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The main function of vacuolar H + ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H + ATPase ( vatB ) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54 874 Da. The predicted protein sequence is highly homologous to previously determined V/H + ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid-phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.

Abstract 3732 Background: The BCR-ABL T315I mutation at the gatekeeper residue is frequent in advanced phases of chronic myeloid leukemia (CML) and is one of the main cause of resistance to Tyrosine Kinase Inhibitors (TKI) by disrupting important contact points between the drugs and the enzyme. Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown. The aim of the study was to develope a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of T315I mutation thus improving both the diagnostic resolution and the knowledge on the behaviour of the mutated clone. Methods: We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring T315I mutation or the wild type form of BCR-ABL. CD34+ cells were enriched from CML patients who were positive for T315I mutation, or from patients who lost T315I mutation after ponatinib treatment. In addition we tested CML patients without T315I mutation as control and BM samples from healthy donors to establish the specificity of the method. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the BCR-ABL T315I sequence. Slides were analyzed by fluorescence confocal microscopy. Results: We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 3 to 90%. In addition these data indicate that fluorescinated BCR-ABL T315I/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of T315I positive cells collected from a patient who lost the mutation (evaluated by sequencing) after ponatinib therapy and acquired T317 mutation a small amount of T315I positive CD34+ cells were still present. This percent did not exceeded 2% of the total CD34+ cell population. Importantly, the lack of positivity detected in CD34 positive cells from CML patient without mutations or in 20 healthy subjects demonstrates a high specificity of this method. Conclusions: BCR/ABL T315I/PNA probe method displays high specificity and reliability in discriminating cell subpopulations harbouring the mutation. In addition, it allows to analyze the CD34+ population at the single cell level and to monitor the behaviour of the clone at the stem/progenitor cell level. This approach allows to monitor longitudinally the evolution of the mutated population over time, the response to specific drugs active against T315I mutants and to characterization the mutated stem/progenitor cell compartment in patients with CML. Disclosures: Saglio: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 4616 Background: The Sprouty family proteins, including Spred, were originally identified in Drosophila melanogaster as an antagonist of Breathless, the ortholog of Fibroblast Growth Factor Receptor (FGFR) in mammals. These proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. Their main function is to deregulate the RAS/MAPK and RAS/RAF/ERK signal pathways by physically interacting. The role of Spread proteins in haematological malignancies is still not been clarified. Aim: The aim of this study was to investigate a possible involvement of Spred in maintaining the aberrant TK signalling in patients affected by acute myeloid leukaemia (AML). Methods: Bone marrow (BM) cells were collected from 82 AML patients at diagnosis and 10 BM samples from healthy donors as control. In addition 20 patients were analyzed during follow-up at the time haematological remission. All the patients included had been characterized at the cytogenetic level by conventional karyotyping, and screened by reverse transcriptase-PCR for the presence of the most frequent fusion transcripts or mutations. We analysed Spread mRNA expression by RQ-PCR and the protein by Western blot and immunofluorescence assay. In addition gain of function experiments were performed by transfecting with Spred coding sequence different leukemic cells lines lacking Spred activity. Results: We found that Spread mRNA levels were significantly decreased in AML compared to healthy subjects (2−DDct = 0,1 in AML compared to 0,6 in controls) (p Conclusions: A decreased expression and activity of Spred, a negative regulator of TK activity through Ras pathway, is implicated in sustaining