Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p–PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers.

The mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia (AML) cell lines through blockade in G0/G1 phase of the cell cycle. Accordingly, 2 downstream effectors of mTOR, 4E-BP1 and p70S6K, are phosphorylated in a rapamycin-sensitive manner in a series of 23 AML cases. Interestingly, the mTOR inhibitor markedly impairs the clonogenic properties of fresh AML cells while sparing normal hematopoietic progenitors. Moreover, rapamycin induces significant clinical responses in 4 of 9 patients with either refractory/relapsed de novo AML or secondary AML. Overall, our data strongly suggest that mTOR is aberrantly regulated in most AML cells and that rapamycin and analogs, by targeting the clonogenic compartment of the leukemic clone, may be used as new compounds in AML therapy.

Activation of the Wnt/β-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether β-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for β-catenin expression by Western blot. β-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, β-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, β-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in β-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/β-catenin pathway only in leukemic cells. Indeed, β-catenin negative leukemic cells were found to increase β-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasitePlasmodium falciparumestablishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile ofP. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. ThePlasmodiumgenome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted inPlasmodium berghei, andin vitrogrowth ofP. falciparumwas sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential inPlasmodiumblood stages. For intraparasitic PI3P localization, transgenicP. falciparumthat expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function inPlasmodiumappear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.

During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110β null mouse model and demonstrated a critical role of PI3Kβ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor γ-chain complex, and its contribution in response to G-protein–coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110β null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein–coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kβ is also acting downstream of αIIbβ3. In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl3 injury of the carotid, suggesting that PI3Kβ is a potential target for antithrombotic drugs.