Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis

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Cyclin dependent kinases as therapeutic agents in
Rheumatoid Arthritis
Professor Janet M Lord Rheumatology Research
Group MRC Centre for Immune Regulation University
of Birmingham
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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
T
B
B
B
B
B
B
B
T
T
T
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Meet the Neutrophil
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Phagocytosis. Degranulation, and activation of
NADPH oxidase
Destruction of microbe
Microbe
Rolling, adhesion and diapedesis.
Phagocytosis by Tissue macrophages
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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

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The Big Question in RA is.
Resolution
Rheumatoid Arthritis
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Chronic inflammation in Rheumatoid Arthritis
X
TNF-a
SDF-1a
IFN-b/a
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Neutrophil apoptosis in synovial fluid from
patients with arthritis
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Prevention versus Treatment
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Understanding the switch to persistence in RA
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The early arthritis clinic
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Ultrasound guided joint aspiration
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Very early RA has a distinct cytokine profile
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Synovial fluid leukocyte apoptosis is inhibited
in very early RA
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Very early RA
Established
Normal
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

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Synovial cytokines prevent Neutrophil and T cell
apoptosis
T cells
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What Next?
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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?
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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 ?

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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

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First generation screen for compounds inducing
neutrophil apoptosis

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Lead LGR compounds and Neutrophil apoptosis
EC50 1 ?M for all 3 compounds
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LGR1406 and 1407 can inhibit GM-CSF induced
survival
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Can LGR 1406/1407 block inflammatory effects of
neutrophils?
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LGR1406 and 1407 inhibit GM-CSF primed neutrophil
superoxide generation
IC50 2 ?M IC50 10 ?M
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LGR1406 and 1407 inhibit GM-CSF primed neutrophil
IL-8 release
IC50 0.1 µM
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Next Steps

• Do the compounds work in vivo?
• Mode of action is it CDK and if so which one?

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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
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LGR1407 reduces the infiltration of neutrophils
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LGR1407 reduces inflammatory cytokines
in a mouse air pouch model system
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How do the LGR compounds work?
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LGR compounds CDK inhibitors

• Roscovitine LGR compounds

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CDK inhibitors have anti-inflammatory activity
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Neutrophils express only the cell cycle
independent CDKs 5, 7 and 9
A B
H N H N H N H N
H N H N H N
CDK1 CDK2 CDK4 CDK5 CDK6
CDK7 CDK9
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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 gt100 1
LGR1407 5.5 1.5 65 2 gt100 1.9
Flavopiridol
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CDK9 is a transcriptional regulator
Cyclin T1
CDK9

RNA Pol II
TF
P
Gene transcription
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CDK9 activity declines as neutrophils age and
enter apoptosis
0h 9h

9h
0h
CDK9
CDK9
Irr
Irr
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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
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Model of CDK9 regulation of neutrophil apoptosis
LGR1406/7
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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

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

• Chris Buckley
• Karim Raza
• Dagmar Scheel-Toellner
• Keqing Wang
• Hema Chahal
• Peter Hampson
• Paul Pechan
• Miroslav Strnad
• Vladimir Krystof
• Libor Havlícek

• ARC
• EU FP6 C3bio
• Wyeth International

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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) plt0.01 CRP mg/l 25 (18-41)
23 (7-56) Anti-CCP 10
2 plt0.0001 RF 10
7 plt0.0001