the oncogenetic signalling and abnormal proliferation induced by tyrosine kinase proteins in acute myeloid leukemia patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3472 Background: The JAK2V617F gene mutation is a common feature of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF); however the disease can occur without the JAK2 mutation suggesting that ET, PV and PMF share a common molecular mechanisms which is still unknown. In addition, in patients with the JAK2V617F mutation a substantial proportion of progenitor cells do not bear the JAK2V617F mutation and it is still unclear whether this population is normal or not. Methods: 64 patients affected by MPNs and 12 healthy subjects were included in the study. Twenty three patients were affected by ET, 25 by PV and 22 by PMF. We analyzed the expression, of the anti-apoptotic gene Bcl-xL, both at mRNA and protein level by qRT-PCR and immunofluorescence assay with specific antibodies. Erythroblasts, CD34+ and CD34+CD38-cell populations were selected and analyzed. Moreover, we designed specific PNA (Peptide Nucleic Acid) for JAK2V617F and for Bcl-xL isoform to identify their presence/expression at single CD34+ cell level. Results: Bcl-xL protein and mRNA were found progressively over-expressed in erythroblasts, in CD34+ and in CD34+ CD38- cells from ET, PV and PMF patients without any differences between JAK2V617F and wild type (WT) patients. The median protein level in erythroblasts increased from 22 Relative Units (RU) in ET, to 68,5 in PV and to 85 in PMF. Similarly, in CD34+ Bcl-xL increased from 30 RU in ET, to 89 RU in PV, and to 135 RU in PMF. Finally, CD34+CD38-increased from 17.4 RU in ET, to 45.5 RU in PV, to 101.7 RU in PMF. In addition, analysis by PNA identified distinct progenitor cells population. In patients bearing the JAK2 mutation, three CD34+ cell sub-populations were found: (a) one JAK2 positive and with high levels of Bcl-xL; (b) one JAK2 negative and with high levels of Bcl-xL. Thus, the cell population showing high levels of Bcl-xL is always greater than the JAK2 positive one; (c) one double negative population. In patients without JAK2 mutation, two CD34+ cell populations are identified: (a) one characterized by high levels of Bcl-xL and (b) one double negative. The distribution of these cell subpopulations is different in the distinct MPN. Conclusions: We have identified a previously undisclosed CD34+ cell population which is characterized by high level of Bcl-xL expression and represents the majority of CD34+ cells both in wild type and mutated samples. Interestingly, in mutated samples, this population encompasses the JAK2 mutated one. These findings suggest that Bcl-xL plays an important role in MPN, although it remains undisclosed whether this activation is dependent from an upstream oncogenetic signal. Finally, with this technique we can now identify a putative residual normal CD34+ cells in MPN which may be useful for monitoring patients undergoing targeted therapy with inhibitors. Altogether, these findings disclose unfolded aspects of pathophysiology of MPN and may contribute to a better understanding of the pathogenesis of MPN. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Chronic Myeloid Leukaemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL. ABL-specific tyrosine kinase inhibitors (TKIs), whilst effective against mature CML cells, induce little apoptosis in stem/progenitor cells. However, in stem/progenitor cells TKIs exert potent anti-proliferative effects through a poorly understood mechanism. We showed that in CD34(+) CML cells FOXO1, 3a and 4 (FOXOs) were phosphorylated, predominantly cytoplasmic and inactive, consequent to BCR-ABL expression. TKIs decreased phosphorylation of FOXOs, leading to their re-localisation from cytoplasm (inactive) to nucleus (active), thus inducing G1 arrest. Of key importance, despite BCR-ABL activity, primitive quiescent CML stem cells showed low levels of FOXO phosphorylation and predominant nuclear localisation, resembling the pattern in normal stem cells. These results demonstrate for the first time that TKI-induced G1 arrest in CML progenitor cells is mediated by re-activation of FOXOs, whilst quiescence of CML stem cells is regulated by sustained FOXO activity. These data contribute to our understanding of CML stem cell quiescence and TKI activity, suggesting new strategies to target CML stem/progenitor cells by preventing or reversing this effect.

