Inhibition of asparagine synthetase effectively retards polycystic kidney disease progression.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.

Primary cilia sense glutamine availability and respond via asparagine synthetase.

Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.

Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness.

Polycystin-1 (PC-1) and 2 (PC-2) are the products of the PKD1 and PKD2 genes, which are mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). They form a receptor/channel complex that has been suggested to function as a mechanosensor, possibly activated by ciliary bending in the renal tubule, and resulting in calcium influx. This model has recently been challenged, leaving the question as to which mechanical stimuli activate the polycystins still open. Here, we used a SILAC/Mass-Spec approach to identify intracellular binding partners of tagged-endogenous PC-1 whereby we detected a class of interactors mediating regulation of cellular actomyosin contraction. Accordingly, using gain and loss-of-function cellular systems we found that PC-1 negatively regulates cellular contraction and YAP activation in response to extracellular stiffness. Thus, PC-1 enables cells to sense the rigidity of the extracellular milieu and to respond appropriately. Of note, in an orthologous murine model of PKD we found evidence of increased actomyosin contraction, leading to enhanced YAP nuclear translocation and transcriptional activity. Finally, we show that inhibition of ROCK-dependent actomyosin contraction by Fasudil reversed YAP activation and significantly improved disease progression, in line with recent studies. Our data suggest a possible direct role of PC-1 as a mechanosensor of extracellular stiffness.

Dissection of metabolic reprogramming in polycystic kidney disease reveals coordinated rewiring of bioenergetic pathways.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder caused by loss-of-function mutations in PKD1 or PKD2. Increased glycolysis is a prominent feature of the disease, but how it impacts on other metabolic pathways is unknown. Here, we present an analysis of mouse Pkd1 mutant cells and kidneys to investigate the metabolic reprogramming of this pathology. We show that loss of Pkd1 leads to profound metabolic changes that affect glycolysis, mitochondrial metabolism, and fatty acid synthesis (FAS). We find that Pkd1-mutant cells preferentially use glutamine to fuel the TCA cycle and to sustain FAS. Interfering with either glutamine uptake or FAS retards cell growth and survival. We also find that glutamine is diverted to asparagine via asparagine synthetase (ASNS). Transcriptional profiling of PKD1-mutant human kidneys confirmed these alterations. We find that silencing of Asns is lethal in Pkd1-mutant cells when combined with glucose deprivation, suggesting therapeutic approaches for ADPKD.

Regulation of the microtubular cytoskeleton by Polycystin-1 favors focal adhesions turnover to modulate cell adhesion and migration.

BACKGROUND: Polycystin-1 (PC-1) is a large plasma membrane receptor, encoded by the PKD1 gene, which is mutated in most cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). The disease is characterized by renal cysts. The precise function of PC-1 remains elusive, although several studies suggest that it can regulate the cellular shape in response to external stimuli. We and others reported that PC-1 regulates the actin cytoskeleton and cell migration. RESULTS: Here we show that cells over-expressing PC-1 display enhanced adhesion rates to the substrate, while cells lacking PC-1 have a reduced capability to adhere. In search for the mechanism responsible for this new property of PC-1 we found that this receptor is able to regulate the stability of the microtubules, in addition to its capability to regulate the actin cytoskeleton. The two cytoskeletal components are acting in a coordinated fashion. Notably, we uncovered that PC-1 regulation of the microtubule cytoskeleton impacts on the turnover rates of focal adhesions in migrating cells and we link all these properties to the capability of PC-1 to regulate the activation state of Focal Adhesion Kinase (FAK). CONCLUSIONS: In this study we show several new features of the PC-1 receptor in modulating microtubules and adhesion dynamics, which are essential for its capability to regulate migration.

Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis.

Several organs, including the lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintenance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1, a large receptor of unknown function. Here we demonstrate that PC-1 has an essential role in the establishment of correct tubular diameter during nephron development. Polycystin-1 associates with Par3 favouring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a programme of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis, and in renal cyst formation. Our data define Polycystin-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis.

Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals.

Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

Polycystin-1 regulates extracellular signal-regulated kinase-dependent phosphorylation of tuberin to control cell size through mTOR and its downstream effectors S6K and 4EBP1.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease characterized by bilateral renal cyst formation. Both hyperproliferation and hypertrophy have been previously observed in ADPKD kidneys. Polycystin-1 (PC-1), a large orphan receptor encoded by the PKD1 gene and mutated in 85% of all cases, is able to inhibit proliferation and apoptosis. Here we show that overexpression of PC-1 in renal epithelial cells inhibits cell growth (size) in a cell cycle-independent manner due to the downregulation of mTOR, S6K1, and 4EBP1. Upregulation of the same pathway leads to increased cell size, as found in mouse embryonic fibroblasts derived from Pkd1-/- mice. We show that PC-1 controls the mTOR pathway in a Tsc2-dependent manner, by inhibiting the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of tuberin in Ser664. We provide a detailed molecular mechanism by which PC-1 can inhibit the mTOR pathway and regulate cell size.

Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion.

Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1-/- mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and beta-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated beta-catenin through activation of GSK3beta. Expression of a nondegradable form of beta-catenin in PC-1 MDCK cells restores strong cell-cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis.

Polycystin-1 induces resistance to apoptosis through the phosphatidylinositol 3-kinase/Akt signaling pathway.

Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K beta activity in PC-1-expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease.