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Abstract 3489 Telomeres are a repetitive DNA sequences associated with a protein complex named shelterin that protect chromosome ends. Two types of mechanisms maintain telomere in cancer cells. The first involves telomerase an enzyme able to copy the telomeric motif that consists of three principal subunits, including the telomerase reverse transcriptase hTERT. The second, named ALT (Alternative Lengthening of Telomere), corresponds to the recombination between telomeres that involves notably a complex formed by the topoisomerase III alpha (hTopoIIIa), BLM, RMI1 and RMI2. Little is known about the involvement of the ALT mechanism in B-chronic lymphocytic leukemia (B-CLL). In fact this leukemic disease shows low telomerase activity, shelterin defect and telomeric dysfunction. In an effort to characterize ALT cells from 31 B-CLL patients, we analyzed their telomere length and telomerase activity. B-CLL patients showed almost no hTERT transcript (detected in three cases), low telomerase activity (detected in 7 cases) and a telomere average size ranging from 3 to 10 kb. Moreover, a strong deregulation of genes encoding three shelterin proteins, TRF1, TRF2, Pot1, and an at least two fold downregulation of hTopoIIIa gene expression in 21 cases were observed, suggesting the presence of a telomere maintenance dysfunction affecting both mechanisms, telomerase dependent and ALT. CpG island methylation has been mapped for both promoters and if hTERT shows a disseminated methylation profile in 22 patients, for hTopoIIIα we identified nine CpG upstream the minimal promoter, being methylated in 19 of our 31 analyzed patients. We then performed luciferase experiments and we showed that methylation in this 9 CpG induced a strong inhibition of hTopoIIIa transcription. Finally we correlated telomere length and hTopoIIIa methylation status as we observed that 25.4% of the hTopoIIIa promoters were methylated in patients with shorter chromosomes and only 11.1 % were methylated in patients with longer telomeres (p As nearly no telomerase activity have been detected in our patients and as downregulation of hTopoIIIa could increase recombination rate between sister chromatid, methylation of hTERT and hTopoIIIa promoter CpG islands may lead to telomere dysfunction and increased genetic instability in B-CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2813 Background: A somatic point mutation in the Janus kinase 2 gene (JAK2) leading to the expression of the JAK2 V617F mutant occurs with high frequency in myeloproliferative neoplasm (MPN) patients (>95 % in polycythemia vera (PV), >50 % in essential thrombocythemia (ET) and primary myelofibrosis (PMF)). It confers constitutive activity to the kinase and results in cytokine hypersensitivity and a proliferative advantage of hematopoietic progenitor cells. These findings suggest that inhibiting JAK2 V617F may be therapeutically beneficial. Several JAK2 inhibitors are currently in clinical trials for the treatment of MPN, and first results show clinical improvements for PMF patients. However, since approximately 50 % of ET and PMF patients do not carry an activating mutation in JAK2, we speculate that the inhibition of signaling proteins other than JAK2 or in combination with JAK2 inhibition could be beneficial for these patients. Methods: We characterized compounds from different chemical classes, which previously have been published to be JAK(2) inhibitors. These compounds were compared in several assays using primary CD34+ cells from PV patients positive for the JAK2 V617F mutation and/or the JAK2 V617F-bearing cell line HEL. We used (quantitative) Western blot detections, in vitro kinase assays, proliferation assays, cell size measurements, cell cycle analyses and colony forming cell (CFC) assays to analyze the efficacy of the different inhibitors. Moreover, the IC50 values of the compounds were determined. Results: In total 15 published JAK2 inhibitors have been characterized in detail. As monitored in an in vitro kinase assay and by Western blot detection of phosphorylated signaling proteins, several compounds previously described as JAK(2) inhibitors did not target JAK2 V617F. However, some compounds, which turned out not to inhibit JAKs, showed growth-inhibitory effects on JAK2 V617F-positive cells. Such compounds could be used in combination with a specific JAK inhibitor in order to achieve beneficial effects on suppression of cell proliferation and induction of apoptosis. We could demonstrate that the combined application of a JAK inhibitor together with an Aurora kinase inhibitor was most promising: application of both Janus and Aurora kinase inhibitors in proliferation assays and CFC assays demonstrated a more effective suppression of growth than achieved by respective single treatments. Interestingly, we observed in the CFC assay that a JAK2 inhibitor seems to preferentially suppress the growth of erythroid colonies, while an Aurora kinase inhibitor preferentially blocks myeloid colony growth. Conclusion: Here we present a comparative analysis and a detailed biochemical characterization of numerous compounds from different chemical classes, all supposed to be JAK(2) inhibitors. We confirmed JAK(2) inhibitory activity for several compounds but not for all. In addition, we identified some compounds, which effectively inhibited the proliferation of JAK2 V617F-bearing cells without targeting JAK2. Thus, combined inhibition of JAK2 and other kinases may represent a promising therapeutic strategy. In particular, we suggest that a combination of Janus and Aurora kinase inhibitors might be beneficial for the treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma is, despite the emergence of new treatment strategies in recent years, still a lethal disease in 2005. Over the last several years, significant insights into the dysregulation of various signal transduction pathways of MM have emerged and have evolved the development of new agents. Histone deacetylase inhibitors as valproic acid are compounds that inhibit HDACs enzymes that, in conjunction with histone acetylases, regulate the acetylation state of histones and, by extension, the conformational status of chromatin. VPA is clinically known to be used in treatment of different types of epileptic disease. Effects and signal transduction pathways of cell death have been studied in cells harvested and purified from routine bone marrow aspirates of several patients with multiple myeloma, as well as on myeloma cell lines (OPM2, U266) treated with a physiologic dose range of valproic acid (1–4 mM). We observe significant in vitro toxicity starting at doses of 1 mM for 24h in cell lines as well as in patient cells using an XTT based cytotoxicity assay. The question we adressed was the mechanisms by which the MM died. Flow cytometric analysis with PI / Annexin V staining does not show apoptosis features, nor do TUNEL staining or DNA fragmentation assays, suggesting non activation of the intrinsic apoptosis pathway. In contrast, cell morphology of treated cells stained with May-Gruenwald-Giemsa staining show increase in multinucleated giant cells. Multinucleation has often been described in cells which die through mitotic catastrophe. Further experiments exploring this hypothesis will be conducted. Effect of 48h valproic ac on myeloma cell lines Effect of 48h valproic ac on myeloma cell lines Effect of 48h valproic ac on CD138 cells from myeloma patients Effect of 48h valproic ac on CD138 cells from myeloma patients