Abstract 3964 Poster Board III-900 Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. By using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we have identified Dab1 and Dab2 as genes involved in CML progression. In Dm the loss of function of these genes induced a worsening of the phenotype generated by hBcr-Abl expression. Moreover, an even stronger phenotype was obtained by silencing Disabled using RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Disabled is a non receptor tyrosine kinase, identified in Dm as an adaptor protein acting downstream of many tyrosine kinases receptors (RTK). One of the human homolog of Disabled, Dab1 is a large common fragile site gene, involved in neural migration and the other homolog Dab2 codes is an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many types of cancer suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM). Among those patients 55 were collected at diagnosis (19 enrolled in TOPS study), 9 patients in CP collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC, In addition 21 healthy donors (10 PB and 11 BM). In 18 patients, genes expression was analyzed during remission as well. Protein expression was investigated by Western Blot and Immunofluorescence. In addition, K562 cells were transfected with Dab plasmids to gain insight about the effects of Dab1/2 on cell proliferation and apoptosis. We found that in CML patients Dab1/2 expression levels were significantly decreased in either BM or PB (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p Disclosures No relevant conflicts of interest to declare.

The FoxO transcription factor promotes apoptosis and triggers cell cycle inhibition through multiple mechanisms. It is inactivated by PI3K/Akt induced phosphorylation thus resulting in nuclear exclusion and degradation. Moreover, several data demonstrated an abnormal activation of PI3K/Akt pathway in acute myeloid leukemia (AML) with contrasting prognostic significance. The aim of this study was to clarify the role of FoxO3 in AML and to investigate alternative pathways eventually responsible for FoxO3 inactivation. Importantly, we explore the effects of FoxO3 re-activation in CD34+ AML cells. Cell cycle was analyzed by FACS in CD34+ cell population as well as the levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization. Protein amount and localization were analyzed by Western blot and immunofluorescence, the DNA binding activity was measured by EMSA. 35 BM samples from AML patients at diagnosis and in 20 healthy donors were analyzed. Furthermore Spred1, known to be a FoxO3 target gene was quantified by RQ-PCR. We previously described the absence of Spred1 in AML patients and demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally BM cells were incubated with a PI3K inhibitor LY294002 and the IKK inhibitor PS1145, alone and in combination. Moreover, the t(8;21) positive Kasumi cell line was transfected with pECE-FoxO3 to evaluate FoxO3 effects on cell growth and apoptosis. We found that, while FoxO3 in control cells is localized in both nucleus (mean value of intensity of 21,4 ±2) and cytoplasm (14,6±1,7), it is completely cytoplasmatic in AML cells (18,1±4,6 in cytoplasm vs 8,2±4 in the nucleus) and enters the nucleus after chemotherapy or in vitro incubation with LY29400. Moreover, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and restored after therapy. Also the mRNA of Spred1 is rather undetectable at diagnosis (2−ΔΔCt= 0,009±0,3) and shows normal levels during remission (2−ΔΔCt = 2±1,5) or after LY29400 incubation (2−ΔΔCt =0,8±0,3). In addition LY294002 and PS1145 treatment results in FoxO3 partial nuclear re-localization while their association induces a complete nuclear shuttle suggesting that both pathways could be implicated in FoxO3 inactivation. The restoring of FoxO3 function by LY29400 in CD34+ cells induces quiescence of this progenitor cell compartment as demonstrated by the comparison of cell cycle kinetics and the decreased expression of CD47 (p=0,01). Finally, FoxO3 overexpression in transfected cells results in a block of proliferation rate (66% of inhibition compared to empty vector transfected cells) and apoptosis induction (35% vs 12%). Taken together these data suggest that FoxO3 inactivation may be crucial for the leukemic progression and demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy. On the other hand, the re-activation of FoxO3 induces quiescence of the stem cell compartment so providing a mechanism of escape from chemotherapy induced apoptosis.

