Publications by authors named "Elizabeth P Henske"

101 Publications

Interleukin-6 mediates PSAT1 expression and serine metabolism in TSC2-deficient cells.

Proc Natl Acad Sci U S A 2021 Sep;118(39)

Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either or Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti-IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.
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http://dx.doi.org/10.1073/pnas.2101268118DOI Listing
September 2021

Genetic risk assessment for hereditary renal cell carcinoma: Clinical consensus statement.

Cancer 2021 Aug 3. Epub 2021 Aug 3.

Massachusetts General Hospital Cancer Center, Boston, Massachusetts.

Background: Although renal cell carcinoma (RCC) is believed to have a strong hereditary component, there is a paucity of published guidelines for genetic risk assessment. A panel of experts was convened to gauge current opinions.

Methods: A North American multidisciplinary panel with expertise in hereditary RCC, including urologists, medical oncologists, clinical geneticists, genetic counselors, and patient advocates, was convened. Before the summit, a modified Delphi methodology was used to generate, review, and curate a set of consensus questions regarding RCC genetic risk assessment. Uniform consensus was defined as ≥85% agreement on particular questions.

Results: Thirty-three panelists, including urologists (n = 13), medical oncologists (n = 12), genetic counselors and clinical geneticists (n = 6), and patient advocates (n = 2), reviewed 53 curated consensus questions. Uniform consensus was achieved on 30 statements in specific areas that addressed for whom, what, when, and how genetic testing should be performed. Topics of consensus included the family history criteria, which should trigger further assessment, the need for risk assessment in those with bilateral or multifocal disease and/or specific histology, the utility of multigene panel testing, and acceptance of clinician-based counseling and testing by those who have experience with hereditary RCC.

Conclusions: In the first ever consensus panel on RCC genetic risk assessment, 30 consensus statements were reached. Areas that require further research and discussion were also identified, with a second future meeting planned. This consensus statement may provide further guidance for clinicians when considering RCC genetic risk assessment.

Lay Summary: The contribution of germline genetics to the development of renal cell carcinoma (RCC) has long been recognized. However, there is a paucity of guidelines to define how and when genetic risk assessment should be performed for patients with known or suspected hereditary RCC. Without guidelines, clinicians struggle to define who requires further evaluation, when risk assessment or testing should be done, which genes should be considered, and how counseling and/or testing should be performed. To this end, a multidisciplinary panel of national experts was convened to gauge current opinion on genetic risk assessment in RCC and to enumerate a set of recommendations to guide clinicians when evaluating individuals with suspected hereditary kidney cancer.
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http://dx.doi.org/10.1002/cncr.33679DOI Listing
August 2021

TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism.

Nat Commun 2021 07 12;12(1):4245. Epub 2021 Jul 12.

Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2 mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.
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http://dx.doi.org/10.1038/s41467-021-24499-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8275687PMC
July 2021

mTORC1 is a mechanosensor that regulates surfactant function and lung compliance during ventilator-induced lung injury.

JCI Insight 2021 Jul 22;6(14). Epub 2021 Jul 22.

Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, and.

The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation (MV) maintains gas exchange in patients with ARDS but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during MV impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during MV. Conversely, treatment with rapamycin at the time MV is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during MV. Our data demonstrate that, in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during MV exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.
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http://dx.doi.org/10.1172/jci.insight.137708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410036PMC
July 2021

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

Mol Cancer Res 2021 Aug 22;19(8):1389-1397. Epub 2021 Apr 22.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.

Tuberous sclerosis complex (TSC) is caused by mutations of either the or 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 . 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.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-1046DOI Listing
August 2021

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex.

Cancer Res 2021 04 16;81(8):2086-2100. Epub 2021 Feb 16.

Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2 mouse embryonic fibroblasts (MEF), with no impact on Tsc2 MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2 MEFs compared with Tsc2 MEFs, and treatment of Tsc2 MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2 MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137542PMC
April 2021

Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex.

