ALBERT

Description: Abstract 2864 Myeloproliferative neoplasms (MPN) are clonal disorders characterized by excessive production of mature blood cells and secondary stromal changes in the bone marrow leading to myelofibrosis. The role of a number of fibrogenic cytokines derived from megakaryocytes or monocytes in driving reactive secondary responses of bone marrow stroma cells (BMSC) in fibrosis has been repeatedly discussed. The aim of this study is the comparative analysis of BMSC from MPN patients and non-MPN donors in regard to hematopoietic stem and progenitor cell supporting capacity, extracellular matrix (ECM) remodeling in 3D collagen-based scaffolds as well as their in situ localization in the bone marrow niche. BMSC from bone marrow routine aspirates were obtained from patients diagnosed with chronic myeloid leukemia (CML; n=5), essential thrombocythemia (ET; n=5) and polycythemia vera (PV; n=5) and compared to BMSC isolated from control BM (untreated initial diagnosis of non-Hodgkin′s lymphoma or from patients undergoing orthopedic joint replacement surgery). BMSC cultures from prefibrotic MPN patients (as determined by reticulin staining) were established and fulfilled MSC criteria according to common consensus comparable to BMSC cultures from control individuals. As BMSC have been shown to support hematopoiesis in vitro, we compared their constitutive production of hematopoietic-affiliated cytokines. BMSC from MPN and non-MPN patients expressed IL-7, M-CSF, FLT3-L, IL-6, TPO and LIF in the following order: control BMSC 〉 ET BMSC 〉 PV BMSC 〉 CML BMSC. In myeloid colony formation unit (CFU) assays using healthy CD34+ hematopoietic stem and progenitor cells as readout, myeloid CFU activity was highest in the supernatant of control BMSC suggesting a decrease of hematopoiesis supporting capacity in BMSC from MPN patients. When activated through contact with the collagenous matrix in 3D scaffolds, only BMSC from ET (in 3 of 5 cases studied) or CML (1/5) patients extensively remodeled and significantly contracted the collagenous matrix. A significant up-regulation of ECM proteins –fibronectin, collagen type I, collagen type IV, laminin and osteopontin - detected by qtRT-PCR and immunohistochemistry was seen in BMSC from MPN-patients. To evaluate if BMSC contribute to ECM remodeling in vivo we analyzed fibronectin deposition in corresponding bone punches of ET patients. Co-stainings with the recently identified BMSC markers CD271 and CD146 revealed that BMSC in ET patients strongly co-expressed fibronectin and were mobilized from their perivascular and endosteal niche. BMSC in ET patients diffusely localized in the bone marrow and seemed to be attracted by dysplastic megakaryocytes. In addition, local accumulation of unbound fibronectin was detected in prefibrotic ET patients. Remarkably, conventional reticulin staining was absent in these patients suggesting a predictive role for fibronectin staining in myelofibrosis. Therefore - and because recent evidence has shown that the amount of reticulin staining does not correlate with disease progression and prognosis - we analyzed matrix remodeling in bone marrow core biopsies on tissue microarrays including CML (n=16), ET (n=15) and PV (n=16) in comparison to primary myelofibrosis (MF, n=12) and non-Hodgkin′s lymphoma (n=19). Clinical data including several laboratory parameters associated with myeloproliferative disorders (spleen size, molecular diagnostics) have been assessed retrospectively and correlated with fibronectin and CD271 expression. There was a positive correlation for higher fibronectin and CD271 expression with lower haemoglobin levels (p = 0.001[both]) and higher blast counts (p = 0.006 [fibronectin]; p = 0.027 [CD271]) at diagnosis, but no correlation with JAK2 V617F or BCR/ABL expression or spleen size. In conclusion, our data indicate that MSC from MPN patients actively participate in the process of myelofibrosis after being recruited and attracted by the malignant hematopoietic clone/dysplastic megakaryocytes. Further analysis will reveal if fibronectin and CD271-staining are suitable routine markers for disease prognosis and potential targets for therapeutic strategies aimed at the prevention of disease progression towards secondary MF. Disclosures: No relevant conflicts of interest to declare.

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: Introduction Telomere length (TL) reflects the replicative potential of a cell and is a useful parameter to assess chronological age of an organism. Aplastic anemia (AA) and dyskeratosis congenita (DKC) are bone marrow failure syndromes (BMFS) characterized by shortened telomeres compared to age-matched healthy individuals in peripheral blood leukocytes. Human aging is associated with DNA methylation (DNAm) changes at specific sites in the genome. The aim of this study was to analyze whether aging specific epigenetic modifications can be used to track donor age and reflect premature aging in patients with AA and DKC. Methods A large set of publically available DNAm profiles were used to identify an Epigenetic-Aging-Signature. The DNAm level of three corresponding CpG sites was then analyzed by pyrosequencing after bisulfite conversion. The results were used for linear regression models for age predictions. This method was applied with peripheral blood leukocytes of a training as well as a validation set. Furthermore, TL analysis by flow-FISH of peripheral blood leukocytes of 106 healthy donors was used to generate age-adapted reference values. Pyrosequencing and telomere length analysis were carried out in 16 AA and 6 DKC patients Results Our analysis revealed that DNAm levels at three AR-CpGs – located in the genes ITGA2B, ASPA and PDE4C – were best suited for age predictions. The mean absolute deviation from chronological age in the validation set of 69 donors was less than 5 years. Age-adapted telomere length analysis revealed significant telomere attrition in AA (- 1.1 kb) and DKC patients (-3.7 kb, all below the 1% percentile). In analogy, the epigenetic changes revealed premature aging in AA patients – particularly in those which revealed extensive telomere attrition. Conclusion Our results indicate that patients with AA and DKC undergo massive premature aging on the epigenetic level comparable to the observed telomere shortening in these diseases. Determination of this newly defined Epigenetic-Aging-Signature in combination with telomere length measurement may be used to further refine diagnosis of AA and DKC in the clinical setting. Disclosures: No relevant conflicts of interest to declare.