Background: Philadelphia positive (Ph+) disorders, such as Chronic Myelogenous Leukemia (CML) are characterized by the presence of abnormal chromosome rising from translocation between chromosome 9 and 22 thus giving birth to a chimeric oncogenic protein named Bcr-Abl. This oncogenic kinase displays constitutive tyrosine kinase activity which leads to tyrosine residues autophosphorylation, in turn recruiting SH2 and/or PTB containing proteins. In the last decade Bcr-Abl targeted therapy has been successfully employed and, among currently available drugs inhibiting Bcr-Abl activity, Imatinb mesylate represents the most efficient. Despite the huge amount of data reporting the effects of Imatinib on signal transduction pathways (for example ERK1/2 and PI3K activation, CrkL phosphorylation etc…) in Ph+ leukemic cells rather few experimental evidences are available on the effects of Imatinib on adapter molecules. Therefore we are currently attempting to investigate such field. Aims and Methods: The significance of interactions occurring between Bcr-Abl and adapter molecules is still matter of debate. Most of the interactions so far described (CrkL, Grb2, PI3K p85 regulatory subunit etc…) appear to play a role in coordinating and integrating a plethora of signals which in turn lead to proliferation, cell survival and/ or cytoskeletal organization. In the last years few, but very interestingly, data supporting the hypothesis that adapter molecules might also act as c-Abl catalytic regulators have been presented. By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl interactions. Results and Conclusions: The data herein presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl and the RNA binding protein Sam68. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta and Sam68, supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68. Interestingly, preliminary data carried-out using RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/ Bcr-Abl might modulates splicing process of a gene encoding for a protein capable of regulating apoptosis events, such as Bcl-X. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam- 68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex mRNA splicing scenario.

The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. PI3K/Akt pathway has been reported to be abnormally activated in AML blast cells. The aim of this study was to investigate the function of FoxO in AML blast cells and the presence of alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 35 AML patients at diagnosis and after chemotherapy and from 20 healthy donors. The expression levels of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Furthermore, downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by FLT3. We have previously described the absence of Spred1 is AML patients and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells were incubated with 5 mM of the PI3K inhibitor LY294002 and 20 mM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation and with the combination LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in AML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in AML cells and it enters the nucleous after chemotherapy. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in AML cells is 8,2±4 in the nucleous and 18.1±4,6 in the cytoplasm. Additionally, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and reappears after therapy. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,009±0,3) and is upregulated during remission (mean value 2−ΔΔ= 2±1,5) or after LY29400 incubation (mean value =0,8±0,3). LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±3 and 12±3 respectively. Interestingly, the association of PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 25±4, suggesting that both pathways are implicated in FoxO inactivation. Taken together these observations suggest that FoxO inactivation may be crucial for the apoptosis arrest observed in AML. These data demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of selective inhibitors such as FLT3 or Akt inhibitors or standard chemotherapy and the IKK inhibitor.

Background Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM. Results RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein. Conclusion The data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

Introduction Multiple myeloma (MM) remains an incurable malignancy despite important recent advances in treatments. Neo-vascularization entails a crucial aspect of interactions between neoplastic plasma cells (PCs) and their microenvironment. Without it, MM would be unable to grow and progress, and would probably regress to a low-mass steady-state comparable to monoclonal gammopathy of undetermined significance (MGUS). To overcome drug resistance and improve clinical response to novel therapeutic approaches halting both PC growth and the increased bone marrow (BM) microvascular density are needed. In this setting, monoclonal antibodies against MM-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate membrane target structures expressed across all MM cells. The Eph receptors, a large family of receptor tyrosine kinases, have been implicated in many processes involved in malignancy, including alteration of the tumour microenvironment, and in angiogenesis, in both of which EphA3 likely plays an active role. Interestingly, the over-expression of EphA3 is sufficient to confer tumorigenic potential, although probably further mechanisms can occur to abnormally activate the receptor. A first-in-class engineered IgG1 antibody targeting the EphA3 was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EphA3 