Targeting WNT/CaMKII in Osteoarthritis

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

Cartilage is the tissue that covers the ends of the bones and that allows the frictionless motion of the joint. Cartilage loss leads to pain, impairment of the joint, and is called Osteoarthritis. Osteoarthritis is, together with cardiovascular disease, the main cause of disability worldwide; it affects over 50% of the population over the age of 65 and therefore is one of the major factors affecting wellbeing in the elderly. It is a disabling disease, costing the UK economy of 36 million working days in 1999-2000 costing the economy nearly 3.2 billion in lost production (NICE 2012 OA guidance draft).

We do not have any treatment that stops progression of osteoarthritis and the only remedies are pain killers and joint replacement. There is a great need for drugs that can stop the degradation of articular cartilage as well as control the pain.

One of the molecular mechanisms known to mediate cartilage breakdown is driven by molecules called WNTs. Therefore, theoretically, blocking WNTs should halt cartilage breakdown in Osteoarthritis. Unfortunately, however, WNTs also mediate important and essential functions for cartilage such as cell proliferation, and therefore such non-selective blockade of WNTs has also resulted in cartilage breakdown.

The opportunity to block the damaging effects of WNTs while preserving the beneficial ones comes from our discovery that a molecule called CaMKII mediates the damaging effects of WNTs, but not the beneficial ones. In addition, we have discovered that CaMKII is strongly activated in osteoarthritis compared with normal cartilage. Finally, it is known that blocking CaMKII in mice results in reduction of pain perception. Therefore blocking CaMKII could represent a terrific strategy to simultaneously reduce cartilage breakdown and reduce the disabling symptoms of osteoarthritis.

In this application we want to understand the function of CaMKII activation in osteoarthritis, and explore if CaMKII blockade can reduce cartilage breakdown as well as pain in an experimental osteoarthritis model in mice.

Although we know that WNT/CaMKII activation leads to deleterious effects in cartilage, we ignore how CaMKII works, and the "big picture" of CaMKII blockade. In a first set of experiments we will identify all the genes activated by WNTs in cartilage and which ones are dependent on CaMKII. From this analysis we will identify possible, perhaps unexpected, consequences of CaMKII blockade and gain insight into the molecular mechanisms of how CaMKII drives cartilage breakdown.

In a second set of experiments we will see whether blocking CaMKII pharmacologically in mice with osteoarthritis will improve the integrity of their joints as well as the symptoms in terms of pain.

These studies will lead to the understanding of the role of WNT signalling in cartilage integrity and will allow us to target CaMKII to prevent cartilage degradation in osteoarthritis. This could not be done with non-specific WNT inhibition because the WNT signal also mediates positive effects, but could be feasible targeting CaMKII, which specifically mediates the damaging ones.

Finally, CaMKII is present in cartilage in two different forms. We will explore which sub-type of CaMKII is most important in cartilage breakdown. This part will be crucial for the design or selection of new drugs that block only the variant of CaMKII that causes cartilage breakdown.

These studies could give new insight into how WNT signalling works, may have important consequences in the therapy of osteoarthritis, and benefit the pharmaceutical industry, since several CaMKII inhibitors are already available and could be tested in pre-clinical and clinical studies. Finally, CaMKII and WNT signalling also play important roles (and are potential targets) in cancer, Alzheimer's disease, and cardiovascular diseases, and knowledge in this field will certainly benefit also these other fields of biomedical research.

Technical Summary

WNT signalling drives cartilage loss in osteoarthritis (OA) but could not be targeted therapeutically because it also mediates essential homeostatic mechanism.

We discovered that CaMKII mediates WNT-induced chondrocyte dedifferentiation and loss of extracellular matrix production, whereas beta catenin supports chondrocyte proliferation (Nalesso et al. 2011 JCB). New data showed that CaMKII phosphorylation was increased in human and experimental murine OA.

Therefore, blocking CaMKII will allow targeting only the pathogenic effects of WNT activation, without affecting its homeostatic functions.

Aim 1. To identify the transcriptional signature of WNT/CaMKII activation. To separate CaMKII-dependent from beta catenin-dependent transcriptional targets we will compare (microarrays) gene expression of chondrocytes treated with WNT3A in the presence of either the CaMKII inhibitor KN93 or DKK1 (inhibitor of the beta catenin-dependent pathway) or the inactive compound KN92. Results will be validated by PCR and WB using alternative CaMKII inhibitors.