Proc Natl Acad Sci U S A 2021 02;118(6)

Department of Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131;

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl/H exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.
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http://dx.doi.org/10.1073/pnas.2020190118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017711PMC
February 2021

MITF is a driver oncogene and potential therapeutic target in kidney angiomyolipoma tumors through transcriptional regulation of CYR61.

Oncogene 2021 01 20;40(1):112-126. Epub 2020 Oct 20.

Cancer Genetics Laboratory, Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by tumor development in multiple organs, including renal angiomyolipoma. Biallelic loss of TSC1 or TSC2 is a known genetic driver of angiomyolipoma development, however, whether an altered transcriptional repertoire contributes to TSC-associated tumorigenesis is unknown. RNA-seq analyses showed that MITF A isoform (MITF-A) was consistently highly expressed in angiomyolipoma, immunohistochemistry showed microphthalmia-associated transcription factor nuclear localization, and Chromatin immuno-Precipitation Sequencing analysis showed that the MITF-A transcriptional start site was highly enriched with H3K27ac marks. Using the angiomyolipoma cell line 621-101, MITF knockout (MITF.KO) and MITF-A overexpressing (MITF.OE) cell lines were generated. MITF.KO cells showed markedly reduced growth and invasion in vitro, and were unable to form xenografted tumors. In contrast, MITF.OE cells grew faster in vitro and as xenografted tumors compared to control cells. RNA-Seq analysis showed that both ID2 and Cysteine-rich angiogenic inducer 61 (CYR61) expression levels were increased in the MITF.OE cells and reduced in the MITF.KO cells, and luciferase assays showed this was due to transcriptional effects. Importantly, CYR61 overexpression rescued MITF.KO cell growth in vitro and tumor growth in vivo. These findings suggest that MITF-A is a transcriptional oncogenic driver of angiomyolipoma tumor development, acting through regulation of CYR61.
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http://dx.doi.org/10.1038/s41388-020-01504-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796905PMC
January 2021

mTORC1 hyperactivation in lymphangioleiomyomatosis leads to upregulation in type II pneumocytes: implications for COVID-19.

Eur Respir J 2021 02 11;57(2). Epub 2021 Feb 11.

Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1183/13993003.02737-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562695PMC
February 2021

Tumour predisposition and cancer syndromes as models to study gene-environment interactions.

Nat Rev Cancer 2020 09 29;20(9):533-549. Epub 2020 May 29.

Thoracic Oncology, University of Hawaii Cancer Center, Honolulu, HI, USA.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.
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http://dx.doi.org/10.1038/s41568-020-0265-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104546PMC
September 2020

Chromophobe renal cell carcinoma: New genetic and metabolic insights.

Urol Oncol 2020 08 20;38(8):678-681. Epub 2020 May 20.

Brigham and Women's Hospital, Boston, MA. Electronic address:

Chromophobe renal cell carcinoma (chRCC) represents 5% of all RCC. ChRCC appears to arise from the distal nephron, in contrast to clear cell RCC that arises from the proximal nephron. ChRCC is distinctive because the tumor cells contain abundant abnormal mitochondria, and frequently have loss of one copy of multiple chromosomes, with a low rate of somatic mutations. Here, we focus on recent discoveries related to genetic and metabolic factors that may promote the progression of chRCC and summarize how these findings may relate to the prognosis and treatment of chRCC.
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http://dx.doi.org/10.1016/j.urolonc.2020.04.035DOI Listing
August 2020

New Developments in the Pathogenesis of Pulmonary Cysts in Birt-Hogg-Dubé Syndrome.

