Justyna Chlebowska

Statins impair glucose uptake in human cells

Objective Considering the increasing number of clinical observations indicating hyperglycemic effects of statins, this study was designed to measure the influence of statins on the uptake of glucose analogs by human cells derived from liver, adipose tissue, and skeletal muscle. Design Flow cytometry and scintillation counting were used to measure the uptake of fluorescently labeled or tritiated glucose analogs by differentiated visceral preadipocytes, skeletal muscle cells, skeletal muscle myoblasts, and contact-inhibited human hepatocellular carcinoma cells. A bioinformatics approach was used to predict the structure of human glucose transporter 1 (GLUT1) and to identify the presence of putative cholesterol-binding (cholesterol recognition/interaction amino acid consensus (CRAC)) motifs within this transporter. Mutagenesis of CRAC motifs in SLC2A1 gene and limited proteolysis of membrane GLUT1 were used to determine the molecular effects of statins. Results Statins significantly inhibit the uptake of glucose analogs in all cell types. Similar effects are induced by methyl-β-cyclodextrin, which removes membrane cholesterol. Statin effects can be rescued by addition of mevalonic acid, or supplementation with exogenous cholesterol. Limited proteolysis of GLUT1 and mutagenesis of CRAC motifs revealed that statins induce conformational changes in GLUTs. Conclusions Statins impair glucose uptake by cells involved in regulation of glucose homeostasis by inducing cholesterol-dependent conformational changes in GLUTs. This molecular mechanism might explain hyperglycemic effects of statins observed in clinical trials.

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

Adenanthin targets proteins involved in the regulation of disulphide bonds

Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II through its functional α,β-unsaturated ketone group serving as a Michael acceptor. A similar group is found in SK053, a compound recently developed by our group to target the thioredoxin-thioredoxin reductase (Trx-TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets additional proteins including thioredoxin-thioredoxin reductase system as well as protein disulfide isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold. Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes containing readily accessible, nucleophilic cysteines. This observation might be of importance in considering potential therapeutic applications of adenanthin to include a range of diseases, where aberrant activity of Prdx, Trx-TrxR and PDI is involved in their pathogenesis.

Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity

Background: The influence of farnesyltransferase inhibitors (FTIs) on CD20 levels is unknown. Results: FTIs increase CD20 expression and improve rituximab-mediated activation of complement-dependent cytotoxicity. Conclusion: FTIs sensitize tumor cells to anti-CD20 mAbs. Significance: The combination of FTIs with anti-CD20 mAbs seems to be a reasonable therapeutic approach worth to be tested in patients with B-cell tumors. Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma

FAM46C is one of the most frequently mutated genes in multiple myeloma. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active non-canonical poly(A) polymerase which enhances mRNA stability and gene expression. Reintroduction of active FAM46C into multiple myeloma cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induces cell death. Moreover, silencing of FAM46C in multiple myeloma cells expressing WT protein enhance cell proliferation. Finally, using a FAM46C-FLAG knock-in mouse strain, we show that the FAM46C protein is strongly induced during activation of primary splenocytes and that B lymphocytes isolated from newly generated FAM46C KO mice proliferate faster than those isolated from their WT littermates. Concluding, our data clearly indicate that FAM46C works as an onco-suppressor, with the specificity for B-lymphocyte lineage from which multiple myeloma originates.FAM46C is one of the most frequently mutated genes in multiple myeloma (MM), but its molecular function remains unknown. Here the authors show that FAM46C is a poly(A) polymerase and that loss of function of FAM46C drives multiple myeloma through the destabilisation of ER response transcripts.

Abstract 5347: SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells

Although differentiation-inducing agents have significantly improved the management of acute promyelocytic leukemia, no significant progress has been made in the treatment of other acute myeloid leukemias (AML). Numerous proteins involved in tumor development have so-called allosteric disulfide bonds amenable to modifications affecting protein structure and function. We have developed SK053, a small molecule and mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI). The aim of our studies was to evaluate anti-leukemic activity of SK053 in human AML cells. To validate if SK053 targets PDI, a binding assay and an insulin turbidimetric activity assay were used. Cytostatic/cytotoxic effects in HL60, NB4, KG-1 and MOLM14 cells as well as in primary AML cells were assessed with trypan blue exclusion. Differentiation of AML cells was studied with May-Grunwald-Giemsa staining, nitro blue tetrazolium reduction assay and flow cytometry analysis of CD11b, CD14 and CD15 levels and by RNA sequencing, qRT-PCR and western blotting (WB). We observed covalent binding of SK053 to PDI and inhibition of its enzymatic activity with IC50 of 10 μM. Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), a transcription factor crucial for neutrophils maturation, we evaluated the potential of SK053 to induce differentiation and cytostatic/cytotoxic effects in human AML cells. SK053 exerts significant cytostatic/cytotoxic activity in human AML cells (HL60, NB4, KG-1 and MOLM14), and induces differentiation of AML blasts into more mature myeloid cells. Incubation of AML cells with SK053 induced expression of CEBPA and hexokinase 3 mRNA in quantitative RT-PCR and increased amount of CEBPA protein in nuclear fraction measured in WB. Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes as well as for histone proteins. Expression of other genes of mature myeloid lineage such as adhesion molecules (collagen type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated. The GeneOntology analysis done with the RNAseq results revealed enrichment of gene transcripts regulating myeloid cells differentiation. SK053 exerts potent anti-leukemic activity and induces differentiation of numerous types of human AML cells. Targeting allosteric disulfide bonds with small molecule inhibitors presents a promising therapeutic strategy in AML. Citation Format: Dominika Nowis, Justyna Chlebowska, Pawel Gaj, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, Pawel Leszczynski, Piotr Stawinski, Szymon Klossowski, Ryszard Ostaszewski, Krzysztof Giannopoulos, Rafal Ploski, Dariusz Plewczynski, Jakub Golab. SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2015-5347