Aim 2. To test whether CaMKII blockade improves experimental OA. OA will be induced by surgical destabilization of the medial meniscus. CaMKII will be inhibited by systemic administration of trifluoperazine or KN93 and cartilage and bone changes will be compared to the respective control groups. Preliminary time course and dose response experiment will be used to fine-tune experimental conditions. Since CaMKII mediates inflammatory pain and allodynia, we will explore if CaMKII blockade improves pain parameters.

Aim 3. To identify isoform-specificity/redundancy. Chondrocytes express CaMKII-gamma and -delta. Kinase activity assays following WNT-3A treatment and immunoprecipitation of the 2 isoforms will be used to assess which isoform is activated by WNT3A. Loss of function experiments with siRNA will be used to assess which isoform is responsible for the WNT-3A/CaMKII-dependent chondrocyte de-differentiation.

Planned Impact

The development of knowledge in the homeostatic mechanisms of adult tissues and cartilage in particular will benefit our ageing population who suffers from degenerative diseases including ostoearthritis, caused by the disruption of such mechanisms, and the phermaceutical industry in search of molecular targets to develop suitable therapeutic strategies. The costs to society are estimated over $185 billion yearly in the USA only for medical care (Kotzlar & Rizzo, Arthritis Rheum 2007). OA caused the loss of 36 million working days in 1999-2000 costing the economy nearly 3.2 billion in lost production (NICE 2012 OA guidance draft).
Our findings that the catabolic affects of WNT activation are specifically mediated by CaMKII offers an opportunity for specific targeting of the branch of WNT signalling responsible for the catabolic effects without harming its essential homeostatic functions such as chondrocyte proliferation. CaMKII blockade, therefore, may be a new tool for chondroprotection and cartilage regeneration. therefore the potential beneficiaries will include:

1) Pharmaceutical industry active in the field of Osteoarthritis and cartilage regeneration. These studies will provide proof of concept of efficacy of CaMKII blockade in OA and will produce specificity data for the design of more selective drugs (e.g. specific for the gamma or delta isoforms, or engineered to target joint tissues) which will reduce foreseeable side effects. PK/PD data obtained through this study will also facilitate translation in human trials.

2) Biotech companies involved in musculoskeletal tissue engineering. The pathway will be similar to that in point one, but in vitro treatment of cellular products (autologous chondrocyte transplantation or by use of mesenchymal stem cells) could be made readily possible and available for clinical practice and human trials.

3) The medical and orthopaedic community, and clinical investigators involved in osteoarthritis and cartilage regeneration

4) Patients with osteoarthritis and cartilage defects

5) Society at large for the spectacular direct and indirect costs related to osteoarthritis, in terms of medical costs, disability and absenteism.

6) veterinary surgeons involved in cartilage repair in race horses

How will they benefit from this research?

If the studies outlined in this application are funded, we will have full information of the biological effects mediated by WNT/CaMKII signalling in cartilage, and also proof of concept that CaMKII blockade in mice is an effective strategy to slow down OA progression. This will represent necessary information to design pre-clinical or early clinical studies to evaluate the effects of CaMKII blockade in osteoarthritis and joint surface repair. Our group have the expertise and the skills to run relevant animal models of osteoarthritis (destabilization of the medial meniscus in mice: this is currently the "gold standard"), and we have recently validated a novel model of joint surface injury and regeneration in mice (Eltawil, Osteoarthritis & Cartilage, 2009). On the other hand, several small compounds are being identified by the pharmaceutical industry with the capacity to block CaMKII in vivo. Trifluoperazine, investigated in this study, is used in humans as an antipsychotic. Although its side effects may be a problem when used for OA, adjustments to drug delivery may make its general side effects negligible.
Our Rheumatology department has a very active clinical trial unit currently running 15 trials.
We have the expertise, the infrastructure, and the contact to drive the translation of these exciting findings into benefit for the industry, society, and health and wellbeing in the elderly.
To facilitate the path to impact, we will ensure protection of intellectual property, and we will seek industrial partnership directly or through the mediation of QM Innovation ldt.