Semin Respir Crit Care Med 2020 04 12;41(2):247-255. Epub 2020 Apr 12.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder caused by germline loss-of-function mutations in gene (FLCN). BHD is characterized by lower lobe-predominant pulmonary cysts with risk of pneumothorax, benign skin tumors (fibrofolliculomas), and renal cell carcinoma, often of an unusual chromophobe/oncocytic hybrid histology. The FLCN protein functions in multiple signaling and metabolic pathways including positive regulation of mechanistic target of rapamycin complex 1 (mTORC1) activity via FLCN's GTPase (GAP) activity for Rag C, positive regulation of Wnt signaling (in mesenchymal cells), and negative regulation of TFE3 nuclear localization. Therefore, FLCN-deficient cells are predicted to have reduced mTORC1 and Wnt activity and enhanced TFE3 activity. Folliculin also has functions in autophagy, mitochondrial biogenesis, cell-cell adhesion, 5' AMP activated protein kinase activity, and other pathways. The specific contributions of these pathways to the lung manifestations of BHD are largely unknown. This review is focused on the pulmonary manifestations of BHD, highlighting selected recent advances in elucidating the cellular functions of FLCN and current hypotheses related to the pathogenesis of cystic lung disease in BHD, including the "stretch hypothesis." We also discuss important knowledge gaps in the field, including the genetic, cellular and physical mechanisms of cyst pathogenesis, and the timing of cyst initiation, which may occur during lung development.
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http://dx.doi.org/10.1055/s-0040-1708500DOI Listing
April 2020

IMPDH inhibitors for antitumor therapy in tuberous sclerosis complex.

JCI Insight 2020 04 9;5(7). Epub 2020 Apr 9.

Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

Recent studies in distinct preclinical tumor models have established the nucleotide synthesis enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) as a viable target for antitumor therapy. IMPDH inhibitors have been used clinically for decades as safe and effective immunosuppressants. However, the potential to repurpose these pharmacological agents for antitumor therapy requires further investigation, including direct comparisons of available compounds. Therefore, we tested structurally distinct IMPDH inhibitors in multiple cell and mouse tumor models of the genetic tumor syndrome tuberous sclerosis complex (TSC). TSC-associated tumors are driven by uncontrolled activation of the growth-promoting protein kinase complex mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which is also aberrantly activated in the majority of sporadic cancers. Despite eliciting similar immunosuppressive effects, the IMPDH inhibitor mizoribine, used clinically throughout Asia, demonstrated far superior antitumor activity compared with the FDA-approved IMPDH inhibitor mycophenolate mofetil (or CellCept, a prodrug of mycophenolic acid). When compared directly to the mTOR inhibitor rapamycin, mizoribine treatment provided a more durable antitumor response associated with tumor cell death. These results provide preclinical support for repurposing mizoribine, over other IMPDH inhibitors, as an alternative to mTOR inhibitors for the treatment of TSC-associated tumors and possibly other tumors featuring uncontrolled mTORC1 activity.
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http://dx.doi.org/10.1172/jci.insight.135071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205253PMC
April 2020

Mesenchymal folliculin is required for alveolar development: implications for cystic lung disease in Birt-Hogg-Dubé syndrome.

Thorax 2020 06 1;75(6):486-493. Epub 2020 Apr 1.

The Saban Research Institute, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

Background: Pulmonary cysts and spontaneous pneumothorax are presented in most patients with Birt-Hogg-Dubé (BHD) syndrome, which is caused by loss of function mutations in the () gene. The pathogenic mechanisms underlying the cystic lung disease in BHD are poorly understood.

Methods: Mesenchymal was specifically deleted in mice or in cultured lung mesenchymal progenitor cells using a Cre/loxP approach. Dynamic changes in lung structure, cellular and molecular phenotypes and signalling were measured by histology, immunofluorescence staining and immunoblotting.

Results: Deletion of in mesoderm-derived mesenchymal cells results in significant reduction of postnatal alveolar growth and subsequent alveolar destruction, leading to cystic lesions. Cell proliferation and alveolar myofibroblast differentiation are inhibited in the knockout lungs, and expression of the extracellular matrix proteins Col3a1 and elastin are downregulated. Signalling pathways including mTORC1, AMP-activated protein kinase, ERK1/2 and Wnt-β-catenin are differentially affected at different developmental stages. All the above changes have statistical significance (p<0.05).