Description: The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment. Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice. In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the BCR-ABL translocation. Telomere length (TL) reflects the replicative history of eukaryotic cells and progressive telomere shortening is associated with genetic instability. Clinically, accelerated telomere shortening has been demonstrated in patients with CML and was found to correlate with disease progression and clinical risk score in the pre-tyrosine kinase inhibitor (TKI) era. The aim of the current study was to investigate whether telomere length (TL) at diagnosis might predict response to treatment in patients receiving nilotinib as first line treatment of chronic phase CML on the ENEST1st study (NCT01061177) Methods and Patients TL analysis of peripheral blood leukocytes was analyzed using monochrome multiplex quantitative PCR in blood samples from 93 newly diagnosed CML patients enrolled on study in Germany. One extreme outlier was excluded from the analysis. 89 healthy controls were used for age-adaption of TL. Median age of the analyzed CML patients was 49.6 years (range: 19-83), Sokal (32 low, 31 intermediate, 13 high risk) and Euro (34 low, 36 intermediate, 6 high risk) scores were available in 76 patients. Response to treatment according to standard criteria was available at 3 month (mo, n=79 patients), 6 mo (n=75), 12 mo (n=71) and 18 mo (n=55) after treatment start. Results Mean age-adjusted TL in CML patients was significantly shortened compared to normal individuals (ΔT/S ratio: -0.30 +/- 0.67, p=

Description: Introduction: AML is an aggressive clonal myeloid neoplasia. Incidence increases with age and age is also the most dominant prognostic indicator for adult patients with AML. Although numerous genetic events have recently been identified in good and intermediate risk AML, understanding of the pathophysiology behind poor risk AML, which is most predominant in elderly patients and characterized by complex karyotypic abnormalities remains limited. Changes in the epigenetic signature and telomere attrition are two well recognized hallmarks of aging and genetic instability. Here, we investigate the hypothesis that poor risk AML arises from a prematurely aged, genetically instable hematopoietic stem cell population. Methods: We analyzed 45 newly diagnosed AML patients who were followed from diagnosis, through achievement of complete cytogenetic remission (CCR) and/or until refractory disease or relapse. Overall, 95 samples (n=88 bone marrow (BM), n=7 peripheral blood, (PB)) were available. Analysis were performed at first diagnosis (Dx, n=20), after two cycles of induction chemotherapy (post-IC, n=42), after three additional cycles of consolidation chemotherapy (post-CC, n=28) and after one year follow up (n=5). Mean age of the cohort was 50.4 years (range 21-75y). A recently developed “epigenetic aging signature (EAS)” capable to predicted to the chronological age of healthy individuals with a mean absolute deviation (MAD) of 5 years (Weidner et al., Genome Biol, 2014) was used to express the epigenetic age of the AML samples. Epigenetic changes in DNA methylation were analyzed by pyrosequencing and monochrome multiplex quantitative-PCR was used for telomere length (TL) analysis. TL was age-adapted based on a control cohort of 87 healthy donors as described previously. Results: A cross-sectional analysis of TL reveals substantial telomere shortening in AML patients at the time of first diagnosis compared to healthy controls (mean±SE: -0.65 ± 0.2 T/S ratio, p=0.001). When studied sequentially, the TL deficit was less pronounced in remission after IC (-0.33 ± 0.1 T/S ratio, n=34) however, still significantly shortened compared to age-adapted healthy subjects (p= 0.001). This tendency continued in patients in remission after CC (-0.18 ± 0.1 T/S ratio, n=24, p=0.03) and one year (-0.25 ± 0.1 T/S ratio, n=5, p=0.28). In comparison, in patients with active leukemia, i.e. persistent AML post-IC (-0.61 ± 0.2 T/S ratio, n=8, p=0.006) or relapsed AML post-CC (-1.0 ± 0.2 T/S ratio, n=4, p=0.04), TL was comparable to untreated, diagnostic samples. By using EAS for age prediction, leukemic cells from AML patients with active disease at first diagnosis (+29.6 ± 8.6 years, p=0.001), in persistence (+28.9 ± 11.2 years, p=0.01) and in relapse (+86.8 ± 29.3 years, p=0.01) were found to be significantly and prematurely aged compared to their respective chronological age. In contrast, premature aging based on EAS was substantially less pronounced in CCR post-IC (+4.5 ± 1.8 additional years, p=0.01) and post-CC (+1.4 ± 2.3 additional years, p=0.52). Conclusion: We demonstrate that hematopoietic cells from AML patients at diagnosis are characterized by premature aging indicated both by accelerated telomere shortening and EAS. Not unexpectedly, this premature aging signature of leukemic cells persists in refractory and/or relapsed patients. However interestingly, this phenomenon (although less pronounced) is not completely reverted once patients reach cytogenetic remission either post induction chemotherapy, post consolidation or one year after diagnosis, i.e. when hematopoiesis has shifted from clonal leukemic to non-clonal cells. These data support the conclusion that either normal cells in patients with AML undergo accelerated aging during conventional treatment or alternatively, AML arises from prematurely aged (yet non-clonal cells) hematopoietic stem cells. Disclosures Wagner: Cygenia GmbH: Employment, Equity Ownership, Patents & Royalties.