Tuberous sclerosis complex (TSC) is characterized by multisystem, low-grade neoplasia involving the lung, kidneys, brain, and heart. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting almost exclusively women. TSC and LAM are both caused by mutations in TSC1 and TSC2 that result in mTORC1 hyperactivation. Here, we report that single-cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid biosynthesis pathway. Accordingly, the expression of acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzymes controlling sphingolipid and ceramide metabolism, was significantly increased in TSC2-null cells. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids, and suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) resulted in markedly decreased TSC2-null cell viability. In vivo, 17a significantly decreased the growth of TSC2-null cell-derived mouse xenografts and short-term lung colonization by TSC2-null cells. Combined rapamycin and 17a treatment synergistically inhibited renal cystadenoma growth in Tsc2+/- mice, consistent with increased ASAH1 expression and activity being rapamycin insensitive. Collectively, the present study identifies rapamycin-insensitive ASAH1 upregulation in TSC2-null cells and tumors and provides evidence that targeting aberrant sphingolipid biosynthesis pathways has potential therapeutic value in mechanistic target of rapamycin complex 1-hyperactive neoplasms, including TSC and LAM.
Abstract Tuberous Sclerosis Complex (TSC) is disorder of multi-system benign neoplasia in the brain, heart, kidneys and lungs. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting exclusively women. Both are caused by mutations in TSC1 and TSC2 , resulting in mTORC1 hyperactivation. Single cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid pathway. Independent validation studies showed that acid ceramidase ( ASAH1 ) and dihydroceramide desaturase ( DEGS1 ), key enzyme for regulating sphingolipid and ceramide metabolism, were significantly increased in TSC2-null cells, and their expression and activity were rapamycin-insensitive. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids. Suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) markedly decreased the viability of TSC2-null cells. In vivo , 17a significantly decreased the growth of Tsc2-null cell derived mouse xenografts. When combined with rapamycin, 17a more strongly inhibited the progression of renal cystadenomas in Tsc2 +/- mice than either agent alone, evaluated by pathology and MRI. Collectively, our studies identify a rapamycin-insensitive disorder of sphingolipid metabolism in TSC2-null cells and tumors and validate the novel hypothesis that TSC2 regulates sphingolipid production and action via ASAH1. Targeting aberrant sphingolipid metabolism pathways may have therapeutic value in TSC and LAM, and possibly in mTORC1-hyperactive neoplasms.
OBJECTIVES/GOALS: Team science competencies are not well-defined for nonfaculty staff of Clinical Research Professionals (CRPs) who conduct research. Using an existing framework, our work has determined skills associated with team science competencies as related to CRPs. Our team also outlined examples of those skills on a fundamental, skilled, and advanced level. METHODS/STUDY POPULATION: The team consists of both CRPs and those working in the Team Science space. This team used a modified Delphi approach to determine the skills and leveling examples of each team science competency. The team broke into four groups and was assigned 3-4 competencies each. Each group determined skills needed to support (exhibit, promote) each competency and then described an example of this skill at the fundamental, skilled, and advanced levels. Once each group was finished with their assigned competencies, they were re-assigned to a different group for review and changes. Finally, team science and CRP experts reviewed the skills and levels. RESULTS/ANTICIPATED RESULTS: Our results are a rubric that defines 3-5 practical skills per described competency. These skills are needed to support and promote each competency as a CRP. An additional outcome from this work includes examples of each skill at the fundamental, skilled, and advanced levels in a CRP’s career. Each leveled example is described in a concise, actionable way using Bloom’s taxonomy. This rubric is meant to be easily understood, very useable and able to be used in conjunction with existing CRP competency frameworks. By using Bloom’s taxonomy, we set the stage for future educational programming in Team Science skill-building for clinical research professionals. DISCUSSION/SIGNIFICANCE: Team science concepts and competencies have been increasingly integrated into translational science teams. However, team science competencies related to CRPs have remained largely undefined. Our work helps to define these competencies for CRPs in a practical way. Our rubric fills gaps in, and builds on, existing CRP competency frameworks.
SUMMARY Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women that causes cystic lung remodeling and progressive respiratory failure. The source of LAM cells that invade the lung and the reasons that LAM targets women have remained elusive. We employed single cell and single nuclei RNA sequencing on LAM lesions within explanted LAM lungs, known to contain smooth muscle like cells bearing mTOR activating mutations in TSC1 or TSC2, and identified a unique population of cells that were readily distinguished from those of endogenous lung cells. LAM CORE cells shared closest transcriptomic similarity to normal uterus and neural crest. Immunofluorescence microscopy demonstrated the expression of LAM CORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers predicted to be secreted by LAM CORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAM CORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM. SIGNIFICANCE Present study identified a novel population of LAM CORE cells, which is likely originated from uterine neural crest; identified novel LAM cell-specific secretome proteins that hold promise as potential biomarkers and therapeutic targets. Advancing the understanding of LAM pathogenesis and metastasis model may yield broader insights into the biology of cancer.
Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.