Conclusions: Mesenchymal Flcn is an essential regulator during alveolar development and maintenance, through multiple cellular and molecular mechanisms. The mesenchymal knockout mouse model provides the first in vivo disease model that may recapitulate the stages of cyst development in human BHD. These findings elucidate the developmental origins and mechanisms of lung disease in BHD.
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http://dx.doi.org/10.1136/thoraxjnl-2019-214112DOI Listing
June 2020

Tumors with TSC mutations are sensitive to CDK7 inhibition through NRF2 and glutathione depletion.

J Exp Med 2019 11 10;216(11):2635-2652. Epub 2019 Sep 10.

Cancer Genetics Laboratory, Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA

Tuberous sclerosis complex (TSC) is characterized by tumor development in the brain, heart, kidney, and lungs. In TSC tumors, loss of the TSC1/TSC2 protein complex leads to activation of mTORC1 with downstream effects on anabolism and cell growth. Because mTORC1 activation enhances mRNA transcription, we hypothesized that aberrant mTORC1 activation might confer TSC-null cell dependence on transcriptional regulation. We demonstrate that TSC1- or TSC2-null cells, in contrast to their wild-type counterparts, are sensitive to pharmacological inhibition of CDK7. Mechanistic studies revealed that CDK7 inhibition markedly reduces glutathione levels and increases reactive oxygen species due to reduced expression of NRF2 and glutathione biosynthesis genes. Treatment of both mice and a TSC1-null bladder cancer xenograft model with a CDK7 inhibitor showed marked reduction in tumor volume and absence of regrowth in the xenograft model. These results suggest that CDK7 inhibition is a promising therapeutic approach for treatment of TSC-associated tumors and cancers with mutations in either or .
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http://dx.doi.org/10.1084/jem.20190251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829598PMC
November 2019

Immunotherapy for Lymphangioleiomyomatosis and Tuberous Sclerosis: Progress and Future Directions.

Chest 2019 12 19;156(6):1062-1067. Epub 2019 Aug 19.

Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA. Electronic address:

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that T cells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating T cells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.
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http://dx.doi.org/10.1016/j.chest.2019.08.005DOI Listing
December 2019

Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ).

Oncogene 2019 12 16;38(49):7367-7383. Epub 2019 Aug 16.

Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.
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http://dx.doi.org/10.1038/s41388-019-0957-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895402PMC
December 2019

Alterations in Polyamine Metabolism in Patients With Lymphangioleiomyomatosis and Tuberous Sclerosis Complex 2-Deficient Cells.

Chest 2019 12 9;156(6):1137-1148. Epub 2019 Jul 9.

Pulmonary and Critical Care Medicine Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA.

Background: Lymphangioleiomyomatosis (LAM), a destructive lung disease that affects primarily women, is caused by loss-of-function mutations in TSC1 or TSC2, leading to hyperactivation of mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Rapamycin (sirolimus) treatment suppresses mTORC1 but also induces autophagy, which promotes the survival of TSC2-deficient cells. Based on the hypothesis that simultaneous inhibition of mTORC1 and autophagy would limit the availability of critical nutrients and inhibit LAM cells, we conducted a phase 1 clinical trial of sirolimus and hydroxychloroquine for LAM. Here, we report the analyses of plasma metabolomic profiles from the clinical trial.

Methods: We analyzed the plasma metabolome in samples obtained before, during, and after 6 months of treatment with sirolimus and hydroxychloroquine, using univariate statistical models and machine learning approaches. Metabolites and metabolic pathways were validated in TSC2-deficient cells derived from patients with LAM. Single-cell RNA-Seq was employed to assess metabolic enzymes in an early-passage culture from an LAM lung.

Results: Metabolomic profiling revealed changes in polyamine metabolism during treatment, with 5'-methylthioadenosine and arginine among the most highly upregulated metabolites. Similar findings were observed in TSC2-deficient cells derived from patients with LAM. Single-cell transcriptomic profiling of primary LAM cultured cells revealed that mTORC1 inhibition upregulated key enzymes in the polyamine metabolism pathway, including adenosylmethionine decarboxylase 1.

