ALBERT

Description: Langmuir DOI: 10.1021/acs.langmuir.5b02907

Description: Introduction: BCR-ABL has been shown to be both necessary and sufficient for the induction of chronic myeloid leukemia (CML) in experimental models. The selective tyrosine kinase inhibitor imatinib has been shown to induce major cytogenetic remissions in 〉80% of CML patients in chronic phase. However, resistance to imatinib develops frequently especially in later stages of the disease. Therefore, investigations on potential synergistic treatment strategies are required in order to be able to overcome clinical resistance. Materials and Methods: Protein expression analysis of K562 leukemic cells was performed using 2-dimensional gel electrophoresis after treatment with imatinib (4 μM) or DMSO (as a solvent control) for 24 hours and 48 hours. Resulting differentially expressed prominent proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential expression was confirmed by western blot analysis for some candidate proteins. Cytotoxicity was measured in K562 cells and primary CD34+ cells from patients with CML at diagnosis using MTT-assay, the fraction of apoptotic cells was measured by flow cytometry using propydium iodide (PI). Results: Using 2-dimensional gel electrophoresis, thirteen candidate proteins were identified to be down-regulated whereas 6 proteins were up-regulated by cell treatment with imatinib. Interestingly, we detected the down-regulation of the eukaryotic translation initiation factor 5A (eif5A), which is essential for cell proliferation. Eif5a represents the only known eukaryotic protein activated by posttranslational hypusination. Formation of hypusine is one of the most specific polyamine-dependent biochemical reactions and its biosynthesis involves two enzymatic steps which can be inhibited with the compounds ciclopirox and GC-7. To assess the effects of the hypusination inhibitors as single agent and in combination with imatinib on Bcr-Abl positive or negative cells, we performed cytotoxicity- and apoptosis-assays. Hypusination inhibition resulted in substantial and dose-dependent cytotoxicity and in an increasing fraction of apoptotic cells in K562, HL-60 and primary Bcr-Abl positiv CD34+ cells. Interestingly, synergistic effects of imatinib and ciclopirox or GC-7 could be detected on cellular cytotoxicity and apoptosis in both Bcr-Abl positive K562 cells and Bcr-Abl positive primary CD34+ cells from four newly diagnosed CML patients. Discussion: We could detect significant differences in protein expression levels in the BCR-ABL-dependent cell line K562 upon treatment with imatinib. Furthermore, we observed down-regulation of eif5a which so far has not been shown to be involved in proliferation control of Bcr-Abl positive leukemias. Eif5a suggests to be a promising target for imatinib-based synergistic treatment strategies. Considering that a number of hypusination inhibitors are clinically approved drugs with acceptable toxicity profiles, our results might have important implications for the design of novel synergistic treatment strategies in patients with Bcr-Abl-positive leukemias and other imatinib-responsive diseases.

Description: Introduction: The selective tyrosine kinase inhibitor imatinib (formerly STI571, Glivecâ) has been shown to block phosphorylation of tyrosine residues by occupying the ATP binding site of the Abl tyrosine kinases Bcr-Abl, c-Abl, v-Abl and Abl-related gene (ARG), as well as platelet-derived growth factor receptor (PDGF) alpha and beta and of the receptor for human stem cell factor (SCF) c-kit. We chose a large scale phospho-proteomics approach to identify novel downstream targets of imatinib which could possibly be utilized for combined treatment strategies. Material and Methods: Phospho-proteomics was performed by comparison of large scale phosphotyrosine-immunoprecipitation of imatinib (10 mM/2 hours) versus DMSO treated K562 cells separated by one-dimensional polyacrylamide gelelectrophoresis. In addition enriched CD34+ cells (〉70%) of a newly diagnosed Bcr-Abl positive CML patient were used immediately after purification and treated in the same way as described above. Resulting differentially immuno-precipitated proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification via peptide mass-fingerprinting and peptide sequencing was performed using Mascot search tool and NCBI nr database. Differential phosphorylation was confirmed by combined immunoprecipitation and western blot analysis of selected candidate proteins. Results: Phospho-proteomics of K562 cells revealed 8 differentially phosphorylated proteins after a two hour treatment with imatinib including the recently identified c-cbl, and Bcr-Abl itself, the latter confirming autophosphorylation. Ship2 which was originally identified as beeing constitutively phosphorylated in chronic myelogenous leukemia progenitor cells showed reduced, imatinib sensitive phosphorylation. Remaining candidates could be classified as being involved in protein folding or in ATPase activities associated with a variety of functions (type II AAA). The analysis of primary CD34+ cells from a CML patient showed a predominance of the nonmuscular myosin heavy chain protein in different molecular weight forms. Discussion: We detected significant imatinib-dependent differences in protein phosphotyrosine-immunoreactivity of the Bcr-Abl -dependent cell line K562. Previously identified down-stream targets of Bcr-Abl could be confirmed and novel candidate proteins were identified. Phosphorylation of Ship2, a previously identified down-stream target of Bcr-Abl, was found to be inhibited by imatinib treatment. Ongoing studies are aimed at the characterization of the role of the identified phospho-proteins, particularly type II AAAs for Bcr-Abl induced signal transduction as well as for the development of resistance to imatinib.

