Dissecting mechanisms of alveolar repair failure and lung destruction in lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare, progressive and incurable disease causing respiratory failure, lymphatic abnormalities and renal tumours which almost exclusively affects women. LAM arises due to loss of function of genes which control mTOR, a key cellular regulator in a specific cell type called the LAM cell. Constant mTOR activation allows LAM cells to multiply, survive in adverse conditions and invade the lungs and lymphatic tissue to cause tissue damage. In LAM, the lungs are continually damaged leading to progressive breathlessness and lung collapse, but the mechanisms causing this lung damage are not understood. The lungs are continually exposed to harmful chemicals and particles in the environment and are normally able to repair low level damage as it occurs. However due to aging or disease, these repair mechanisms become less efficient resulting in impaired lung function. In this work we will test the idea that senescence, a biological process that normally reduces the capacity of abnormal cells which develop during aging, occurs prematurely in LAM as a result of mTOR activation. We think that senescent LAM cells produce factors that induce senescence in surrounding cells, including alveolar type II cells which are normally responsible for replacing damaged alveolar epithelial cells. This mTOR induced senescence therefore reduces alveolar repair contributing to lung damage in LAM. We have already shown that cells within both LAM lung nodules and the surrounding alveoli express multiple genes and protein markers associated with senescence. In our animal model of LAM, senescence markers increase over time and are associated with progressive lung damage. In this work we will systematically examine how mTOR activation leads to senescence in LAM. To do this we will examine alveolar epithelial cell gene expression in LAM and control lungs using single cell RNA sequencing which allows measurement of gene expression in individual cell populations from LAM lungs taken at lung transplants. To study early LAM we will also measure gene expression in alveolar cells and LAM nodules by laser capture microdissection in diagnostic biopsy tissue to isolate specific cell populations. By using gene expression analysis tools we will examine which signaling pathways are active in epithelial cells in LAM and how these relate to cell death and repair. Next, using upstream regulator analysis, a tool which predicts which mediators result in specific gene changes we will examine which soluble proteins from LAM nodules affect epithelial cell repair and how this is related to mTOR activation. Using 40 individual lung biopsies with linked clinical data we will confirm these gene changes in human tissue and importantly, how these proteins are related to duration of disease and disease progression measured by prospective loss of lung function. Once we have understood which LAM cell derived factors result in reduced alveolar repair we will use novel 3D tissue culture systems we have developed to study how these processes are regulated and how they affect alveolar cell repair in vitro by using drugs or gene editing to block individual proteins. We will then examine these processes in a mouse model of LAM we have developed in our lab. The model will allow us to examine these changes in a whole organism and assess how blocking key proteins and processes, including mTOR activation and senescence affects lung damage. These experiments will shed new light on LAM and other lung diseases where mTOR activation contributes to lung damage. Using these data to understand how lung damage occurs we hope to improve treatments for LAM and potentially other destructive lung diseases.

Lymphangioleiomyomatosis (LAM) is a rare lung disease. Loss of TSC2 causes mTOR activation in LAM cells which accumulate in the lungs causing cysts by an unknown mechanism. We hypothesise that cysts form when mTOR activation causes LAM cell senescence in turn inducing senescence in surrounding alveolar type II (ATII) cells reducing alveolar repair. Using single cell sequencing we have shown that ATII cells in LAM express a senescence gene signature and in both human LAM and an animal model, senescence protein markers parallel lung damage. In the proposed work we will systematically examine how senescence leads to lung damage in LAM. We will examine alveolar epithelial cell gene transcription using single cell RNA sequencing of LAM lung cell populations from lung transplants and study early LAM biopsies by laser capture microdissection / RNA sequencing. Using gene expression and upstream regulator analysis tools we will examine cell death and repair pathways in epithelial cells and how they are regulated by LAM nodule derived proteins. Using LAM biopsies with linked clinical data we will confirm how these proteins are related to disease duration and disease activity measured by prospective loss of lung function. We will then study how senescence is regulated in vitro using novel 3D LAM nodule spheroids co-cultured with alveolar epithelial cells with individual mediators inhibited using small molecules, blocking antibodies and CRISPRcas9. We will validate these findings and test pharmacologic strategies on senescence, air space enlargement and nodule size in an immunocompetent murine TSC2 null xenograft model. Drugs affecting mTOR mediated senescence including metformin, resveratrol and mediators identified above amenable to intervention will be compared with rapalogues. These experiments will inform how lung damage occurs in LAM and other lung diseases where mTOR activation occurs, with the aim of improving treatment of LAM and potentially other lung diseases.

Publicising the project to maximise clinical resources.
The UK Centre for lymphangioleiomyomatosis (LAM) has one of the largest cohorts of patients with LAM under active follow up in the world. Women with LAM are recruited to the clinical study through the LAM centre. Making patients aware of the research protocol and the benefits of contributing will be one of our first impact initiatives. We have already had the grant proposal reviewed by patients from LAM Action, the UK LAM patients' charity who are positive about the study. The LAM research group in Nottingham work closely with the patients group in their research and the team regularly present both upcoming studies and research results to the UK LAM patients meeting held in Nottingham once a year, and also through patient newsletter distributed by LAM Action. The LAM Centre clinical day, held yearly, will also focus on maximising patient recruitment.

Ensuring a platform to develop clinical studies based on our findings
In the longer term, the wider LAM research and patient community will hear the study outcomes, hopefully with a view to taking forward positive findings in clinical trials. This will initially comprise safety and tolerability studies and in the future randomised studies examining the efficacy of senesence inhibitors, and senolytics as alternatives or adjuncts to mTOR inhibitors to prevent lung function decline in LAM. In addition to the standard presentations of scientific research at appropriate International conferences, dissemination of research data for rare diseases must aim to facilitate the necessary collaborations for follow up studies. As it is likely that definitive intervention studies would require a multi-national randomised controlled trials and we will ensure that groups with the infra-structure and willingness to contribute will be targeted for dissemination of our findings. A clinical trial group would be likely to include the north American LAM clinics with the input of the pharmaceutical industry and other relevant stakeholders. To this end, data and future aims will be presented at the International LAM Research Conference, the American Thoracic Society and appropriate European rare disease groupings including EuroLAM. We have strong existing collaborations with these groups but will disseminate our findings appropriately to take the studies forward in future.
Patients with LAM are keen to participate in research and are generally knowledgeable about their disease and upcoming treatments. Our findings will be presented to patients through the American and UK and European LAM patients' groupings as described above. Incorporating patients in these plans is important as there is a strong history of patient advocacy for research in LAM particularly and rare diseases generally.

mTOR driven senescence in pathological tissue remodelling other than LAM.
The role of senescence and alveolar repair failure in lung destruction including emphysema has been hinted at in animal studies. Should our findings suggest a role for mTOR driven senescence in pathological lung remodelling in other destructive lung diseases we will consider this and discuss with appropriate stakeholders including researchers with interest in COPD and possibly cancer biologists interested in stromal / neoplastic cell interactions. We are well placed to perform exploratory clinical studies as principal investigators at the Nottingham BRC and as co-lead of the COPD theme we have access to primary COPD and control derived fibroblasts and other appropriate reagents. As senolytic drugs are in phase 3 studies for various indications we would expect significant interest for these indications from the pharmaceuticals industry with whom we will liaise through existing collaborations and contacts.