Conclusions: Our data demonstrate that polyamine metabolic pathways are targeted by the combination of rapamycin and hydroxychloroquine, leading to upregulation of 5'-methylthioadenosine and arginine in the plasma of patients with LAM and in TSC2-deficient cells derived from a patient with LAM upon treatment with this drug combination.

Trial Registry: ClinicalTrials.gov; No.: NCT01687179; URL: www.clinicaltrials.gov. Partners Human Research Committee, protocol No. 2012P000669.
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http://dx.doi.org/10.1016/j.chest.2019.05.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904859PMC
December 2019

Loss of FLCN inhibits canonical WNT signaling via TFE3.

Hum Mol Genet 2019 10;28(19):3270-3281

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/β inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/β inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression-two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/β inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/β inhibition. Finally, we found that a constitutively active β-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.
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http://dx.doi.org/10.1093/hmg/ddz158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859437PMC
October 2019

Allosteric and ATP-Competitive Inhibitors of mTOR Effectively Suppress Tumor Progression-Associated Epithelial-Mesenchymal Transition in the Kidneys of Tsc2 Mice.

Neoplasia 2019 08 14;21(8):731-739. Epub 2019 Jun 14.

Institute of Medical Genetics, Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. Electronic address:

In tuberous sclerosis (TSC)-associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2 mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2 mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.
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http://dx.doi.org/10.1016/j.neo.2019.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580094PMC
August 2019

The Codon 72 Polymorphism Contributes to TSC Tumorigenesis through the Notch-Nodal Axis.

Mol Cancer Res 2019 08 14;17(8):1639-1651. Epub 2019 May 14.

Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Science Center, Abilene, Texas.

We discovered that 90.3% of patients with angiomyolipomas, lymphangioleiomyomatosis (LAM), and tuberous sclerosis complex (TSC) carry the arginine variant of codon 72 (R72) of and that R72 increases the risk for angiomyolipoma. R72 transactivates and better than the proline variant of codon 72 (P72); therefore, the expression of and is increased in angiomyolipoma cells that carry R72. The loss of and within nestin-expressing cells in mice resulted in the development of renal cell carcinomas (RCC) with high and expression, suggesting that similar downstream mechanisms contribute to tumorigenesis as a result of p53 loss in mice and p53 polymorphism in humans. The loss of murine or expression of human R72 contributes to tumorigenesis via enhancing epithelial-to-mesenchymal transition and motility of tumor cells through the Notch and Nodal pathways. IMPLICATIONS: This work revealed unexpected contributions of the p53 polymorphism to the pathogenesis of TSC and established signaling alterations caused by this polymorphism as a target for therapy. We found that the codon 72 TP53 polymorphism contributes to TSC-associated tumorigenesis via Notch and Nodal signaling.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-1292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677621PMC
August 2019

A genome-wide association study implicates in lymphangioleiomyomatosis pathogenesis.

Eur Respir J 2019 06 27;53(6). Epub 2019 Jun 27.

Division of Pulmonary and Critical Care Medicine and of Genetics, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA

Introduction: Lymphangioleiomyomatosis (LAM) occurs either associated with tuberous sclerosis complex (TSC) or as sporadic disease (S-LAM). Risk factors for development of S-LAM are unknown. We hypothesised that DNA sequence variants outside of / might be associated with susceptibility for S-LAM and performed a genome-wide association study (GWAS).

Methods: Genotyped and imputed data on 5 426 936 single nucleotide polymorphisms (SNPs) in 426 S-LAM subjects were compared, using conditional logistic regression, with similar data from 852 females from COPDGene in a matched case-control design. For replication studies, genotypes for 196 non-Hispanic White female S-LAM subjects were compared with three different sets of controls. RNA sequencing and immunohistochemistry analyses were also performed.