Description: In normal somatic cells telomeres shorten with each cell division because of the end-replication problem. The ribonucleoprotein enzyme telomerase is able to prevent replicative telomere shortening and to maintain or elongate telomere length. In 90 % of tumour cells the enzyme telomerase is found to be upregulated. Chronic myeloid leukemia is a disorder characterized by a reciprocal translocation between Chromosome 9 and 22, leading to the so called Philadelphia chromosome harbouring the BCR-ABL translocation. BCR-ABL positive leukemic stem cells are characterized by increased turnover leading to accelerated telomere shortening as opposed to their normal counterparts. It is unclear to date whether accelerated telomere shortening in Bcr-Abl-positive cells is linked to genetic instability eventually leading to the acquisition of secondary clonal events that might propagate acceleration of the disease to blast crisis. Therefore we aimed to characterize Bcr-Abl positive chronic myeloid leukemia cell line K562 with or without inhibition of telomerase activity under long-term culture conditions. K652 cells were expanded for 400 populations doublings (PD) with or without treatment with the small molecule telomerase inhibitor BIBR1532 in vitro. While telomeres in untreated control cells remained relatively constant, telomeres in BIBR1532 treated cells underwent replicative shortening from 10 kb to 3 kb (as measured by flow FISH), reflecting a rate of 22 base pairs (bp) lost per PD. No difference in growth kinetics were observed until that stage. We next characterized treated K562 with short telomeres (K562-S) in contrast to control cells with long telomeres (K562-L) for the expression of telomere and telomerase-binding proteins. No difference in mRNA expression for any of the candidate proteins were observed by RT-PCR. Comparative analysis of global protein expression was performed by 2D gel electrophoresis. Taken together, 23 protein spots were found to be differentially expressed between treated and untreated cells, fifteen of which were already identified by mass spectometry. Additionally, we analysed the cells for the acquisition of additional cytogenetic abnormalities by M-FISH. Interestingly, in this ongoing study, we consistently found acquisition of genetic material on chromosome 7 in treated as compared to untreated cells. To study radiation sensitivity under BIBR1532 treatment, K562 cells were exposed to increasing doses of irradiation. Interestingly, despite of a dose-dependent increase in the fraction of apoptotic cells in the pre-treated as opposed to control cells, no accumulation in the number of double strand breaks or lethal aberrations were detected. Interestingly, telomere shortening after telomerase inhibition translated to increased sensitivity to Imatinib (IC50 0.6 μM vs. IC50 1.2 μM). Taken together, telomerase inhibition represent a attractive new therapeutic strategy in Bcr-Abl positive leukemias. However, careful evaluation of side effects need to be studied on the proteomics and cytogenetic level.

