Dysregulation of sphingolipid metabolism and signaling in LAM

Project Summary Abstract Lymphangioleiomyomatosis (LAM) is a devastating rare lung disease almost exclusively affecting young women. LAM is characterized by smooth muscle cell growth and emphysema-like lung destruction. LAM is associated with mutations in the TSC2 resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). Although the mTORC1 inhibitor, Sirolimus, stabilizes lung function and improves symptoms in some LAM patients, lung function declines after drug discontinuation and the long-term safety and efficacy of the drug are unknown. There is therefore an urgent unmet need to understand disease-promoting mechanisms for development of optimal interventions. Despite many advances in understanding the role of mTORC1-dependent pathways in LAM progression, the fundamental mechanisms underlying the female predominance of LAM primarily are not understood. In the Multicenter International LAM Efficacy of Sirolimus (MILES) trial, lung function declined five times more rapidly in premenopausal than postmenopausal patients, suggesting that estrogen plays a major role in LAM progression. Considering the actions of estrogen may be mediated by changes in sphingolipid metabolism, our hypothesis is that estrogen promotes sphingolipid metabolism in TSC2-deficient cells, leading to abnormal LAM lung cell growth and emphysema-like destruction of lung parenchyma and lung function decline. In Aim 1, we will use biochemical and single-cell multiome approaches to determine the molecular mechanisms through which TSC2 inactivation and estrogen promote sphingolipid- mediated cell survival. In Aim 2, we will determine the impact of pharmacologic suppression of ASAH1, S1PR1/3, ERα/β, or PR, on E2-induced lung remodeling in vivo. We postulate that that the combined suppression of sphingolipid signaling with Fingolimod, an S1PR1/3 modulator, or estrogen receptor inhibition with Faslodex or a SERM Raloxifene, or PR suppression with Mifepristone, and Sirolimus will, unlike cytostatic treatments involving Sirolimus alone, induce regression of E2-promoted lung remodeling and attenuate lung function decline. In Aim 3, we will test the hypothesis that the combinatorial treatment with 17a, Fingolimod, Faslodex, Raloxifene, or Mifepristone, plus Sirolimus will be well-tolerated and more effective than single agent in a uterine-specific Tsc2 knockout mouse model that closely resembles the human LAM. We will utilize innovative, state-of-the-art technology including single-cell multiome to identify estrogen/progesterone-regulated transcription factor activation, epigenomic alterations, and non-invasive optical and MRI imaging. The significance of this project is that it will reveal for the first time the combined roles of sphingolipid metabolism and E2 in LAM progression. This study will have high impact because current therapies for LAM are cytostatic and limited to stabilizing lung function. As Fingolimod and anti-hormonal compounds are already FDA approved for the treatment of multiple sclerosis and breast cancer, these cell-based and preclinical studies could be rapidly translated into human clinical trials for patients with LAM and other mTORC1 hyperactive diseases.