Results: Two noncoding genotyped SNPs met genome-wide significance: rs4544201 and rs2006950 (p=4.2×10 and 6.1×10, respectively), which are in the same 35 kb linkage disequilibrium block on chromosome 15q26.2. This association was replicated in an independent cohort. (nuclear receptor subfamily 2 group F member 2), a nuclear receptor and transcription factor, was the only nearby protein-coding gene. expression was higher by RNA sequencing in one abdominal LAM tumour and four kidney angiomyolipomas, a LAM-related tumour, compared with all cancers from The Cancer Genome Atlas. Immunohistochemistry showed strong nuclear expression in both LAM and angiomyolipoma tumours.

Conclusions: SNPs on chromosome 15q26.2 are associated with S-LAM, and chromatin and expression data suggest that this association may occur through effects on expression, which potentially plays an important role in S-LAM development.
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http://dx.doi.org/10.1183/13993003.00329-2019DOI Listing
June 2019

Serum endostatin levels are associated with diffusion capacity and with tuberous sclerosis- associated lymphangioleiomyomatosis.

Orphanet J Rare Dis 2019 03 29;14(1):72. Epub 2019 Mar 29.

Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Endostatin is a naturally occurring collagen fragment with anti-angiogenic properties. We investigated the association between serum endostatin levels and DLCO in a cohort of patients with lymphangioleiomyomatosis (LAM). Associations of endostatin levels to clinical features of LAM were explored using logistic regression models. Endostatin levels were associated with DLCO and were higher in subjects with TSC-associated LAM compared to sporadic LAM. These data suggest that endostatin could be a predictive biomarker of decline in DLCO and that germline mutational inactivation of the TSC1 or TSC2 gene is associated with higher endostatin levels. These findings could offer novel insights into the pathogenesis of LAM.
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http://dx.doi.org/10.1186/s13023-019-1050-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440133PMC
March 2019

The Genetics of Pneumothorax.

Am J Respir Crit Care Med 2019 06;199(11):1344-1357

5 Pulmonary Genetics Center, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; and.

A genetic influence on spontaneous pneumothoraces-those occurring without a traumatic or iatrogenic cause-is supported by several lines of evidence: ) pneumothorax can cluster in families (i.e., familial spontaneous pneumothorax), ) mutations in the gene have been found in both familial and sporadic cases, and ) pneumothorax is a known complication of several genetic syndromes. Herein, we review known genetic contributions to both sporadic and familial pneumothorax. We summarize the pneumothorax-associated genetic syndromes, including Birt-Hogg-Dubé syndrome, Marfan syndrome, vascular (type IV) Ehlers-Danlos syndrome, alpha-1 antitrypsin deficiency, tuberous sclerosis complex/lymphangioleiomyomatosis, Loeys-Dietz syndrome, cystic fibrosis, homocystinuria, and cutis laxa, among others. At times, pneumothorax is their herald manifestation. These syndromes have serious potential extrapulmonary complications (e.g., malignant renal tumors in Birt-Hogg-Dubé syndrome), and surveillance and/or treatment is available for most disorders; thus, establishing a diagnosis is critical. To facilitate this, we provide an algorithm to guide the clinician in discerning which cases of spontaneous pneumothorax may have a genetic or familial contribution, which cases warrant genetic testing, and which cases should prompt an evaluation by a geneticist.
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http://dx.doi.org/10.1164/rccm.201807-1212CIDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543724PMC
June 2019

Vitamin D binding protein: a new biomarker of disease severity in lymphangioleiomyomatosis.

Eur Respir J 2018 11 1;52(5). Epub 2018 Nov 1.

Division of Pulmonary and Critical Care Medicine, Dept of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1183/13993003.01886-2018DOI Listing
November 2018

Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis.

Sci Rep 2018 09 21;8(1):14161. Epub 2018 Sep 21.

Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5 MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.
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http://dx.doi.org/10.1038/s41598-018-32256-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155086PMC
September 2018
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