over-expressing hematological myeloid malignancies refractory to conventional treatment. Methods We investigated the EphA3 role in MM patients in order to define whether it may represent a potential new molecular target for a novel therapeutic approach with a specific anti EphA3 monoclonal antibody. The EphA3 expression was studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with MGUS (MGECs), of control subjects (normal ECs). Moreover, the effects of anti EphA3 antibody in MM were studied in vitro and in vivo in a MM xenograft mouse model. After written informed consent, BM aspirates were collected from 26 MM and 6MGUS patients. Normal ECs were derived from 5 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed both mRNA and protein levels of EphA3 in normal ECs, MGECs and MMECs and in MM cell lines by absolute RT-PCR and by WB coupled to immunofluorescence and FACS analysis respectively. Immunoistochemistry was also performed on MM BM biopsies. The biological effects of EphA3 targeting were studied in vitro silencing (siRNA) the EphA3 mRNA in MMECs and using the anti EphA3 antibody testing them in series of in vitro functional assays including viability, apoptosis, adhesion, migration, wound healing and angiogenesis tests. We further examined the inhibitory capacity of anti-EphA3 Ab on tumor growth in SCID mice bearing MM tumor cell xenografts. Finally, we assessed morphology, vessel density, and apoptosis of excised xenotransplanted tumors. Results Briefly, our data showed that EphA3 mRNA and protein levels are progressively increased from ECs to MGECs, reaching the highest values in MMECs. EphA3 stained intensely and diffusely MM microvessels and PC in MM BM biopsies. The EphA3siRNA MMECs revealed a protein level reduction of approximately 80% when compared to the control. We not detected viability or apoptotic defects, whereas in vitro adhesion, migration and angiogenesis inhibition was evident when compared to the not silenced counterpart. The anti EphA3 antibody inhibited MMECs migration and reduced in vitro MM angiogenesis. In particular, tumour masses developed in xenograft mice treated with anti-EphA3 Abs were smaller in size and showed foci of ischemic-hemorrhagic necrosis, in association with a significant (P < 0.05) reduction in the number of intact tumor microvessels. The proliferative activity was not significantly different from that observed in tumors from untreated or control isotype treated mice, while the apoptotic index was significantly (P < 0.05) increased in comparison with tumors from both groups of mice. Conclusions In this study we have characterized the role of the EphA3in MM patients, providing in vitro and in vivo experimental evidences that support the possibility of using EphA3 as a new molecular target for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 2500 Wilms' tumor gene 1 (WT1) is a tumor suppressor gene coding for a zinc finger transcriptional factor which was found overexpressed in acute myeloid leukemias (AML). In AML WT1 functions as an oncogene rather than a tumor suppressor. This may depend on the unbalanced expression of its different isoforms derived from alternative splicing in exon 5 (EX5+/−) or the presence of KTS in exon 9 ( KTS+/KTS-). In normal hematopoietic cells the physiological ratio between KTS+/KTS- isoforms is 1:1. KTS are inserted between the third and fourth zinc fingers thus influencing the DNA binding and therefore the transcriptional activity. The aim of the study was to investigate the possibility of an unbalanced ratio between the isoforms thus leading to the disruption of WT1 function. In addition we correlated KTS+ isoform with clinical and biological features. Methods: we analyzed KTS+/KTS- isoforms in 120 AML patients ( 102 BM samples, 18 PB), 63 chronic phase CML patients (48 BM and 15 PB). In addition we evaluated 5 samples of selected blast cells from AML patients. In a subset of 38 adult acute myeloid leukemia (AML) patients we measured all WT1 isoforms (A[EX5 -/KTS -], B[+/−], C[-/+] and D[+/+]. Quantitative PCR was used for the WT1 isoforms detection and measurement. Results: KTS+ isoform is significantly overexpressed with a ratio KTS+/KTS− 2:1 in both AML and CML patients and it is confirmed in blast cell samples. In the subgroup of patients analyzed for the expression pattern of all WT1 isoforms we detected a significant overexpression of isoform D (EX5+/KTS+) (median value 821) while the isoform A (EX5−/KTS−) is the less represented ( median value 303). In this subgroup the expression of KTS+ isoforms (B+D) is higher than KTS- ( A+C) with a median value of 642 compared to 421. The ratio of WT1 isoforms was not significantly different among FAB subgroups or according to cytogenetic risk, FLT3 (fms-like tyrosine kinase receptor-3) internal tandem duplication or exon 17 mutation, NPM (nucleophosmin) gene mutations and BAALC (brain and acute leukemia, cytoplasmic) gene expression. Conclusion: Although WT1 is overexpressed in AML and CML patients it lacks its typical tumor suppressor function. In this study we have demonstrated the overexpression of WT1 KTS+ isoforms. In addition we have previously demonstrated that KTS+ isoform does not have transcriptional activity since it is mainly localized within the cytoplasm. In conclusion, WT1 is overexpressed in AML but the unbalanced ratio between the isoforms may probably influence the transcriptional activity. Any possible correlation between the unbalanced ratio of WT1 isoforms and clinical outcome requires further prospective studies to be established. Disclosures: No relevant conflicts of interest to declare.