Description: Abstract 3011FN2 Background: Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at high risk of acquiring fungal infections. Post-transplant immune deficiency, additional immunosuppression, viral infection, persistent fever during neutropenia, and GvHD are known risk factors for fungal infections. Antifungal prophylaxis early after allogeneic stem cell transplantation is therefore indicated. Oral antifungal prophylaxis with extended-spectra azoles, e.g. voriconazole or itraconazole, is preferentially used in pediatric patients after allogeneic HSCT, although only few studies have been published on the matter. Due to the absence of controlled studies for oral antimycotic prophylaxis in children after BMT, we retrospectively analyzed patients who received posaconazole based on data in adult patients. Cornely and colleagues published that posaconazole was more effective than fluconazole or itraconazole in the prevention of invasive fungal infections in adult patients undergoing chemotherapy for myelodysplastic syndrome or acute myeloid leukemia. Retrospectively, we assessed the safety, feasibility and initial data of efficacy for itraconazole, voriconazole and posaconazole with regard to pediatric patients and adolescents after high-dose chemotherapy and HSCT. Patients and Methods: The study is a single centre, retrospective survey on antimycotic prophylaxis in pediatric patients under eighteen years of age undergoing allogeneic HSCT between January 2004 and June 2011 at the University Children's Hospital Tübingen, Germany. 50 pediatric patients received posaconazole, 50 pediatric patients received voriconazole and 50 pediatric patients received itraconazole after HSCT as oral antifungal prophylaxis. The observation period was defined as the time from the start of oral prophylactic treatment with itraconazole, voriconazole or posaconazole until the end of oral antimycotic prophylaxis, a maximum of 200 days post transplant. Results: The median observation period in the itraconazole group (median age 8.5 years) was 109 days (range 20 – 200 days), 105 days (range 18 – 200 days) in the voriconazole group (median age 7.5 years) and 117 days (range 16 – 200 days) in the posaconazole group (median age of 8.5 years), respectively. No incidences of proven or probable invasive mycosis according to EORTC definitions (De Pauw et al., 2008) occurred under itraconazole, voriconazole, and posaconazole during the observation period. Cases of possible fungal infections according to EORTC definitions in the voriconazole group (10%) and itraconazole group (5%) were higher, but still not significantly different from those in the posaconazole group (0%). The percentage of patients with adverse events potentially related to clinical drugs was higher in the voriconazole (17.5%) and itraconazole (15.0%) group than posaconazole (9.8%) group, but not statistically significant. Laboratory investigations were comparable during itraconazole, voriconazole, and posaconazole treatment. Statistical analysis by the Wilcoxon Matched-Pairs Signed Rank Test showed a significant increase beyond the upper normal limit of ALT between baseline before conditioning and maximum during oral antimycotic prophylaxis in all three groups, itraconazole (P = 0.000015), voriconazole (P = 0.0033), posaconazole (P = 0.000013), and a significant increase of AST during itraconazole (P = 0.00023), voriconazole (P = 0.0004), and posaconazole (P = 0.00026) treatment. Significant decreases of potassium (P = 0.000000024) and bicarbonate (P = 0.0026) below the lower limit of the normal range were observed during voriconazole treatment. Moderate elevation of CsA levels were observed during voriconazole and posaconazole treatment, but were also not statistically significant. Conclusion: Taken together, itraconazole, voriconazole and posaconazole showed comparable efficacy as antifungal prophylaxis in pediatric patients after allogeneic HSCT. With regard to the clinical tolerability, posaconazole was superior to itraconazole and voriconazole. Disclosures: No relevant conflicts of interest to declare.

Description: Telomere length has been linked to disease stage and degree of (pan-)cytopenia in patients with bone marrow failure syndromes. The aim of the current study was to analyze the impact of replicative stress on telomere length in residual glycosylphosphatidylinositol-positive (GPI+) versus GPI– hematopoiesis in patients with paroxysmal nocturnal hemoglobinuria (PNH). Peripheral blood granulocytes from 16 patients and 22 healthy individuals were analyzed. For this purpose, we developed proaerolysin flow-FISH, a novel methodology that combines proaerolysin staining (for GPI expression) with flow-FISH (for telomere length measurement). We found significantly shortened telomeres in GPI– granulocytes (mean ± SE: 6.26 ± 0.27 telomere fluorescence units [TFU]), both compared with their GPI+ counterparts (6.88 ± 0.38 TFU; P = .03) as well as with age-matched healthy individuals (7.73 ± 0.23 TFU; P 〈 .001). Our findings are in support of a selective growth advantage model of PNH assuming that damage to the GPI+ hematopoietic stem-cell (HSC) compartment leads to compensatory hyperproliferation of residual GPI–HSCs.

Description: Imatinib mesylate (STI571) is a competitive Bcr-Abl tyrosine kinase inhibitor and has yielded encouraging results in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stroma tumors (GISTs). Apart from inhibition of the Abl protein tyrosine kinases, it also shows activity against platelet-derived growth factor receptor (PDGF-R), c-Kit, Abl-related gene (ARG), and their fusion proteins while sparing other kinases. In vitro studies have revealed that imatinib mesylate can inhibit growth of cell lines and primitive malignant progenitor cells in CML expressing Bcr-Abl. However, little is known about the effects of imatinib mesylate on nonmalignant hematopoietic cells. In the current study we demonstrate that in vitro exposure of mobilized human CD34+ progenitors to therapeutic concentrations of imatinib mesylate (1-5 μM) inhibits their differentiation into dendritic cells (DCs). DCs obtained after 10 to 16 days of culture in the presence of imatinib mesylate showed concentration-dependent reduced expression levels of CD1a and costimulatory molecules such as CD80 and CD40. Furthermore, exposure to imatinib mesylate inhibited the induction of primary cytotoxic T-lymphocyte (CTL) responses. The inhibitory effects of imatinib mesylate were accompanied by down-regulation of nuclear localized RelB protein. Our results demonstrate that imatinib mesylate can act on normal hematopoietic cells and inhibits the differentiation and function of DCs, which is in part mediated via the nuclear factor κB signal transduction pathway.