Dissecting mechanisms of alveolar repair failure and lung destruction in lymphangioleiomyomatosis

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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.