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

T

B

T

T

T T

T

T T

T

TT

TT

T

T

TT T

T

B

B

BB

BB

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