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Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis

Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis. Professor Janet M Lord Rheumatology Research Group MRC Centre for Immune Regulation University of Birmingham. Lecture content. What is Rheumatoid Arthritis? Neutrophils and their role in RA

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Cyclin dependent kinases as therapeutic agents in Rheumatoid Arthritis

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  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. T T T T T T T T T T T T T T T Inflammatory Response and Rheumatoid arthritis B T B B B B B B T T T

  4. Meet the Neutrophil

  5. Apoptosis Phagocytosis. Degranulation, and activation of NADPH oxidase Destruction of microbe Microbe 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. Early synovitis The Big Question in RA is……. Resolution Rheumatoid Arthritis

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

  9. 20 15 % Apoptotic Neutrophils 10 5 0 Crystal RA Arthritis Neutrophil apoptosis in synovial fluid from patients with arthritis

  10. Prevention versus Treatment

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

  12. The early arthritis clinic RA Non-RA persistent Resolving 0 3 18 months from symptom onset

  13. Tibia Talus Ultrasound guided joint aspiration

  14. 0 1 2 3 IL-13 Decrease in classification accuracy IL-2 IL-15 bFGF IL-4 0.3 EGF Eotaxin IL-1β 0.2 MIP1β GM-CSF IL-12 0.1 MIP1α MCP-1 IL-17 0.0 IL-10 IFN G-CSF -0.1 VEGF TNFα RANTES -0.2 IL-8 IL-6 IL-5 -0.4 -0.2 0.0 0.2 -0.6 Early RA Other early arthritis Very early RA has a distinct cytokine profile

  15. 3 2 RA non-RA persistent resolving ** 30 1 0 20 % lymphocyte apoptosis % neutrophil apoptosis 10 0 RA non-RA persistent resolving Synovial fluid leukocyte apoptosis is inhibited in very early RA

  16. synovium synovial fluid Very early RA Normal Established 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. Control 10ng/ml 50 ng/ml GM-CSF Synovial cytokines prevent Neutrophil and T cell apoptosis T cells

  18. What Next?

  19. Normal Very early synovitis Established RA 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?

  20. Therapy in very early RA • Treat patients at very high risk of the subsequent development of RA • Small scale pilot studiesto testthe 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 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 Sampleinflamed site + blood

  30. P= 0.0075 2 ) 6 1 Cell counts (x10 0 sal/1407 Carr/ 1407 Sal/ DMSO Carr/ DMSO LGR1407 reduces the infiltration of neutrophils

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

  32. How do the LGR compounds work?

  33. LGR compounds: CDK inhibitors RoscovitineLGR compounds

  34. CDK inhibitors have anti-inflammatory activity

  35. 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 CDK7CDK9

  36. 120 100 80 60 % Apoptosis 40 20 0 50 0 5 10 100 500 Concentration (nM) CDK9 is the likely target LGR1406 Roscovitine NU6102 120 100 80 60 % Apoptosis 40 20 0 0 0.01 0.1 1 10 50 100 Concentration( µM) Flavopiridol

  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 9h * 0h 9h CDK9 CDK9 Irr Irr

  39. 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

  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. 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 Acknowledgements

  44. 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<0.0001 RF 10 7 p<0.0001

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