Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Glutaredoxin-1 promotes lymphangioleiomyomatosis progression through inhibiting Bim-mediated apoptosis via COX2/PGE2/ERK pathway.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease, characterized by progressive cyst formation and respiratory failure. Clinical treatment with the mTORC1 inhibitor rapamycin could relieve partially the respiratory symptoms, but not curative. It is urgent to illustrate the fundamental mechanisms of TSC2 deficiency to the development of LAM, especially mTORC1-independent mechanisms. Glutaredoxin-1 (Glrx), an essential glutathione (GSH)-dependent thiol-oxidoreductase, maintains redox homeostasis and participates in various processes via controlling protein GSH adducts. Redox signalling through protein GSH adducts in LAM remains largely elusive. Here, we demonstrate the underlying mechanism of Glrx in the pathogenesis of LAM. METHODS: 1. Abnormal Glrx expression in various kinds of human malignancies was identified by the GEPIA tumour database, and the expression of Glrx in LAM-derived cells was detected by real-time quantitative reverse transcription (RT-qPCR) and immunoblot. 2. Stable Glrx knockdown cell line was established to evaluate cellular impact. 3. Cell viability was determined by CCK8 assay. 4. Apoptotic cell number and intracellular reactive oxygen species (ROS) level were quantified by flow cytometry. 5. Cox2 expression and PGE2 production were detected to clarify the mechanism of Bim expression modulated by Glrx. 6. S-glutathionylated p65 was enriched and detected by immunoprecipitation and the direct regulation of Glrx on p65 was determined. 7. The xenograft animal model was established and photon flux was analyzed using IVIS Spectrum. RESULTS: In LAM, TSC2 negatively regulated abnormal Glrx expression and activation in a mTORC1-independent manner. Knockdown of Glrx increased the expression of Bim and the accumulation of ROS, together with elevated S-glutathionylated proteins, contributing to the induction of apoptotic cell death and inhibited cell proliferation. Knockdown of Glrx in TSC2-deficient LAM cells increased GSH adducts on nuclear factor-kappa B p65, which contributed to a decrease in the expression of Cox2 and the biosynthesis of PGE2. Inhibition of PGE2 metabolism attenuated phosphorylation of ERK, which led to the accumulation of Bim, due to the imbalance of its phosphorylation and proteasome degradation. In xenograft tumour models, knockdown of Glrx in TSC2-deficient LAM cells inhibited tumour growth and increased tumour cell apoptosis. CONCLUSIONS: Collectively, we provide a novel redox-dependent mechanism in the pathogenesis of LAM and propose that Glrx may be a beneficial strategy for the treatment of LAM or other TSC-related diseases.

Lymphangioleiomyomatosis: where endocrinology, immunology and tumor biology meet.

Abstract: Lymphangioleiomyomatosis (LAM) is a cystic lung disease found almost exclusively in genetic females and caused by small clusters of smooth muscle cell tumors containing mutations in one of the two tuberous sclerosis genes (TSC1 or TSC2). Significant advances over the past 2-3 decades have allowed researchers and clinicians to more clearly understand the pathophysiology of LAM, and therefore better diagnose and treat patients with this disease. Despite substantial progress, only one proven treatment for LAM is used in practice: mechanistic target of rapamycin complex 1 (mTORC1) inhibition with medications such as sirolimus. While mTORC1 inhibition effectively slows LAM progression in many patients, it is not curative, is not effective in all patients, and can be associated with significant side effects. Furthermore, the presence of established and accurate biomarkers to follow LAM progression is limited. That said, discovering additional diagnostic and treatment options for LAM is paramount. This review will describe recent advances in LAM research, centering on the origin and nature of the LAM cell, the role of estrogen in LAM progression, the significance of melanocytic marker expression in LAM cells, and the potential roles of the microenvironment in promoting LAM tumor growth. By appreciating these processes in more detail, researchers and caregivers may be afforded novel approaches to aid in the treatment of patients with LAM.

Cell survival pathways targeted in rare lung disease affecting women.

Novel drug targets are identified in lymphangioleiomyomatosis (LAM), a rare disease in women. These targets focus on uterine transcription factors necessary for LAM cell survival.

Single-cell multiomic analysis identifies a HOX-PBX gene network regulating the survival of lymphangioleiomyomatosis cells.

