MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia.

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules-but not their wild-type counterparts-rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.

Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model.

BACKGROUND: Triple-negative breast cancers (TNBC) are considered the most aggressive type of breast cancer, for which no targeted therapy exists at the moment. These tumors are characterized by having a high degree of chromosome instability and often overexpress the spindle assembly checkpoint kinase TTK. To explore the potential of TTK inhibition as a targeted therapy in TNBC, we developed a highly potent and selective small molecule inhibitor of TTK, NTRC 0066-0. RESULTS AND CONCLUSIONS: The compound is characterized by long residence time on the target and inhibits the proliferation of a wide variety of human cancer cell lines with potency in the same range as marketed cytotoxic agents. In cell lines and in mice, NTRC 0066-0 inhibits the phosphorylation of a TTK substrate and induces chromosome missegregation. NTRC 0066-0 inhibits tumor growth in MDA-MB-231 xenografts as a single agent after oral application. To address the effect of the inhibitor in breast cancer, we used a well-defined mouse model that spontaneously develops breast tumors that share key morphologic and molecular features with human TNBC. Our studies show that combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in doubling of mouse survival and extended tumor remission, without toxicity. Furthermore, we observed that treatment efficacy is only achieved upon co-administration of the two compounds, which suggests a synergistic in vivo effect. Therefore, we propose TTK inhibition as a novel therapeutic target for neoadjuvant therapy in TNBC.

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes.

BACKGROUND AND PURPOSE: The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers. EXPERIMENTAL APPROACH: CXCR3-CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity. KEY RESULTS: We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific ß-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation. CONCLUSIONS AND IMPLICATIONS: This study indicates that heteromeric CXCR3-CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

Fluorescence assays for high-throughput screening of protein kinases.

Protein kinases comprise one of the most important group of targets for drug discovery research today. Methods to identify novel kinase inhibitors by high-throughput screening have evolved rapidly in recent years. An important aspect is the availability of fluorescent probes that can be applied in a homogeneous, or mix-and-measure, assay format. Here, we illustrate the application of fluorescence read-out technologies for kinase targets in light of our own experiences in assay development and high-throughput screening.

The elusive factor Xa receptor: failure to detect transcripts that correspond to the published sequence of EPR-1.

The coagulation protease factor Xa induces cellular responses implicated in cardiovascular and inflammatory disease. Effector-cell protease receptor 1 (EPR-1) is a functionally characterized receptor of factor Xa, and the EPR-1 complementary DNA (cDNA) was published. Remarkably, the cDNA encoding an inhibitor of apoptosis, survivin, is reportedly identical to that of EPR-1 except for a few nucleotide differences and its orientation opposite to EPR-1. To isolate the EPR-1 cDNA and gene, we surveyed gene databases for expressed sequence tags (ESTs) that could be derived from EPR-1. All ESTs with strong homology to EPR-1/survivin were derived from survivin and could not encode EPR-1. By polymerase chain reaction and Southern blot hybridization, EPR-1 was not detectable in the human or murine genome, but survivin was. Our data suggest that EPR-1 is either highly cell-specific or the published EPR-1 cDNA includes sequences from clones derived from survivin messenger RNA. The means by which factor Xa mediates its cellular effects requires further evaluation.

Tyrosine kinase activity of purified recombinant cytoplasmic domain of platelet-derived growth factor beta-receptor (beta-PDGFR) and discovery of a novel inhibitor of receptor tyrosine kinases.

Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.

Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.

The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Transport of the glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP.

The multidrug resistance protein MRP has been shown to mediate the transport of glutathione S-conjugates across membranes. In this study we demonstrate that the glutathione S-conjugate of the diuretic drug ethacrynic acid, which is an efficient inhibitor of glutathione S-transferases, is a high-affinity substrate and inhibitor of the glutathione S-conjugate pump associated with MRP. This implies that ethacrynic acid may modulate drug resistance of tumor cells not only by inhibiting glutathione S-transferase activity, but also by inhibiting the export of drug conjugates from the cell by MRP.