Abstract 2186 Poster Board II-163 Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression remain largely unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed a genome-wide genetic screening using our set-up model of human p210 Bcr-Abl transgenic Drosophila melanogaster (Dm) in which the expression of hBcr-Abl in a tissue specific manner is able to induce a severe eye-glazed phenotype or the formation of melanotic tumors, (clusters of hemocytes) when expressed into the fly lymph gland which represent the Dm hematopoietic system. A wide modifier screening of the whole fly genome containing approx 14.000 genes was performed using 278 fly stocks commercially available and carrying well characterized chromosome deletions. The resulting progeny was screened using the eye phenotype as first read-out system. Furthermore each deletion responsible for phenotype changes was analyzed either by expressing it into lymph gland as second read-out system, in order to analyze their function into a haematopoietic background and to exclude genes involved in eye development, such as genes able to modify the eye phenotype even without being directly involved in Bcr-Abl oncogenic signalling (false positives). Data obtained from primary and secondary screen were first analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified candidate genes were tested and validated analyzing either BM or PB samples from CML patients and healthy donors. 14.000 Dm genes were analyzed for their capability to genetically interact with hBcr-Abl in the fly model. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screening crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. We found that 42% of the 4000 Dm genes mapping in the deleted regions able to modify Bcr-Abl phenotype, displayed a known human counterpart. GeneOntology profiles of these genes included oncogenes, tumor suppression genes and human genes encoding proteins involved in the regulation of transcriptions, signal transduction, proliferation and cell growth, differentiation, apoptosis and splicing processes. Moreover, a computational comparison of our results with gene expression signatures of CML from Microarray data, showed only a partially overlap between genes identified in fly screen and genes obtained from Microarray analysis. The 72% of identified genes in fact was not known to be involved in human leukaemia. However, further confirmation of our findings into fly comes from the validation in human samples in which 1250 genes were found to be significantly associated with human CML; among these genes, we found not only an alteration of their expression profiles in CML patients with respect to the healthy donors, but also protein alterations, such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach able to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study point to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression Disclosures: No relevant conflicts of interest to declare.

Abstract 187 First line therapy for CML involves tyrosine kinase inhibitors (TKIs) which can induce rapid cytogenetic responses in the majority of patients in chronic phase (CP), but do not eliminate BCR-ABL transcripts in the majority, suggesting persistence of residual disease. These findings, together with the rapid kinetics of relapse in patients who discontinue TKIs, suggest the presence of a reservoir of TKI-resistant leukaemic stem cells, although the mechanism for TKI-insensitivity of CML stem cells remains unclear. The FoxO family of transcription factors is mainly regulated by PI3K/Akt induced phosphorylation, resulting in nuclear exclusion and degradation. FOXO activity is implicated in maintaining haemopoietic stem cell (HSC) quiescence. Its transcriptional activity in normal HSC results in cell cycle arrest by expression of p27, p130, p21, down-regulation of Cyclin D and protection from oxidative stress. Cell line studies suggest that FOXOs may play a central role in the anti-proliferative effects of TKIs, but their role in primary CML stem cells has not been previously investigated. Methods: Quiescent CML stem cells were isolated phenotypically (Lin−CD34+38−) by FACS from the total CD34+ stem/progenitor cell population and D-FISH analysis performed to determine the percentage of Ph+ cells. The expression levels of Spred1, FOXO1, FOXO3a, FOXO4 and Cyclin D1 were evaluated by Real-Time-PCR. Protein levels and localization were studied by Western blot, immunofluorescence and FACS. EMSA assay was used to evaluate FOXO3a