Selected list of putative interacting TSC2 partners identified by yeast 2-hybrid analysis.

Ritanserin inhibits the proliferation of multiple TSC2-deficient cell lines, independently of autophagy.

Gene expression of DGKs and pentose phosphate pathway genes.

Intrinsically disordered regions in TSC2.

Ritanserin inhibits macropinocytosis and acidic organelle homeostasis in TSC2-deficient cells

Purine metabolism following ritanserin treatment in Tsc2-deficient cells

Lipidomic analysis of ritanserin treated Tsc2-deficient cells

In tuberin-deficient cells, SYK regulates ETV2 nuclear translocation, where it regulated Parpbp expression. Silencing Etv2 or Parpbp induces ER stress–mediated cell 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.

PAH is a devastating and incurable disease characterized by abnormal pulmonary vascular phenotypes and progressive right ventricular failure, but the underlying mechanisms are incompletely understood. There is an urgent need of new therapeutic approaches, and a better understanding of disease pathogenesis is essential in identifying novel pathways that can be therapeutically targeted. Stress granules (SGs) are small intracellular structures that form in response to stress and contain mRNA molecules and mRNA‐binding proteins. These granules allow cells to temporarily halt translation by “parking” or sequestering certain mRNAs in a protected zone (the stress granule) and this allows the cells to survive in conditions of stress. The role of SGs in abnormal vascular and cardiac homeostasis in PAH is not known.

Pulmonary arterial hypertension (PAH) is a serious, progressive, and often fatal disease that is in urgent need of improved therapies that treat it. One of the remaining therapeutic challenges is the increasingly recognized skeletal muscle dysfunction that interferes with exercise tolerance. Here we report that in the adult rat Sugen/hypoxia (SU/Hx) model of severe pulmonary hypertension (PH), there is highly significant, almost 50%, decrease in exercise endurance, and this is associated with a 25% increase in the abundance of type II muscle fiber markers, thick sarcomeric aggregates and an increase in the levels of FoxO1 in the soleus (a predominantly type I fiber muscle), with additional alterations in the transcriptomic profiles of the diaphragm (a mixed fiber muscle) and the extensor digitorum longus (a predominantly Type II fiber muscle). In addition, soleus atrophy may contribute to impaired exercise endurance. Studies in L6 rat myoblasts have showed that myotube differentiation is associated with increased FoxO1 levels and type II fiber markers, while the inhibition of FoxO1 leads to increased type I fiber markers. We conclude that the formation of aggregates and a FoxO1-mediated shift in the skeletal muscle fiber-type specification may underlie skeletal muscle dysfunction in an experimental study of PH.

Abstract 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.

Abstract 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.

Background: Right ventricular (RV) performance is a key determinant of mortality in pulmonary arterial hypertension (PAH). RV failure is characterized by metabolic dysregulation with unbalanced anaerobic glycolysis, oxidative phosphorylation, and fatty acid oxidation (FAO). We previously found that acetazolamide (ACTZ) treatment modulates the pulmonary inflammatory response and ameliorates experimental PAH. Objective: To evaluate the effect of ACTZ treatment on RV function and metabolic profile in experimental PAH. Design/Methods: In the Sugen 5416/hypoxia (SuHx) rat model of severe PAH, RV transcriptomic analysis was performed by RNA-seq, and top metabolic targets were validated by RT-PCR. We assessed the effect of therapeutic administration of ACTZ in the drinking water on hemodynamics by catheterization [right and left ventricular systolic pressure (RVSP and LVSP, respectively)] and echocardiography [pulmonary artery acceleration time (PAAT), RV wall thickness in diastole (RVWT), RV end-diastolic diameter (RVEDD), tricuspid annular plane systolic excursion (TAPSE)] and on RV hypertrophy (RVH) by Fulton's index (FI) and RV-to-body weight (BW) ratio (RV/BW). We also examined myocardial histopathology and expression of metabolic markers in RV tissues. Results: There was a distinct transcriptomic signature of RVH in the SuHx model of PAH, with significant downregulation of metabolic enzymes involved in fatty acid transport, beta oxidation, and glucose oxidation compared to controls. Treatment with ACTZ led to a pattern of gene expression suggestive of restored metabolic balance in the RV with significantly increased beta oxidation transcripts. In addition, the FAO transcription factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α) was significantly downregulated in untreated SuHx rats compared to controls, and ACTZ treatment restored its expression levels. These metabolic changes were associated with amelioration of the hemodynamic and echocardiographic markers of RVH in the ACTZ-treated SuHx animals and attenuation of cardiomyocyte hypertrophy and RV fibrosis. Conclusion: Acetazolamide treatment prevents the development of PAH, RVH, and fibrosis in the SuHx rat model of severe PAH, improves RV function, and restores the RV metabolic profile.

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.

Actin remodeling is indispensable for dendritic spine development, morphology and density which signify learning, memory and motor skills. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic spine density and neuronal dendritic length were altered in Cap2gt/gt. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. In membrane depolarization assays, Cap2gt/gt synaptosomes exhibit an impaired F/G actin ratio, indicating altered actin dynamics. We show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and is cofilin ser3 phosphorylation dependent. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.

Cyclase associated protein (CAP) is a highly conserved protein with roles in actin dynamics and many cellular processes. Two isoforms exist in higher eukaryotes, CAP1 and CAP2. CAP1 is ubiquitously expressed whereas CAP2 shows restricted tissue distribution. In mice, ablation of CAP2 leads to development of cardiomyopathy. CAP2 is expressed in skin. In human skin its expression is increased in wounds. To elucidate the role of CAP2 in skin upon injury, we studied the wound healing in CAP2 deficient mice and found altered wound healing response presumably resulting from reduced levels of α-SMA, decreased macrophage infiltration and slower neovascularization. In vitro cultured Cap2 deficient keratinocytes showed reduced velocity and a delay in scratch closure. The analysis of primary mutant fibroblasts also showed reduced velocity and less contractibility. They had extended protrusions and more focal adhesions. In addition the F-actin content was increased keeping the total actin content unaltered. Mutant fibroblasts furthermore exhibited an altered response during recovery from drug-induced disruption of the actin cytoskeleton. Interestingly, CAP1 was upregulated in knockout unwounded skin and in wounds which might partially compensate for the loss of CAP2. Taken together, our studies reveal a role for CAP2 in wound healing which may be based on its function as a regulator of the actin cytoskeleton.