Jean Piero Margaria

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Phosphoinositide 3-Kinase Gamma Inhibition Protects from Anthracycline Cardiotoxicity and Reduces Tumor Growth.

Background: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase &ggr; (PI3K&ggr;) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3K&ggr; inhibition. Methods: Mice expressing a kinase-inactive PI3K&ggr; or receiving PI3K&ggr;-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3K&ggr; inhibition was assessed in mouse mammary tumor models. Results: PI3K&ggr; kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3K&ggr; inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3K&ggr; kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3K&ggr; was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3K&ggr;/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3K&ggr; blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. Conclusions: Blockade of PI3K&ggr; may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Rab11 activity and PtdIns(3) P turnover removes recycling cargo from endosomes

AbstractDirectional transport of recycling cargo from early endosomes (EE) to the endocytic recycling compartment (ERC) relies on phosphatidylinositol 3-phosphate (PtdIns(3)P) hydrolysis and activation of the small GTPase Rab11. However, how these events are coordinated is yet unclear. By using a novel genetically-encoded FRET biosensor for Rab11, we report that generation of endosomal PtdIns(3)P by the clathrin-binding phosphoinositide 3-kinase class 2 alpha (PI3K-C2α) controls the activation of Rab11. Active Rab11, in turn, prompts the recruitment of the phosphatidylinositol 3-phosphatase myotubularin 1 (MTM1), eventually enabling the release of recycling cargo from the EE and its delivery toward the ERC. Our findings thus define that delivery of recycling cargo toward the ERC requires spatial and sequential coupling of Rab11 activity with PtdIns(3)P turnover.A Rab11 FRET biosensor reveals a spatiotemporal interplay between Rab11 and PtdIns(3)P turnover during the removal of recycling cargo from the endosome.

Therapeutic Targeting of PDEs and PI3K in Heart Failure with Preserved Ejection Fraction (HFpEF)

Purpose of ReviewHeart Failure with preserved Ejection Fraction (HFpEF) is a prevalent disease with considerable individual and societal burden. HFpEF patients often suffer from multiple pathological conditions thatcomplicate management and adversely affect outcome, including pulmonary hypertension and chronic obstructive pulmonary disease (COPD). To date, no treatment proved to be fully effective in reducing morbidity and mortality in HFpEF, possibly due to an incomplete understanding of the underlying molecular mechanisms.Recent FindingsThe emerging view proposes chronic systemic inflammation, leading to endothelial dysfunction and interstitial fibrosis, as a prominent cause of HFpEF, rather than a mere co-existent disease. In the last decade, efforts from pharmaceutical companies attempted to target pharmacologically enzymes which play key roles in systemic and lung inflammation, such as the cyclic nucleotide-degrading enzymes phosphodiesterases (PDEs) and phosphoinositide-3 phosphate kinases (PI3Ks), especially to limit COPD.SummaryIn this review, we will summarize major successes and drawbacks of hitting these enzymes to tackle inflammation in HFpEF-associated co-morbidities, with a major focus on the results of completed and ongoing clinical trials. Finally, we will discuss the potential of repurposing and/or developing new PDE and PI3K inhibitors for HFpEF therapy.

Identification of a Potent Phosphoinositide 3-Kinase Pan Inhibitor Displaying a Strategic Carboxylic Acid Group and Development of Its Prodrugs

Activation of the phosphoinositide 3‐kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on‐target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life‐threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so‐far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan‐PI3K inhibitor.

The Guareschi Pyridine Scaffold as a Valuable Platform for the Identification of Selective PI3K Inhibitors

A novel series of 4-aryl-3-cyano-2-(3-hydroxyphenyl)-6-morpholino-pyridines have been designed as potential phosphatidylinositol-3-kinase (PI3K) inhibitors. The compounds have been synthesized using the Guareschi reaction to prepare the key 4-aryl-3-cyano-2,6-dihydroxypyridine intermediate. A different selectivity according to the nature of the aryl group has been observed. Compound 9b is a selective inhibitor against the PI3Kα isoform, maintaining a good inhibitory activity. Docking studies were also performed in order to rationalize its profile of selectivity.

PI3K class II α: a novel regulator of vesicular trafficking at the base of the primary cilium

The class II phosphoinositide 3-kinase PI3K-C2α is a protein of the early endocytic compartment and the trans-Golgi network. It produces PtdIns-3-P and is characterized by a clathrin-binding site which confers to this enzyme an important role in modulating clathrin distribution and activity in the cell. However, implication of PI3K-C2α in the primary cilium biology has never been described. Through the generation of a Pik3c2a knock-out mouse strain, we discovered that PI3K-C2α was fundamental during embryonic development and that its loss conferred features of ciliopathy. In particular, homozygous mutant embryos died at midgestation and displayed laterality defects and impaired Hedgehog signaling, while heterozygous adults showed renal cysts susceptibility after kidney injury. Cilia of Pik3c2a deficient embryos were shorter and swollen and displayed a defect in accumulating Smo and Polycystin-2. In primary mouse embryonic fibroblasts, PI3K-C2α was highly enriched on vesicles at the basal body of primary cilia. In Pik3c2a-deficient cells, absence of the protein specifically caused a reduction of vesicular trafficking at the cilium base, suggesting that PI3K-C2α, through its ability to recruit clathrin and produce PtdIns-3-P, is required for the correct exchange of structural proteins and signaling molecules between the cilium compartment and the cytoplasm. Thus, our data indicate PI3K-C2α as a novel regulator of vesicular trafficking at the base of the primary cilium.