The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.

Membrane topology and glycosylation of the human multidrug resistance-associated protein.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Tissue distribution of the multidrug resistance protein.

The human multidrug resistance protein (MRP) is a 190 kd membrane glycoprotein that can cause resistance of human tumor cells to various anticancer drugs, by extruding these drugs out of the cell. Three different monoclonal antibodies, directed against different domains of MRP, allowed us to determine the localization of MRP in a panel of normal human tissues and malignant tumors. Whereas in malignant tumors strong plasma membrane MRP staining was frequently observed, in normal human tissues MRP staining was predominantly cytoplasmatic. Here, MRP was detected in several types of epithelia, muscle cells, and macrophages. From the presence of MRP in many epithelia we infer that MRP, like MDR1 P-glycoprotein, may have an excretory function in protecting the organism against xenobiotics. Recent studies indicate a role for MRP as a carrier for transport of glutathione-conjugated endo- and xenobiotics. The presence of MRP in bronchiolar epithelium, heart muscle, and macrophages would agree with the glutathione S-conjugate carrier activity previously detected in these cells. Furthermore, in 46 of 119 untreated tumors from various histogenetic origins MRP staining was seen. In these tumors MRP may contribute to the intrinsic resistance against treatment with chemotherapeutic drugs.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.

The human Dubin-Johnson syndrome and its animal model, the TR(-) rat, are characterized by a chronic conjugated hyperbilirubinemia. TR(-) rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homolog of the human multidrug resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR(-) rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR(-) phenotype.

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers.

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon.

Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon. In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation. In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA. Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR). By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length. Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification. Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.

Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.

The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

High expression of the multidrug resistance-associated protein (MRP) in chronic and prolymphocytic leukaemia.

The expression of the multidrug resistance-associated protein (MRP), a new glycoprotein involved in drug resistance, was investigated in tumour samples from 80 patients with chronic B-cell malignancies by a quantitative RNase protection assay. In B-cell chronic lymphocytic leukaemia (B-CLL) (n = 32), either treated (n = 18) or untreated (n = 14), a high percentage of patients (20/32: 63%) had relatively high expression levels of the MRP gene (25U or more). In addition, hyperexpression of the MRP gene was demonstrated in 4/10 (40%) untreated patients with B-cell prolymphocytic leukaemia (B-PLL). In contrast, low MRP mRNA expression levels were detected in hairy cell leukaemia (n = 7), non-Hodgkin's lymphoma (n = 13) and multiple myeloma (n = 18). Statistical analysis of MRP expression in untreated CLL (mean +/- SD 29.2 +/- 18.5 U) versus treated CLL (mean +/- SD 26.7 +/- 13.7 U) did not show significant differences in MRP expression between the two groups. Southern blot analysis did not reveal amplification of the MRP gene in the leukaemia samples with elevated MRP mRNA levels. We conclude that B-PLL and B-CLL frequently display high MRP expression and that this hyperexpression is probably due to transcriptional activation and/or increased mRNA stability.

Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies.

We have generated rat and murine monoclonal antibodies against multidrug resistance-associated protein (MRP), a M(r) 180,000-195,000 membrane glycoprotein involved in a non-P-glycoprotein multidrug resistance of human tumor cells. The antibodies were raised against two different segments of MRP and found to be suitable for protein blot analyses, immunohistochemical and cytochemical studies, as well as flow cytometry of permeabilized cells. The antibodies do not cross-react with the human P-glycoproteins. Immunocytochemistry using MRP-overexpressing tumor cells of different histogenetic origins showed that MRP is predominantly located in the plasma membrane. Immunoelectron microscopy confirmed the plasma membrane location of MRP. The MRP antibodies provide a sensitive and specific tool for studies on MRP-mediated multidrug resistance.