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women. LAM cells carry TSC1/TSC2 mutations, causing mTORC1 hyperactivation and uncontrolled cell growth. mTORC1 inhibitors stabilize lung function; however, sustained efficacy requires long-term administration, and some patients fail to tolerate or respond to therapy. Although the genetic basis of LAM is known, mechanisms underlying LAM pathogenesis remain elusive. We integrated single-cell RNA sequencing and single-nuclei ATAC-seq of LAM lungs to construct a gene regulatory network controlling the transcriptional program of LAM cells. We identified activation of uterine-specific HOX-PBX transcriptional programs in pulmonary LAMCORE cells as regulators of cell survival depending upon HOXD11-PBX1 dimerization. Accordingly, blockage of HOXD11-PBX1 dimerization by HXR9 suppressed LAM cell survival in vitro and in vivo. PBX1 regulated STAT1/3, increased the expression of antiapoptotic genes, and promoted LAM cell survival in vitro. The HOX-PBX gene network provides promising targets for treatment of LAM/TSC mTORC1-hyperactive cancers.

Estradiol Augments Tumor-Induced Neutrophil Production to Promote Tumor Cell Actions in Lymphangioleiomyomatosis Models.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease caused by smooth muscle cell-like tumors containing tuberous sclerosis (TSC) gene mutations and found almost exclusively in females. Patient studies suggest LAM progression is estrogen dependent, an observation supported by in vivo mouse models. However, in vitro data using TSC-null cell lines demonstrate modest estradiol (E2) responses, suggesting E2 effects in vivo may involve pathways independent of direct tumor stimulation. We previously reported tumor-dependent neutrophil expansion and promotion of TSC2-null tumor growth in an E2-sensitive LAM mouse model. We therefore hypothesized that E2 stimulates tumor growth in part by promoting neutrophil production. Here we report that E2-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that E2 induces granulopoiesis via estrogen receptor α in male and female bone marrow cultures. With our novel TSC2-null mouse myometrial cell line, we show that factors released from these cells drive E2-sensitive neutrophil production. Last, we analyzed single-cell RNA sequencing data from LAM patients and demonstrate the presence of tumor-activated neutrophils. Our data suggest a powerful positive feedback loop whereby E2 and tumor factors induce neutrophil expansion, which in turn intensifies tumor growth and production of neutrophil-stimulating factors, resulting in continued TSC2-null tumor growth.

Lymphangioleiomyomatosis: a metastatic lung disease.

Lymphangioleiomyomatosis (LAM) is a rare disease affecting women, caused by somatic mutations in the TSC1 or TSC2 genes, and driven by estrogen. Similar to many cancers, it is metastatic, primarily to the lung. Despite its monogenetic nature, like many cancers, LAM is a heterogeneous disease. The cellular constituents of LAM are very diverse, including mesenchymal, epithelial, endothelial, and immune cells. LAM is characterized by dysregulation of many cell signaling pathways, distinct populations of LAM cells, and a rich microenvironment, in which the immune system appears to play an important role. This review delineates the heterogeneity of LAM and focuses on the metastatic features of LAM, the deregulated signaling mechanisms and the tumor microenvironment. Understanding the tumor-host interaction in LAM may provide insights into the development of new therapeutic strategies, which could be combinatorial or superlative to Sirolimus, the current U.S. Food and Drug Administration-approved treatment.

Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM).

Lymphangioleiomyomatosis (LAM), a progressive pulmonary disease exclusively affecting females, is caused by defects or mutations in the coding gene tuberous sclerosis complex 1 (TSC1) or TSC2, causing the mammalian target of rapamycin complex 1 (mTORC1) activation and autophagy inhibition. Clinically, rapamycin shows limited cytocidal effects, and LAM recurs after drug withdrawal. In this study, we demonstrated that TSC2 negatively regulated the sphingolipid metabolism pathway and the expressions of sphingosine kinase 1 (SPHK1) and sphingosine-1-phosphate receptor 3 (S1PR3) were significantly elevated in LAM patient-derived TSC2-deficient cells compared to TSC2-addback cells, insensitive to rapamycin treatment and estrogen stimulation. Knockdown of SPHK1 showed reduced viability, migration and invasion in TSC2-deficient cells. Selective SPHK1 antagonist PF543 potently suppressed the viability of TSC2-deficient cells and induced autophagy-mediated cell death. Meanwhile, the cognate receptor S1PR3 was identified to mediating the tumorigenic effects of sphingosine-1-phosphate (S1P). Treatment with TY52156, a selective antagonist for S1PR3, or genetic silencing using S1PR3-siRNA suppressed the viability of TSC2-deficient cells. Both SPHK1 and S1PR3 inhibitors markedly exhibited antitumor effect in a xenograft model of TSC2-null cells, restored autophagy level, and triggered cell death. Together, we identified novel rapamycin-insensitive sphingosine metabolic signatures in TSC2-null LAM cells. Therapeutic targeting of aberrant SPHK1/S1P/S1PR3 signaling may have potent therapeutic benefit for patients with TSC/LAM or other hyperactive mTOR neoplasms with autophagy inhibition.

Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

LAM Cells as Potential Drivers of Senescence in Lymphangioleiomyomatosis Microenvironment.

Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-ß galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-ß galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.

Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis.

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

ETV2 regulates PARP-1 binding protein to induce ER stress-mediated death in tuberin-deficient cells.

Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2) and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). Here, we report that E26 transformation-specific (ETS) variant transcription factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. ETV2 nuclear localization in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP) mRNA and protein expression, partially reversing the observed down-regulation of PARPBP expression induced by mTORC1 blockade during treatment with both Syk and mTORC1 inhibitors. In addition, silencing Etv2 or Parpbp in Tsc2-deficient cells induced ER stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2, lending support to the translational relevance of our findings. In conclusion, we report a novel ETV2 signaling axis unique to Syk inhibition that promotes a cytocidal response in Tsc2-deficient cells and therefore maybe a potential alternative therapeutic target in LAM.

Vasohibin-1 and -2 in pulmonary lymphangioleiomyomatosis (LAM) cells associated with angiogenic and prognostic factors.

Lymphangioleiomyomatosis (LAM) is a rare pulmonary neoplasm, clinically associated with dyspnea and respiratory failure. Current therapeutic modalities do not necessarily reach satisfactory outcome and novel therapeutic approaches are currently warranted. Therefore, in this study, we focused on vasohibin-1 (VASH1) and -2 (VASH2); VASH1 terminated and VASH2 promoted angiogenesis. In addition, both VASH1/2 were reported to influence the progression of various human malignancies. We first performed hierarchical clustering analysis to attempt to classify 36 LAM cases into three different clusters according to immunoreactivity of VASH1/2 and other angiogenic and prognostic factors of LAM; VEGFR1/2/3, p-mTOR, p-S6, p-4EBP, ERα, PgR, MMP2, and MMP9. The cluster harboring higher angiogenic factors had higher VASH1/2 status. VASH1 was significantly positively correlated with VEGFR2, MMP9, and p-mTOR (p-value <0.05), and VASH2 with both angiogenic and prognostic factors including VEGFR1, PgR, MMP9, p-mTOR, p-S6, and p-4EBP (p-value <0.05). Subsequent PCR array of angiogenic genes demonstrated that high VASH1 mRNA was significantly positively associated with the status of SPHK1 and TYPM, lower EGF and EFNB2 (p-value <0.05), and high VASH2 mRNA negatively with MMP2 (p-value <0.05). VASH1 was considered to be up-regulated by activation of angiogenesis, whereas VASH2 could influence the angiogenesis and progression of LAM.

Cross talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Cathepsin K is Superior to HMB45 for the Diagnosis of Pulmonary Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare cystic lung disease affecting predominantly young women. Classified as a low-grade malignant soft tissue neoplasm from the family of perivascular epithelioid cell (PEC) tumors or PEComas, it is characterized by a proliferation of abnormal smooth muscle-like cells (LAM cells), coexpressing myogenic and melanocytic markers, with HMB45 as the gold-standard immunohistochemical diagnostic marker. Cathepsin K, a papain-like cysteine protease with high matrix degrading activity, is commonly used in the pathologic diagnosis of other PEComa tumors, but there are few data regarding its expression in pulmonary LAM. This study compares the sensitivity of cathepsin K with that of HMB45 as immunohistochemical diagnostic markers for pulmonary LAM. Twenty-one (n=21) specimens of pulmonary LAM were retrieved from the archives of the Department of Pathology of the Cleveland Clinic. All cases were evaluated for protein expression of HMB45 and cathepsin K, on consecutive sections of formalin-fixed, paraffin-embedded tissue. The intensity and the total area of the immunostaining were quantified using an Aperio Scan Scope and analyzed with imaging software (Spectrum). Statistical analysis was performed using GraphPad software. The probability of a positive stained lesion on a transbronchial biopsy for each antibody was calculated. The percentage of LAM cells expressing cathepsin K was significantly higher than for HMB45 and overall expression was statistically significantly higher (P=0.0116). Our findings conclude that cathepsin K is a significantly more sensitive immunohistochemical marker than HMB45 in diagnosing pulmonary LAM.

Sirolimus Suppresses Phosphorylation of Cofilin and Reduces Interstitial Septal Thickness in Sporadic Lymphangioleiomyomatosis.