DNA binding activity. K562 cells were transfected with wt FOXO3a and the constitutively active triple mutant (TM) form of FOXO. After transfection, proliferation and apoptosis were tested using incorporation of H3 thymidine and annexin V detection by FACS, respectively. Results: We found that BCR-ABL, through activation of the PI3K/Akt pathway, induces phosphorylation and cytoplasmic localization of FOXO in CD34+ CML cells, thereby blocking its transcriptional activity as demonstrated by EMSA and by the inhibition of FOXO target genes, including Spred1. Incubation with TKIs decreased phosphorylation and induced re-localization of FOXO to the nucleus in CD34+ CML cells, thus restoring FOXO transcriptional activity. This resulted in decreased levels of Cyclin D1 and reduced ROS. Similar effects and a dramatic reduction of cell proliferation, accompanied by significant apoptosis, were observed by forcing the expression of FOXO3a in K562 through transfection of the TM form, confirming the role of FOXO in inducing cell cycle arrest. Incubation experiments using LY294002, rapamycin and TKIs established that the reactivation of FOXO by TKI is mediated by the PI3K/Akt pathway. Interestingly, we found that phosphorylation of FOXO1, 3a and 4 was higher (i.e. cytoplasmic and inactive) in proliferating CD34+38+ CML cells, as compared to more primitive and quiescent CD34+38−90+ CML cells, although the activity of BCR-ABL, measured by analysis of p-CrKL was found to be similar in both populations, indicating that although present, BCR-ABL was not inducing phosphorylation of FOXO in the quiescent population. In this sense the quiescent CML stem cells resembled normal HSC in terms of FOXO regulation. In conclusion our data indicate that TKIs initiate a process in CML stem and progenitor cells that maintains their quiescence and therefore potential resistance to TKIs themselves. The anti-proliferative activity of TKIs against primary CML CD34+ cells is mediated, at least in part, by the re-activation of FOXO1, 3a and 4. BCR-ABL appears to play a different role in more mature progenitor cells compared to primitive quiescent stem cells, suggesting the possibility of an incomplete activity of BCR-ABL at the stem cell level or alternatively, the possibility that FOXO activity at this level provides the dominant signal responsible for intrinsic quiescence. Disclosures: No relevant conflicts of interest to declare.

Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p

The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. In CML cells the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth and the effect of imatinib (IM) induced re-activation of FoxO3 activity in CML progenitor cells. BM cells were collected from 52 CML patients and 20 healthy donors. The expression level of FoxO3 was tested by RQ-PCR. The protein amount and localization was analyzed by Western blot and immunofluorescence, DNA binding activity was measured by EMSA. In addition, FoxO3 was analyzed in CML primary cells and CD34+ cells after IM incubation. Cell cycle and the expression levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization, was measured by FACS in CD34+ cell population. In addition K562 cells was transfected with pECE-FoxO3 to clarify FoxO3 effects on cell growth and apoptosis. Finally we used our already set up model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl to study the pathway leading to FoxO3 inactivation. We found that, despite either FoxO3 mRNA levels or protein amount are similar in CML cells compared to controls, FoxO3 protein is equally distributed in the nucleus and cytoplasm in controls but it is completely cytoplasmatic in CML cells and it enters the nucleus during in vivo IM treatment or in vitro IM incubation. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears after IM treatment. Moreover FoxO3 overexpression in transfected cells results into a 49±9 % reduction of proliferation which was further reduced of 75±5 % after IM incubation. Furthermore, we demonstrated that IM incubation results into the reactivation of FoxO3 in Ph+ CD34+ cells inducing quiescence into this population as demonstrated by the comparison of cell cycle kinetics and by a decreased expression of CD47. Finally, the progeny obtained from the crossbreeding of Bcr-Abl flies and flies transgenic for FoxO showed a rescue of FoxO phenotype demonstrating that FoxO inactivation is Bcr-Abl mediated. Overall, these in vitro and in vivo experiments suggest that FoxO3 is inactivated in CML cells and its delocalization is mainly dependant from Bcr-Abl activity. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. On the other side, FoxO3 re-activation induced by IM results into a quiescence of Bcr-Abl CD34+ progenitor cells, which raises a hypothesis that FoxO3 could play a role in IM resistance. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