Publications

10 25 50
 
Description classification criteria for early osteoarthritis of the knee
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description A new drug delivery system for joint diseases
Amount £266,699 (GBP)
Organisation UCB Pharma 
Sector Private
Country United Kingdom
Start 10/2014 
End 10/2017
 
Description Modulation of calcium calmodulin kinase II (CaMKII) pathway as a new therapeutic strategy for the treatment of osteoarthritis
Amount £24,769 (GBP)
Organisation William Harvey Research Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 01/2017
 
Title Histomorphometric assessment of osteoarthritis in mice 
Description We have identified a more sensitive method to objectively quantify osteoarthritis in mice. This histomorphometrical method is described in Nalesso, G., Thomas, B. L., Sherwood, J. C., Yu, J., Addimanda, O., Eldridge, S. E., Dell'Accio, F. (2016). WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis. Annals of the Rheumatic Diseases. https://doi.org/10.1136/annrheumdis-2015-208577. The increased sensitivity and consistency significantly increases the po 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2016 
Provided To Others? Yes  
Impact The increased sensitivity and consistency significantly increases the power compared to traditional scoring system thereby reducing the use of experimental animals. In addition, compared to other methods, which are subjective in nature, this method is objective and more reliable. 
 
Description A new delivery system in arthritis 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution The experiments are run in my laboratory by one of my post-doc.
Collaborator Contribution UCB are funding a project designed by us to develop a new delivery system for cartialge. Half of the grant has been awarded and the other half will be awarded depending on the results of the first half. In addition to the funding, UCB and some collaborators at MIT are providing additional expertise and are performing some of the experiments.
Impact The outcome of this collaboration is confidential.
Start Year 2014
 
Description Peter van de Kraan 
Organisation Radboud University Nijmegen
Country Netherlands 
Sector Academic/University 
PI Contribution Successful award of the UK regenerative Medicine Platform Application for additional 2 grants to Arthritis Research UK This group will contribute technology for the generation of a conditional transgenic.
Collaborator Contribution Successful award of the UK regenerative Medicine Platform Application for additional 2 grants to Arthritis Research UK We contribute knowledge in the signalling mechanisms that drive homeostatic responses in Osteoarthritis and animal models.
Impact Successful award of the UK regenerative Medicine Platform and derived papers including 1 Eldridge S, Nalesso G, Ismail H, et al. Agrin mediates chondrocyte homeostasis and requires both LRP4 and a-dystroglycan to enhance cartilage formation in vitro and in vivo. Ann Rheum Dis 2015;:annrheumdis - 2015-207316. doi:10.1136/annrheumdis-2015-207316 2 Nalesso G, Thomas B, Sherwood J, et al. WNT16 antagonizes excessive canonical WNT activation and protects cartilage in osteoarthritis. Ann Rheum Dis 2016 in press
Start Year 2014
 
Description jessica bertrand 
Organisation University Hospital Magdeburg
Country Germany 
Sector Hospitals 
PI Contribution studies on ROR2 KO. We put the animals in models of osteoarthritis
Collaborator Contribution They bred the animals and aged them
Impact none yet
Start Year 2018
 
Description Participation as scientist to the "Research into Arthritis Summer BBQ" 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact Scientists at the William Harvey Research Institute describe their science to the general public (patients, children, general public). Subsequently the scientists took them through a tour in the labs and answered questions while having a barbecue.
Year(s) Of Engagement Activity 2017
 
Description Patient engagement Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact In this meeting we presented our results in the context of the Centre of Excellence for osteoarthritis Pathogenesis Oxford and discussed further research plans.
Year(s) Of Engagement Activity 2020
 
Description Talk at fund-raising event Arthritis Research UK - Farnham branch - "REPAIRING CARTILAGE USING TE PATIENT'S OWN MECHANISMS" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Supporters
Results and Impact The event took place in Farnham on 13-5-2014. I was invited by AR-UK. About 80 fund raisers attended.

I received several emails of congratulations and I was told that, before, they were not aware that AR-UK and MRC were funding this research in regenerative medicine. In fact most of them were not aware of regenerative medicine in the musculoskeletal system and found this field exciting.
Year(s) Of Engagement Activity 2014