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.

Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex.

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.

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.

Inhibition of the mechanistic target of rapamycin induces cell survival via MAPK in tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is a genetic disorder that cause tumors to form in many organs. These lesions may lead to epilepsy, autism, developmental delay, renal, and pulmonary failure. Loss of function mutations in TSC1 and TSC2 genes by aberrant activation of the mechanistic target of rapamycin (mTORC1) signaling pathway are the known causes of TSC. Therefore, targeting mTORC1 becomes a most available therapeutic strategy for TSC. Although mTORC1 inhibitor rapamycin and Rapalogs have demonstrated exciting results in the recent clinical trials, however, tumors rebound and upon the discontinuation of the mTORC1 inhibition. Thus, understanding the underlying molecular mechanisms responsible for rapamycin-induced cell survival becomes an urgent need. Identification of additional molecular targets and development more effective remission-inducing therapeutic strategies are necessary for TSC patients. RESULTS: We have discovered an Mitogen-activated protein kinase (MAPK)-evoked positive feedback loop that dampens the efficacy of mTORC1 inhibition. Mechanistically, mTORC1 inhibition increased MEK1-dependent activation of MAPK in TSC-deficient cells. Pharmacological inhibition of MAPK abrogated this feedback loop activation. Importantly, the combinatorial inhibition of mTORC1 and MAPK induces the death of TSC2-deficient cells. CONCLUSIONS: Our results provide a rationale for dual targeting of mTORC1 and MAPK pathways in TSC and other mTORC1 hyperactive neoplasm.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Rapalog resistance is associated with mesenchymal-type changes in Tsc2-null cells.

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased ß-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

Activation of choline kinase drives aberrant choline metabolism in esophageal squamous cell carcinomas.

Esophageal squamous cell carcinoma (ESCC) is a major health threat worldwide. Research focused on molecular events associated with ESCC carcinogenesis for diagnosis, treatment and prevention is needed. Our goal is to discover novel biomarkers and investigate the underlying molecular mechanisms of ESCC progression by employing a global metabolomic approach. Sera from 34 ESCC patients and 32 age and sex matched healthy controls were profiled using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS). We identified 120 differential metabolites in ESCC patient serums compared to healthy controls. Several amino acids, serine, arginine, lysine and histidine were significantly changed in ESCC patients. Most importantly, we found dysregulated lipid metabolism as an important characteristic in ESCC patients. Several free fat acids (FFA) and carnitines were found down-regulated in ESCC patients. Choline was significantly increased and phosphatidylcholines (PC) were significantly decreased in ESCC serum. The high expression of choline and low expression of total PC in patient serum were associated with the high expression of choline kinase (Chok) and activated Kennedy pathway in ESCC cells. Chok expression can serve as a significant biomarker for ESCC prognosis. In conclusion, metabolite profiles in the ESCC patient serum were significantly different from those in the healthy controls. Phosphatidylcholines and Chok, the key enzyme in the PC metabolism pathway, may serve as novel biomarkers for ESCC.

Proapoptotic protein Bim attenuates estrogen-enhanced survival in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing TSC2 mutations migrate to the lungs, proliferate, and cause cystic remodeling. The female predominance indicates that estrogen plays a critical role in LAM pathogenesis, and we have proposed that estrogen promotes LAM cell metastasis by inhibition of anoikis. We report here that estrogen increased LAM patient-derived cells' resistance to anoikis in vitro, accompanied by decreased accumulation of the proapoptotic protein Bim, an activator of anoikis. The resistance to anoikis was reversed by the proteasome inhibitor, bortezomib. Treatment of LAM patient-derived cells with estrogen plus bortezomib promoted anoikis compared with estrogen alone. Depletion of Bim by siRNA in TSC2-deficient cells resulted in anoikis resistance. Treatment of mice with bortezomib reduced estrogen-promoted lung colonization of TSC2-deficient cells. Importantly, molecular depletion of Bim by siRNA in Tsc2-deficient cells increased lung colonization in a mouse model. Collectively, these data indicate that Bim plays a key role in estrogen-enhanced survival of LAM patient-derived cells under detached conditions that occur with dissemination. Thus, targeting Bim may be a plausible future treatment strategy in patients with LAM.

Rapamycin-resistant poly (ADP-ribose) polymerase-1 overexpression is a potential therapeutic target in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.

Smooth muscle hypertrophy following partial bladder outlet obstruction is associated with overexpression of non-muscle caldesmon.

Partial bladder outlet obstruction (PBOO) induces remodeling of urinary bladder smooth muscle (detrusor). We demonstrate an increase in bladder wall mass, muscle bundle size, and a threefold increase in the cross-sectional area of detrusor myocytes following PBOO in male New Zealand White rabbits compared to that of controls. Some bladders with detrusor hypertrophy function close to normal (compensated), whereas others were dysfunctional (decompensated), showing high intravesical pressure, large residual urine volume, and voiding difficulty. We analyzed the expression of smooth muscle-specific caldesmon (h-CaD) and non-muscle (l-CaD) by Western blotting, RT-PCR, and real-time PCR. The expression of l-CaD is increased significantly at the mRNA and protein levels in the decompensated bladders compared to that of normal and compensated bladders. The CaD was also co-localized with myosin containing cytoplasmic fibrils in cells dissociated from obstructed bladders and cultured overnight. Our data show that the inability of decompensated bladders to empty, despite detrusor hypertrophy, is associated with an overexpression of l-CaD. The level of l-CaD overexpression might be a useful marker to estimate the degree of detrusor remodeling and contractile dysfunction in PBOO.