Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs gemcitabine, fludarabine and cladribine, but not to other classes of anti-lymphoma agents.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.

Proteomic and transcriptomic analysis of heart failure due to volume overload in a rat aorto-caval fistula model provides support for new potential therapeutic targets - monoamine oxidase A and transglutaminase 2.

BACKGROUND: Chronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF. METHODS: We analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip. RESULTS: In the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs. CONCLUSIONS: The differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.

Quinone reductase acts as a redox switch of the 20S yeast proteasome.

The proteasome has an essential function in the intracellular degradation of protein in eukaryotic cells. We found that the dimeric quinone reductase Lot6 uses the flavin mononucleotide (FMN)-binding site to bind to the 20S proteasome with a 1:2 stoichiometry-that is, one 20S proteasome molecule can associate with two quinone reductases. Furthermore, reduction of the FMN cofactor by either NADH or light irradiation results in the binding of the b-Zip transcription factor Yap4 to the Lot6-proteasome complex, indicating that recruitment of the transcription factor depends on the redox state of the quinone reductase. Here, we show that binding of Yap4 to the complex not only protects it from ubiquitin-independent proteasomal degradation, but also regulates its cellular localization. In non-stressed wild-type cells, we did not detect any Yap4 in the nucleus, whereas Yap4 was present in the nuclei from quinone-stressed yeast cultures. Thus, the Lot6-proteasome complex can be regarded as a redox switch in which the quinone reductase acts as a sensor for oxidative stress.

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

Resistance to TRAIL in mantle cell lymphoma cells is associated with the decreased expression of purine metabolism enzymes.

Mantle cell lymphoma (MCL) is a rare aggressive type of B-cell non-Hodgkin's lymphoma. Response to chemotherapy tends to be short and virtually all patients sooner or later relapse. The prognosis of relapsed patients is extremely poor. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered one of the novel experimental molecules with strong antitumor effects. TRAIL triggers extrinsic apoptotis in tumor cells by binding to TRAIL 'death receptors' on the cell surface. Recombinant TRAIL has shown promising pro-apoptotic effects in a variety of malignancies including lymphoma. However, as with other drugs, lymphoma cells can develop resistance to TRAIL. Therefore, the aim of this study was to identify the molecular mechanisms responsible for, and associated with TRAIL resistance in MCL cells. If identified, these features may be used as molecular targets for the effective elimination of TRAIL-resistant lymphoma cells. From an established TRAIL-sensitive mantle cell lymphoma cell line (HBL-2) we derived a TRAIL-resistant HBL-2/R subclone. By TRAIL receptor analysis and differential proteomic analysis of HBL-2 and HBL-2/R cells we revealed a marked downregulation of all TRAIL receptors and, among others, the decreased expression of 3 key enzymes of purine nucleotide metabolism, namely purine nucleoside phosphorylase, adenine phosphoribosyltransferase and inosine-5'-monophosphate dehydrogenase 2, in the resistant HBL-2/R cells. The downregulation of the 3 key enzymes of purine metabolism can have profound effects on nucleotide homeostasis in TRAIL-resistant lymphoma cells and can render such cells vulnerable to any further disruption of purine nucleotide metabolism. This pathway represents a 'weakness' of the TRAIL-resistant MCL cells and has potential as a therapeutic target for the selective elimination of such cells.

Decreased concentrations of retinol-binding protein 4 in sera of epithelial ovarian cancer patients: a potential biomarker identified by proteomics.

Ovarian cancer is the fifth leading cause of cancer death in women. Absence of a reliable biomarker precludes early diagnosis of the disease. To identify new proteins with potential diagnostic or prognostic value for the therapy of ovarian cancer we performed comparative proteomic analysis of sera from ovarian cancer patients and healthy women. We analyzed serum samples from 10 patients diagnosed with epithelial ovarian cancer and 10 age-matched healthy women. To decrease the extremely wide dynamic range of protein concentrations in serum we used combinatorial hexapeptide libraries. Serum samples were then subjected to proteomic 2-DE analysis. Three proteins with differential abundance were found and identified by mass spectrometry: α-1-antitrypsin, apolipoprotein A-IV and retinol-binding protein 4. Identification of α-1-antitrypsin and apolipoprotein A-IV confirms previous studies but the identification of significantly decreased levels of RBP4 in ovarian cancer patients represents a novel observation. We verified the decrease of RBP4 levels in ovarian cancer patient sera by two independent methods and determined absolute RBP4 concentrations in patients and healthy women. We excluded possible non-cancer factors that could be responsible for the observed RBP4 decrease. We propose a connection of RBP4 with epithelial ovarian cancer and advocate the potential of RBP4 as a candidate diagnostic or prognostic biomarker.