Sporadic lymphangioleiomyomatosis (S-LAM) is a rare lung disease characterized by the proliferation of smooth muscle-like LAM cells and progressive cystic destruction. Sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has a proven efficacy in patients with LAM. However, the therapeutic mechanisms of sirolimus in LAM remain unclear. We aimed to evaluate sirolimus-related lung parenchymal changes and the potential effect in LAM cells and modulating pathological cystic destruction. Lung specimens were examined for histopathological changes by HMB45 staining and compared the LAM patients treated with and without sirolimus. We detected the overexpression of mTOR, HMB45, and phosphorylation of cofilin (p-cofilin) in LAM patients. Sirolimus showed efficacy in patients with LAM, who exhibited a reduced expression of mTOR and p-cofilin as well as reduced interstitial septal thickness. In addition, sirolimus suppresses mTOR and p-cofilin, thus suppressing the migration and proliferation of LAM cells isolated from the patient's lung tissue. This study demonstrates that interstitial septal thickness, as determined by histological structural analysis. Sirolimus effectively reduced the expression of p-cofilin and interstitial septal thickness, which may be a novel mechanism by sirolimus. Moreover, we develop a new method to isolate and culture the LAM cell, which can test the possibility of medication in vitro and impact this current study has on the LAM field. The development of approaches to interfere with mTOR-cofilin1-actin signaling may result in an option for S-LAM therapy.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation.

Tuberous sclerosis complex (TSC) is caused by mutations of either the TSC1 or TSC2 tumor suppressor gene. TSC causes tumors of the brain, heart, kidney, skin and lymphangioleiomyomatosis (LAM). Here we report that the TSC2 protein physically binds to high-density lipoprotein binding protein (HDLBP), also called vigilin, a core stress granule (SG) protein, and that TSC2 localizes to SGs. SGs contain mRNAs and translation initiation complexes, and regulate gene expression by sequestering specific transcripts, thereby serving a cytoprotective role. TSC2 has never before been shown to localize to SGs and knocking down vigilin impacts SG translocation of TSC2. TSC2-deficient cells showed a striking increase in the number of SGs after thermal shock and arsenite treatment relative to Tsc2-expressing cells. Our findings also show that murine kidney lysates from a model of TSC have increased levels of SG components including G3BP1 and Caprin1. G3BP1 and Caprin are elevated in renal angiomyolipomas (a renal tumor common in patients with TSC) compared with control normal kidney. G3BP1 is also elevated in TSC-associated subependymal giant cell astrocytomas. We found that genetic inhibition of G3BP1 inhibits the proliferation of TSC2-deficient cells in vitro. Finally, in a mouse model of TSC, genetic inhibition of SGs suppresses cell growth, suggesting that targeting SGs may have efficacy in the therapy of TSC. IMPLICATIONS: This study demonstrates that TSC2 physically interacts with HDLBP/vigilin, a component of SGs, that TSC2 localizes to SG and that TSC2-deficient cells have more SGs, suggesting that SGs represent a novel therapeutic target in TSC.

Connectivity Map Analysis of a Single-Cell RNA-Sequencing -Derived Transcriptional Signature of mTOR Signaling.

In the connectivity map (CMap) approach to drug repositioning and development, transcriptional signature of disease is constructed by differential gene expression analysis between the diseased tissue or cells and the control. The negative correlation between the transcriptional disease signature and the transcriptional signature of the drug, or a bioactive compound, is assumed to indicate its ability to "reverse" the disease process. A major limitation of traditional CMaP analysis is the use of signatures derived from bulk disease tissues. Since the key driver pathways are most likely dysregulated in only a subset of cells, the "averaged" transcriptional signatures resulting from bulk analysis lack the resolution to effectively identify effective therapeutic agents. The use of single-cell RNA-seq (scRNA-seq) transcriptomic assay facilitates construction of disease signatures that are specific to individual cell types, but methods for using scRNA-seq data in the context of CMaP analysis are lacking. Lymphangioleiomyomatosis (LAM) mutations in TSC1 or TSC2 genes result in the activation of the mTOR complex 1 (mTORC1). The mTORC1 inhibitor Sirolimus is the only FDA-approved drug to treat LAM. Novel therapies for LAM are urgently needed as the disease recurs with discontinuation of the treatment and some patients are insensitive to the drug. We developed methods for constructing disease transcriptional signatures and CMaP analysis using scRNA-seq profiling and applied them in the analysis of scRNA-seq data of lung tissue from naïve and sirolimus-treated LAM patients. New methods successfully implicated mTORC1 inhibitors, including Sirolimus, as capable of reverting the LAM transcriptional signatures. The CMaP analysis mimicking standard bulk-tissue approach failed to detect any connection between the LAM signature and mTORC1 signaling. This indicates that the precise signature derived from scRNA-seq data using our methods is the crucial difference between the success and the failure to identify effective therapeutic treatments in CMaP analysis.