a
SDF-1a
Death
IFN-b/a

9. Neutrophil apoptosis in synovial fluid from patients with arthritisRA 5 10 15 20
Crystal
Arthritis
% Apoptotic Neutrophils

10. Prevention versus Treatment

11. Understanding the switch to persistence in RA
Very early
synovitis
Established RA
Normal
synovium
synovial fluid ?

12. months from symptom onset
The early arthritis clinic 3 18
months from symptom onset RA
Non-RA
persistent
Resolving

13. Ultrasound guided joint aspiration
Tibia Talus

14. Very early RA has a distinct cytokine profile1 2 3
IL-13
IL-2
IL-15
bFGF
IL-4
EGF
Eotaxin
IL-1β
MIP1β
GM-CSF
IL-12
MIP1α
MCP-1
IL-17
IL-10
IFN
G-CSF
VEGF
TNFα
RANTES
IL-8
IL-6
IL-5
Decrease in
classification accuracy
-0.6
-0.4
-0.2
0.0
0.2
0.3
0.1
-0.1
Early RA
Other early arthritis

15. Synovial fluid leukocyte apoptosis is inhibited in very early RA1 2 3 RA
non-RA
persistent
resolving
% lymphocyte apoptosis 10 20 30
% neutrophil apoptosis **

16. Very early RA Normal Established
synovium
synovial fluid
IL-2, IL-4, IL-13, IL-15
GM-CSF, bFGF, EGF
Cytokine profile that is distinct & transient
This response may generate the microenvironment required for persistent disease:
IL13 + bFGF promote synoviocyte proliferation and survival
IL4 promotes DC maturation for T cell priming and B cell differentiation and secondary lymphoid tissue formation
Several factors promote neutrophil survival and priming

17. Synovial cytokines prevent Neutrophil and T cell apoptosis
Control ng/ml ng/ml
GM-CSF
T cells

18. What Next?

19. Therapy in very early RA
synovitis
Established RA
Normal
synovium
synovial fluid
Does this phase represent a window in which treatment can modify the subsequent course of disease?
What are the appropriate therapeutic targets?

20. Therapy in very early RA
Treat patients at very high risk of the subsequent development of RA
Small scale pilot studies to test the therapeutic value of specific agents:
Anti-TNF – etanercept B cells – rituximab
T cells – CTLA4 Ig Fibroblasts and neutrophils ?

21. Neutrophils and inflammation
Neutrophils are the most abundant leukocytes but are short lived (24h)
First line of defense against bacterial and fungal infection
Removal of apoptotic neutrophils is important for inflammation resolution
Dysregulation of neutrophil apoptosis has been implicated in many inflammatory diseases
Neutrophils help maintain inflammation, cause tissue damage and promote survival of autoimmune B cells

22. First generation screen for compounds inducing
neutrophil apoptosis *

23. Lead LGR compounds and Neutrophil apoptosis
EC50 ~ 1 M for all 3 compounds

24. LGR1406 and 1407 can inhibit GM-CSF induced survival

25. Can LGR 1406/1407 block inflammatory effects of neutrophils?

26. LGR1406 and 1407 inhibit GM-CSF primed neutrophil superoxide generation
IC50 ~ 2 M IC50 ~ 10 M

27. LGR1406 and 1407 inhibit GM-CSF primed neutrophil IL-8 release
IC50 = 0.1 µM

28. Next Steps Do the compounds work in vivo?
Mode of action – is it CDK and if so which one?

29. Testing in vivo efficacy: Air pouch model
LSHB-CT
4/20/2017
Testing in vivo efficacy: Air pouch model
Air is injected under the skin on the back of the mouse and 6 days later either saline or 1% carrageenan are injected into the pouch.
Inflammatory cells are recruited into the air pouch and can be collected over a period of a week.
Systemic inflammation is minimal in this model which represents a good model of localised inflammation.
Sterile air
6 days
Saline or 1% Carrageenan
0-3 days
Sample inflamed site + blood
20 April 2017

30. LGR1407 reduces the infiltration of neutrophils
Carr/ DMSO
Carr/ 1407
Sal/ DMSO
sal/1407 1 2 P=
Cell counts (x10 6 )

31. LGR1407 reduces inflammatory cytokines in a mouse air pouch model system

32. How do the LGR compounds work?

33. LGR compounds: CDK inhibitors
LGR compounds: CDK inhibitors
Roscovitine LGR compounds

34. CDK inhibitors have anti-inflammatory activity

35. Neutrophils express only the cell cycle independent CDKs 5, 7 and 9B
H N H N H N H N H N H N H N
CDK CDK CDK CDK5 CDK CDK7 CDK9

36. CDK9 is the likely target
Inhibitor
CDK1/
Cyclin B
CDK2/
Cyclin E
CDK4/
Cyclin D
CDK5/
P25
CDK7/
Cyclin H
CDK9/
Cyclin T
LGR1406 3
0.1 15
0.45
>100 1
LGR1407
5.5
1.5 65 2
1.9 20 40 60 80
100
120
0.01
0.1 1 10 50
% Apoptosis
Concentration (
µM)
LGR1406
Roscovitine
NU6102
Flavopiridol 20 40 60 80
100
120 5 10 50
500
% Apoptosis
Concentration (nM)

37. CDK9 is a transcriptional regulator
Cyclin T1
CDK9 +
RNA Pol II TF P
Gene transcription

38. CDK9 activity declines as neutrophils age and enter apoptosis
0h h * 0h 9h
Irr
CDK9
Irr
CDK9

39. CDK9 is a transcriptional regulator: LGR1407 decreases Mcl-1 levels
CDK9 is a transcriptional regulator: LGR1407 decreases Mcl-1 levels
hours
Mcl-1
-Actin
Control
+1407

40. Model of CDK9 regulation of neutrophil apoptosis
LGR1406/7

41. Summary Neutrophils play a key role in the early and late stage of RA
Neutrophils are rational therapeutic targets
CDK9 appears to regulate neutrophil apoptosis
CDK inhibitors reduce inflammation in vivo and represent a novel anti-inflammatory agent

42. Ongoing work - How is Neutrophil function inhibited?
Flavopiridol Purvalonol B

43. Acknowledgements ARC EU FP6 – C3bio Wyeth International Chris Buckley
Karim Raza
Dagmar Scheel-Toellner
Keqing Wang
Hema Chahal
Peter Hampson
Paul Pechan
Miroslav Strnad
Vladimir Krystof
Libor Havlíček
ARC
EU FP6 – C3bio
Wyeth International

44. Age is a risk factor for conversion to persistent RA
Persistent RA Resolving non-RA N
Female
Age (59-74) (32-54) p<0.01
CRP mg/l (18-41) (7-56)
Anti-CCP p<0.0001
RF p<0.0001