The Role of Cyclin-Dependent Kinase-9 Inhibition in Promoting the Resolution of Chronic Inflammation

Disordered inflammation is responsible for most of the diseases afflicting western society, including hardening of the arteries (causing heart attacks, stroke and peripheral vascular disease), lung diseases (including chronic bronchitis and asthma), crippling rheumatoid and osteoarthritis, and the commonest forms of liver and kidney disease necessitating transplantation. Despite the huge burden of illness, loss of work and ultimately death, there is yet no effective drug therapy for most of these conditions.
Previous approaches to drug development have been targeted at interfering at single points within a hugely complex process that involves thousands of mediators, many of which duplicate and triplicate each other's effects. With MRC support over the last twenty years we have been taking an entirely different approach. While some forms of inflammation (pneumonia and acute gout, for example) cause dramatic inflammatory diseases they have the capacity to resolve completely with no residual damage to surrounding tissues. Starting from this observation we have been studying how these conditions resolve in order to generate new therapies for the vast majority of inflammatory diseases that fail to resolve 'in the fashion nature intended' and therefore result in chronic inflammation and ill-health.
Our major early discovery was that certain damaging white blood cells need to undergo a form of 'silent suicide' and be quietly removed by local scavenger cells in order for inflammation to resolve effectively. We spent several years working out the detailed mechanisms of this process in order to attempt to harness them for therapeutic gain. In the 'test-tube' we found many ways of 'driving' these suicide and clearing-up processes, but whenever we tried to reproduce the results in mouse models of human disease the beneficial effects were counteracted by the presence of powerful cell-survival factors in live tissues.
The 'breakthrough' came at the beginning of our current MRC programme grant when we showed that agents called cyclin-dependent kinase (CDK) inhibitor drugs not only drove the cell suicide process in the presence of survival factors in the test tube, but greatly accelerated the rate of resolution in mouse lung models of human inflammatory/scarring diseases. This effectively 'squared the circle' on twenty years of preparatory work.
In the past four years we have homed in on the specific molecules involved in this exciting discovery in order to build a strong scientific foundation for better targeted, less toxic new drugs. We have now clearly identified the molecule CDK9 for close attention in our new research programme, and shown that CDK inhibitor drugs not only drive cell suicide but also cause the 'scavenger' cells to release anti-inflammatory agents, thus adding to the drug's potential therapeutic benefit. Furthermore, we have shown that CDK inhibitor drugs cause resolution of inflammation not only in the lung but also in a mouse model of the inflammatory process responsible for hardening of the arteries and its consequences. Thus in our new programme of research we will extend our studies to clinically relevant models of vascular, liver and kidney disease, in the likelihood that our work will have widespread relevance for the development of new treatments for inflammatory/scarring diseases of all organs.

By demonstrating that CDK inhibitor drugs (CDKi) promote the resolution of lung inflammation through a process which involves 'driving' neutrophil apoptosis, we achieved a key aim of many years of MRC-funded research - showing that resolution mechanisms could be harnessed for potential therapeutic benefit. While CDKi (e.g., R-roscovitine) are beginning to be trialled in humans for various cancers, it is recognised that their specificity and toxicity profiles are not yet ideal.
We have now generated persuasive in vitro and in vivo preliminary data that CDK9 is the key target of the CDKi in a process that involves down-regulation of Mcl-1. The importance of CDK9 in vivo is supported by pilot data using transgenic zebrafish treated with morpholino antisense oligimers to specifically knockdown CDK9. We have also discovered that CDKi inhibit release of pro-inflammatory mediators from stimulated macrophages (without inhibiting efferocytosis) in what is likely to represent an additional pro-resolution benefit. We have recently shown identical pro-resolution effects of CDKi in eosinophilic inflammation, in bleomycin-induced fibrosis and, excitingly, in a mouse model of atherogenesis.
In the proposed programme we will interrogate the hypothesis that CDK9 is pivotal in maintaining chronic inflammation and their inhibition induces the resolution of chronic inflammatory/scarring diseases of the lungs and other organs. Specifically we will:

1. Test our hypothesis in zebrafish, employing a novel model of chronic atheromatous inflammation and using specific molecular knockdown approaches in
genetically modified fish.
2. Confirm the pivotal status of CDK9 in models of chronic inflammation in mice, prioritising the use of a myeloid-specific inducible CDK9 knockout mouse.
3. Establish proof-of-concept assays of pro-resolution effects of CDK9 inhibition in human inflammatory macrophages to provide a platform for drug discovery.

The worldwide healthcare burden of inflammatory/scarring diseases of the lung and other organs is undeniable, as is the fact that most of these are therapeutically intractable and ultimately fatal. Our MRC-supported research showing that the processes by which inflammation resolves 'naturally' can be harnessed in a new approach to treating these diseases has already generated considerable impact. Indeed our twenty-year 'discovery pathway' is often used as an example of the cellular>animal model>human disease translation paradigm.
Our work has already resulted in considerable academic and commercial impact. It has attracted large numbers of high-quality clinical and non-clinical training fellows and continues to do so. Our proposed research programme is positioned directly at the translational interface, in terms of both clinical and commercial exploitation:
1. Our work on CDK inhibitor drugs in lung inflammation (Nature Medicine, 2006 which has been cited at least 164 times) has paved the way for a new therapeutic approach to treating inflammatory disease. We have already filed a patent application entitled 'Use of CDK inhibitor for the treatment of granulocyte mediated disorders' (No 0325931), but our proposed research on CDK9 will lead to better targeted drugs with better toxicity profiles. These will also be patented and commercially exploited.
2. The extension of our findings into a platform of potential new treatments for atheromatous vascular disease, liver and kidney fibrosis will add enormously to the impact of our current and proposed research in chronic inflammatory lung disease.
3. The research of our group has generated considerable academic impact in the inflammation and apoptosis fields, and has influenced the research of numerous groups worldwide. Our publications are widely read and highly cited. Our original observation of neutrophil apoptosis (J Clin Invest, 1989) has been cited more than 1100 times and has stimulated new research by academic groups worldwide. Since 1989 a total of >9583 academic papers have been published on 'neutrophil', 'eosinophil' or 'granulocyte' apoptosis, or programmed cell death, and most of Haslett's (>18,239) citations relate to granulocyte apoptosis. The metric for the applicants are as follows: AG Rossi (H-factor-36, total citations >4542) and C Haslett (H-factor-65; total citations >18,224. R. Duffin, the named senior scientist, has an H-factor of 24 with total citations of >2,933). It is predicted that these metrics will continue to grow proportionately as new publications derive from our proposed work.
4. The group continues to enjoy an excellent record of attracting high-quality scientific trainees from home and abroad and for guiding them on successful career paths. Haslett has been responsible for the supervision/sponsorship of more that twenty-five (MRC and WT) clinical training fellows at different levels of seniority, many of whom now run their own research groups in branches of this field. Two new Training Fellows are now associated with the current programme. The UoE CMVM has a strong history of appointing Programme Grant senior scientists to permanent positions at the end of the grant period. Previous examples include Professors Ian Dransfield and Adriano Rossi (who now heads the 'Inflammation Resolution' programme in the CIR). If the current proposal is awarded, CMVM is committed to the same plan for Dr Rodger Duffin. Thus the research programme will continue to have an important impact on scientific training and career development.

Our Research proposal therefore addresses a major area of unmet human need and fits with a number of strategic objectives in the MRC five-year Strategic Plan (2009-2014):-
- Improve human health
- Promote translation of basic research discoveries into new/improved healthcare
- Encourage commercial exploitation, for the benefit of the UK's health and economy
- Attract and train first-rate researchers.