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Cyclin dependent kinases as therapeutic Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis agents in Rheumatoid Arthritis
Professor Janet M LordProfessor Janet M LordRheumatology Research GroupRheumatology Research Group
MRC Centre for Immune RegulationMRC Centre for Immune RegulationUniversity of BirminghamUniversity of Birmingham
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
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Inflammatory Response and Rheumatoid arthritis
Meet the Neutrophil
Rolling, adhesion and
diapedesis.
Phagocytosis. Degranulation, and activation of NADPH oxidase
Microbe
Destructionof microbe
Phagocytosis byTissue macrophages
Apoptosis
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
ResolutionEarly synovitis
Rheumatoid Arthritis
The Big Question in RA is…….
Chronic inflammation in Rheumatoid Chronic inflammation in Rheumatoid ArthritisArthritis
IFN-
DeathDeath
DivisionDivision
EmigrationEmigrationRecruitmentRecruitment
X
SDF-1TNF-
Neutrophil apoptosis in synovial fluid
from patients with arthritis
RA0
5
10
15
20
CrystalArthritis
% A
po
pto
tic
Neu
tro
ph
ils
Prevention versus Treatment
Very earlysynovitis
EstablishedRA
Normal
synovium
synovial fluid?
Understanding the switch to persistence in RA
0 3 18months from symptom onset
RA
Non-RApersistent
Resolving
The early arthritis clinic
Ultrasound guided joint aspiration
Tibia Talus
Very early RA has a distinct cytokine profile
Early RA
Other early arthritis
0 1 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
IFNG-CSF
VEGF
TNFα
RANTES
IL-8
IL-6
IL-5
Decrease inclassification accuracy
-0.6 -0.4 -0.2 0.0 0.2
0.3
0.2
0.1
0.0
-0.1
-0.2
Synovial fluid leukocyte apoptosis is inhibited in very early RA
0
1
2
3
RA non-RApersistent
resolving%
lym
ph
ocy
te a
po
pto
sis
0
10
20
30
% n
eutr
op
hil
apo
pto
sis
RA non-RApersistent
resolving
**
Very early RA
•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
EstablishedNormal
synovium
synovial fluid
IL-2, IL-4, IL-13, IL-15GM-CSF, bFGF, EGF
Synovial cytokines prevent Neutrophil and T cell apoptosis
Control NAC Desf.0
25
50
75
% a
po
pto
tic
neu
tro
ph
ils
Control 10ng/ml 50 ng/ml
GM-CSF
T cells
What Next?
Very earlysynovitis
EstablishedRA
Normal
synovium
synovial fluid
Therapy in very early RA
Does this phase represent a window in which treatment can modify the subsequent course of disease?
What are the appropriate therapeutic targets?
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 ?
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
LGR samples Apoptosis IC50 : EC50 LGR-169 √ 2M : 12.5M LGR-290 √ 4.5M : 50M LGR-406 √ M : 50M LGR-517 √ 0.8M : 2.5M LGR-521 √ 0.07M: 1M LGR-273 √ 0.2M: 2.5M LGR-310 √ 1.5M : 1.5M LGR-556 √ 0.023M: 5M LGR-561 - LGR-580 - LGR-849 - LGR-461 √
LGR-390 -
LGR-348 -
LGR-540 √
LGR-668 -
LGR-1091
First generation screen for compounds inducing neutrophil apoptosis
*
Lead LGR compounds and Neutrophil apoptosis
EC50 ~ 1 M for all 3 compounds
LGR1406 and 1407 can inhibit GM-CSF induced survival
Can LGR 1406/1407 block inflammatory effects of neutrophils?
LGR1406 and 1407 inhibit GM-CSF primed neutrophil superoxide
generation
IC50 ~ 2 M IC50 ~ 10 M
LGR1406 and 1407 inhibit GM-CSF primed neutrophil IL-8 release
IC50 = 0.1 µM
Next Steps
• Do the compounds work in vivo?
• Mode of action – is it CDK and if so which one?
Testing in vivo efficacy: Air pouch model
April 21, 2023
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
Saline or 1% Carrageenan
6 days
Sample inflamed site + blood
0-3 days
LGR1407 reduces the infiltration of neutrophils
Carr/
DMSO
Carr/
1407
Sal/ D
MSO
sal/1
407
0
1
2
P= 0.0075
Cel
l co
un
ts (
x106
)
LGR1407 reduces inflammatory cytokines in a mouse air pouch model system
MCP-1
salin
e/ D
MSO
Salin
e/ 1
407
Carr/
DMSO
Carr/
1407
0
250
500
750**
*M
ean
MC
P-1
(p
g/m
l)
VEGF
salin
e/ D
MSO
Salin
e/ 1
407
Carr/
DMSO
Carr/
1407
0
25
50
75
100
125**
Mea
n V
EG
F (
pg
/ml)
IL-6
salin
e/ D
MSO
Salin
e/ 1
407
Carr/
DMSO
Carr/
1407
0
25
50
75
100***
Mea
n I
L-6
(p
g/m
l)
IL-5
salin
e/ D
MSO
Salin
e/ 1
407
Carr/
DMSO
Carr/
1407
0
50
100
150
200
250 ***
n/s
Mea
n I
L-5
(p
g/m
l)
How do the LGR compounds work?
21.04.23
LGR compounds: CDK inhibitors
Roscovitine LGR compounds
N
N
NNH
R3R5
R7
CDK inhibitors have anti-inflammatory activity
0
0.2
0.4
0.6
0.8
1
1.2
1.4
CDK1 CDK2 CDK4 CDK5 CDK6 CDK7 CDK9
CD
K:B
-act
in r
atio
H N H N H N H N H N H N H N A
B
CDK1 CDK2 CDK4 CDK5 CDK6 CDK7 CDK9
Neutrophils express only the cell cycle independent CDKs 5, 7 and 9
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 >100 1.9
0
20
40
60
80
100
120
0 0.01 0.1 1 10 50 100
% A
popt
osis
Concentration ( µM)
LGR1406 Roscovitine NU6102
0
20
40
60
80
100
120
0 5 10 50 100 500
% A
pop
tosi
s
Concentration (nM)
Flavopiridol
CDK9 is a transcriptional regulator
Gene transcription
Cyclin T1 CDK9
RNA Pol II
P
+TF
9h0h
Irr
CD
K9
Irr
CD
K9
0h 9h
*
CDK9 activity declines as neutrophils age and enter apoptosis
21.04.23
CDK9 is a transcriptional regulator: LGR1407 decreases Mcl-1 levels
0 2 4 6 9 12 20 hours
Mcl-1
-Actin
Mcl-1
-Actin
Control
+1407
Model of CDK9 regulation of neutrophil apoptosis
LGR1406/7
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
Ongoing work - How is Neutrophil function inhibited?
Flavopiridol Purvalonol B
Acknowledgements
• 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
Age is a risk factor for conversion to persistent RA
Persistent RA Resolving non-RA
N 19 49
Female 11 20
Age 67 (59-74) 41 (32-54) p<0.01
CRP mg/l 25 (18-41) 23 (7-56)Anti-CCP 10 2 p<0.0001RF 10 7 p<0.0001