Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)β(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Prostaglandins are involved in a wide variety of physiological and pathophysiological processes, but the mechanism of prostaglandin release from cells is not completely understood. Although poorly membrane permeable, prostaglandins are believed to exit cells by passive diffusion. We have investigated the interaction between prostaglandins and members of the ATP-binding cassette (ABC) transporter ABCC [multidrug resistance protein (MRP)] family of membrane export pumps. In inside-out membrane vesicles derived from insect cells or HEK293 cells, MRP4 catalyzed the time-and ATP-dependent uptake of prostaglandin E 1 (PGE1) and PGE2. In contrast, MRP1, MRP2, MRP3, and MRP5 did not transport PGE 1 or PGE 2. The MRP4-mediated transport of PGE1 and PGE2 displayed saturation kinetics, with K m values of 2.1 and 3.4 M, respectively. Further studies showed that PGF 1␣, PGF2␣, PGA1, and thromboxane B 2 were high-affinity inhibitors (and therefore presumably substrates) of MRP4. Furthermore, several nonsteroidal antiinflammatory drugs were potent inhibitors of MRP4 at concentrations that did not inhibit MRP1. In cells expressing the prostaglandin transporter PGT, the steady-state accumulation of PGE 1 and PGE2 was reduced proportional to MRP4 expression. Inhibition of MRP4 by an MRP4-specific RNA interference construct or by indomethacin reversed this accumulation deficit. Together, these data suggest that MRP4 can release prostaglandins from cells, and that, in addition to inhibiting prostaglandin synthesis, some nonsteroidal antiinflammatory drugs might also act by inhibiting this release. P rostaglandins are key mediators in the regulation of many physiological processes. They are involved in inflammatory responses and tumorigenesis, and their synthesis and metabolism are tightly regulated (1). The first step in prostaglandin synthesis is the production of arachidonic acid, which is released from membrane lipid primarily by cytosolic phospholipase A 2 (1). Arachidonic acid is then oxidized to the intermediate prostaglandin H 2 (PGH 2 ) by PGH synthases, also known as cyclooxygenase (COX)-1 and -2, and the recently identified COX-3 (2). These enzymes are known clinically as the targets of aspirin and other nonsteroidal antiinf lammatory drugs (NSAIDs) (3). Moreover, several recent studies have shown a link between COX-2 expression and carcinogenesis. Prostaglandins are overproduced by a variety of tumors, leading to the suggested prophylactic use of COX-2 inhibitors to decrease the incidence of colon cancer (4, 5). After COX-mediated synthesis, PGH 2 is further converted by tissue-specific prostaglandin synthases into PGE 2 , PGF 2␣ , PGD 2 , prostacyclin, or thromboxane B 2 , the biologically active molecules (1).Prostaglandins are formed and secreted by most cells, and act as autocrine-or paracrine-signaling molecules. In many cases they exert their effects extracellularly via interaction with a family of G protein-coupled membrane receptors (reviewed in ref. 6), although some prostaglandins interact with the nuclear horm...

Human multidrug-resistance protein (MRP) 4 transports cyclic nucleotides and when overproduced in mammalian cells mediates resistance to some nucleoside analogues. Recently, it has been shown that Mrp4 is induced in the livers of Fxr ((-/-)) mice, which have increased levels of serum bile acids. Since MRP4, like MRP1-3, also mediates transport of a model steroid conjugate substrate, oestradiol 17-beta-D-glucuronide (E(2)17betaG), we tested whether MRP4 may be involved in the transport of steroid and bile acid conjugates. Bile salts, especially sulphated derivatives, and cholestatic oestrogens inhibited the MRP4-mediated transport of E(2)17betaG. Inhibition by oestradiol 3,17-disulphate and taurolithocholate 3-sulphate was competitive, suggesting that these compounds are MRP4 substrates. Furthermore, we found that MRP4 transports dehydroepiandrosterone 3-sulphate (DHEAS), the most abundant circulating steroid in humans, which is made in the adrenal gland. The ATP-dependent transport of DHEAS by MRP4 showed saturable kinetics with K (m) and V (max) values of 2 microM and 45 pmol/mg per min, respectively (at 27 degrees C). We further studied the possible involvement of other members of the MRP family of transporters in the transport of DHEAS. We found that MRP1 transports DHEAS in a glutathione-dependent manner and exhibits K (m) and V (max) values of 5 microM and 73 pmol/mg per min, respectively (at 27 degrees C). No transport of DHEAS was observed in membrane vesicles containing MRP2 or MRP3. Our findings suggest a physiological role for MRP1 and MRP4 in DHEAS transport and an involvement of MRP4 in transport of conjugated steroids and bile acids.

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-␤-D-glucuronide (E 2 17␤G), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E 2 17␤G transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Halfmaximal transport was obtained at 120 M E 2 17␤G, in contrast to values < 20 M for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E 2 17␤G (halfmaximal transport rates at 65 and 16 M E 2 17␤G, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E 2 17␤G transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E 2 17␤G. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.

Key Points MCL cells are mobilized into the peripheral blood of patients treated with the BTK inhibitor ibrutinib. Ibrutinib dose-dependently inhibits BCR- and chemokine-mediated adhesion and migration of MCL cells.

No abstract

Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methylthio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Glucuronidation is a major detoxification pathway for endogenous and exogenous compounds in mammals that results in the intracellular formation of polar metabolites, requiring specialized transporters to cross biological membranes. By using morphine as a model aglycone, we demonstrate that multidrug resistance protein 3 (MRP3͞ABCC3), a protein present in the basolateral membrane of polarized cells, transports morphine-3-glucuronide (M3G) and morphine-6-glucuronide in vitro. Mrp3 (؊/؊) mice are unable to excrete M3G from the liver into the bloodstream, the major hepatic elimination route for this drug. This results in increased levels of M3G in liver and bile, a 50-fold reduction in the plasma levels of M3G, and in a major shift in the main disposition route for morphine and M3G, predominantly via the urine in WT mice but via the feces in Mrp3 (؊/؊) mice. The pharamacokinetics of injected morphine-glucuronides are altered as well in the absence of Mrp3, and this results in a decreased antinociceptive potency of injected morphine-6-glucuronide.analgesia ͉ glucuronides ͉ morphine-3-glucuronide ͉ transport