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Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.

Organic anion-transporting polypeptides (OATPs) are important drug uptake transporters, mediating distribution of substrates to several pharmacokinetically relevant organs. Doxorubicin is a widely used anti-cancer drug extensively studied for its interactions with various drug transporters, but not OATPs. Here, we investigated the role of OATP1A/1B proteins in the distribution of doxorubicin. In vitro, we observed ∼ 2-fold increased doxorubicin uptake in HEK293 cells overexpressing human OATP1A2, but not OATP1B1 or OATP1B3. In mice, absence of Oatp1a/1b transporters led to up to 2-fold higher doxorubicin plasma concentrations and 1.3-fold higher plasma AUC. Conversely, liver AUC and liver-to-plasma ratios of Oatp1a/1b(-/-) mice were 1.4-fold and up to 4.1-fold lower than in wild-type mice, respectively. Decreased doxorubicin levels in the small intestinal content reflected those in the liver, indicating a reduced biliary excretion of doxorubicin in Oatp1a/1b(-/-) mice. These results demonstrate important control of doxorubicin plasma clearance and hepatic uptake by mouse Oatp1a/1b transporters. This is unexpected, as the fairly hydrophobic weak base doxorubicin is an atypical OATP1A/1B substrate. Interestingly, transgenic liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3 could partially rescue the increased doxorubicin plasma levels of Oatp1a/1b(-/-) mice. Hepatic uptake and bile-derived intestinal excretion of doxorubicin were completely reverted to wild-type levels by OATP1A2, and partially by OATP1B1 and OATP1B3. Thus, doxorubicin is transported by hepatocyte-expressed OATP1A2, -1B1 and -1B3 in vivo, illustrating an unexpectedly wide substrate specificity. These findings have possible implications for the uptake, disposition, therapy response and toxicity of doxorubicin, also in human tumors and tissues expressing these transporters.

ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ~65% was conjugated and ~35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na+-taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels. Conclusions ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome.

Combination of a CAR and a chimeric costimulatory receptor augments cytotoxicity and durability of T cells and elimination of antigen-low tumors.

Chimeric antigen receptor (CAR) T cells are highly successful in the treatment of hematologic malignancies. We recently generated affinity-optimized CD38CAR T cells, which effectively eliminate multiple myeloma (MM) cells with little or no toxicities against nonmalignant hematopoietic cells. The lack of universal donors and long manufacturing times however limit the broad application of CAR T cell therapies. Natural killer (NK) cells generated from third party individuals may represent a viable source of “off the shelf” CAR-based products, as they are not associated with graft-versus-host disease unlike allogeneic T cells. We therefore explored the preclinical anti-MM efficacy and potential toxicity of the CD38CAR NK concept by expressing affinity-optimized CD38CARs in KHYG-1 cells, an immortal NK cell line with excellent expansion properties. KHYG-1 cells retrovirally transduced with the affinity-optimized CD38CARs expanded vigorously and mediated effective CD38-dependent cytotoxicity towards CD38high MM cell lines as well as primary MM cells ex vivo. Importantly, the intermediate affinity CD38CAR transduced KHYG-1 cells spared CD38neg or CD38int nonmalignant hematopoietic cells, indicating an optimal tumor nontumor discrimination. Irradiated, short living CD38CAR KHYG-1 cells also showed significant anti-MM effects in a xenograft model with a humanized bone marrow-like niche. Finally, CD38CAR KHYG-1 cells effectively eliminated primary MM cells derived from patients who are refractory to CD38 antibody daratumumab. Taken together, the results of this proof-of-principle study demonstrate the potential value of engineering affinity-optimized CD38CARs in NK cells to establish effective anti-MM effects, with an excellent safety profile, even in patients who failed to response to most advanced registered myeloma therapies, such as daratumumab.

ObjectiveCoupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown.Methods and resultsHere, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles. Both gain- and loss-of-function approaches are used to show that hepatic Cyp17a1 is induced by fasting, catalyzes the production of at least one hormone-ligand (DHEA) for the nuclear receptor PPARα, and is ultimately required for maintaining euglycemia and ketogenesis during nutrient deprivation. The feedback-loop that terminates Cyp17a1-PPARα activity, and re-establishes anabolic liver metabolism during re-feeding is mapped to postprandial bile acid-signaling, involving the receptors FXR, SHP and LRH-1.ConclusionsTogether, these findings represent a novel paradigm of homeostatic control in which nutritional cues feed-forward on to metabolic pathways by influencing extragonadal steroidogenesis.

P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe Cholestatic liver disease. Mutation of ATP8B1 cause progressive familial Intrahepatic Cholestasis type 1 (PFIC1)and benign recurrent intrahepatic cholestasis type 1 (BRIC 1). From our observations we hypothesized that ATP8B1 deficiency causes a phospholipids randomization at the canalicular membrane, which results in extraction of cholesterol due to increase sensitivity of the canalicular membrane. Deficiency of ATP11C causes conjugated hyperbilirubinemia. In our preliminary result we observed accumulation of unconjugated bile salts in Atp11c deficient mice probably because of regulation in the expression or function of OATP1B2. Similar to ATP8B1, ATP11C have regulation on membrane transporters.