Project Summary Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. We profiled a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM- like phenotype. Our study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype. Based on these data, we propose to test our hypothesis that mTORC1 hyperactivation in LAM cells dysregulates mTORC1-WNT signaling crosstalk which drives cystic airspace enlargement due to chronic activation of resident mesenchymal and alveolar epithelial cells. Our translational hypothesis states that dampening hyperactive mTORC1 and WNT signaling to basal physiological levels represents a potential opportunity to enhance the efficacy of rapalogs and potentially provide novel therapy for LAM patients. To test our hypothesis, we will specifically ask in: Aim 1: How does the small subset of LAM cells induce global pathological changes in the lung centered around lung mesenchymal cell hub? Aim 2: Does activation of mTORC1-WNT/?-catenin pathways in LAM lung mesenchyme dysregulate alveolar epithelial cell fitness? Aim 3: Will combined therapeutic targeting of the mTORC1-WNT/?-catenin pathways provide new treatment opportunities for LAM? The proposed studies will yield not only essential new insights into the role of TSC2-dependent mTORC1 activation and WNT signaling in LAM but also advance our understanding of how LAM lung mesenchymal cells orchestrate cystic lung damage. Our translational studies may also provide novel strategies for therapeutic intervention targeting hyperactive mTORC1 and WNT signaling pathways, which have not been tested for treatment of LAM.