Thursday, April 3, 2025
4/3/2025
Cellular determinants of responsiveness to mTOR inhibitors in LAM - Project Summary Abstract Lymphangioleiomyomatosis (LAM) is a destructive lung disease associated with cystic lung remodeling and progressive respiratory failure. LAM is caused by TSC1 or TSC2 germline mutations that activate the mechanistic target of rapamycin complex 1 (mTORC1). Sirolimus, an mTORC1 inhibitor, is the only FDA-approved drug for LAM, stabilizes lung function and improves symptoms in LAM patients, but disease progression resumes when sirolimus is discontinued and some patients do not respond to therapy. Despite the important clinical observations of distinct therapeutic responses to sirolimus in LAM, the cellular mechanisms underlying sirolimus insensitivity are not understood. The objective of this proposal is to elucidate the mechanisms underlying sirolimus insensitivity to improve LAM treatment. This proposal is supported by novel preliminary data derived from both bioinformatics and laboratory findings. Our single cell RNA-sequencing analysis identified a sub- population of cells that are insensitive to sirolimus in heterogenous cells from Tsc2-null xenografts on sirolimus. The sub-population of cells was characterized by the increased expression of YAP-WNT pathway genes and stemness cell markers. The findings were validated using ELT3-245, a Tsc2-null sirolimus-resistant cell line. In addition, we discovered that YAP-WNT activated downstream target gene expression independently of mTORC1 in LAM lung explants and LAM cells in vitro. Our central hypothesis is that the activation of YAP-WNT crosstalk in LAM lung cells promotes the “LAM cell stemness state” that drives sirolimus insensitivity. We postulate the combination of sirolimus and YAP/WNT antagonists will overcome the primary cytostatic effects and enhance sensitivity to sirolimus. Three specific aims are proposed: Aim 1. Identify the molecular mechanisms underlying sirolimus insensitivity in LAM. Aim 2. Evaluate the role of YAP-WNT pathways on genes and processes underlying sirolimus insensitivity in vitro. Aim 3. Determine the effect of YAP-WNT inhibition on restoration of sirolimus sensitivity in LAM cell survival and lung remodeling in vivo. The completion of this proposal will provide for the first time: 1) a full spectrum of genomic/epigenomic resources of differential sirolimus responses in LAM. 2) molecular determinants of acquired sirolimus insensitivity and unique LAM cell subtype insensitive to sirolimus, 3) molecular insights into the role of YAP-WNT crosstalk in sirolimus insensitivity, and 4) preclinical proof-of-principle evidence for a novel regimen of YAP-WNT suppression in restoration of sirolimus sensitivity and inducing remission. Innovative concepts of direct clinical relevance on YAP-WNT crosstalk leading to LAM cell stemness state and sirolimus insensitivity will be assessed at single cell resolution coupled with preclinical models using non-invasive optical imaging to longitudinally monitor treatment responses in mouse lungs. Our work is highly significant and has positive impact as a comprehensive understanding of the molecular mechanisms underlying sirolimus insensitivity will reveal novel targets and inform remission-inducing therapeutic strategies for LAM patients and other mTORC1-hyperactive diseases.
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