Background: Mutations occurring within the kinase domain of the tyrosine kinase c-Abl, and its oncogenic counterpart Bcr-Abl, strongly affects the outcome of clinical treatment with tyrosine kinase inhibitors (TKIs). Although a second and third generation of TKIs has been developed and these molecules are currently available some mutations (i.e. T315I) confer insensitivity to the drugs. Therefore, a method allowing quick detection of these mutations still represents a challenge for most of clinicians because it would allow them to rapidly switch to alternative therapies instead of TKIs. Preliminary data of a novel method allowing easy and rapid detection of the mutation affecting the codon 315 (T315I), which is nowadays the most severe and resistant mutation to TKIs, is presented. Rationale & Methods: The innovative strategy we propose is based on a variant of a single tube PNA-PCR clamping. Peptide Nucleic Acid (PNA) are synthetic molecules sharing physical-chemical features with DNA. Indeed, they possesses a structural backbone formed by N-(2-amino-ethyl)-glycine, instead of deoxy-ribose molecules linked together by phosphodiester bond, covalently conjugated with the four bases (A, T, G, C). This peculiar structure confers to the molecule exceptionally high stability when matches a single complementary DNA strand. Interestingly, the binding of PNA to nucleic acids is not affected by ionic strength and, compared to identical homo-duplex DNA-DNA, displays higher melting temperature (Tm). The latter feature allows detection of point mutations because a single mismatch within the sequences would lower the Tm of approximately 20°C. Taking advantage of these unique PNA properties, together with the fact that PNA can not prime DNA polymerase job, we utilized sequence specific PNA to block PCR amplification. The approach makes use of a specific clamping mechanism, the “primer exclusion”, in which PNA and oligonucleotide primer compete for the same target sequence. As consequence when PNA binds to template DNA the DNA polymerase can not synthesize new DNA strand. The experimental design we propose schedules the use of a PNA harboring a sequence which perfectly matches within the wild-type (wt) c-Abl tyrosine kinase domain. Therefore, when a single base pair mismatch occurs, such as it happens in presence of mutation conferring TKIs resistence, the hetero-duplex PNA-DNA stability is strongly impaired. The expected PCR clamping readout would be as follow: in case of wt template PCR reaction will not work because DNA polymerase activity is impaired by the formation of PNA-DNA hetero-duplex; on the other hand when a mutation occurs the stability between PNA and DNA complementary sequence is compromised allowing oligonucleotide primer to displace PNA. So far we focused our efforts on the mutation affecting codon 315 (T315I). Results & Conclusions: cDNA synthesized from total RNA isolated from patients harboring either wt or mutated Bcr-Abl alleles have been amplified by nested PCR. During the first step a specific pair of primers designed to discriminate between the kinase domain of c-Abl and Bcr-Abl have been utilized. The second step involves the use of sequence specific 19-mer PNA matching within the region around the codon 315 (T315I), so far the most severe mutation described, in a “PNA primer exclusion” PCR clamping reaction. Initially, we aimed to study patients which have previously been analyzed by direct sequencing in which either the wt or the mutated allele were to be predominant. Encouraging results show amplification in patients harboring the point mutation at position 315 which determine the substitution of a threonine for isoleucine (T315I). These results were further corroborated by carrying-out a blind analysis, in which samples were subsequently sequenced. These data show that, in future, the technique herein reported might be extremely useful in detecting the presence of all known mutations within the Bcr-Abl kinase domain with simple, rapid, reliable and very cheap methodology.

The abnormal activation of tyrosine kinases are a common finding in chronic myeloproliferative disorders. Perturbation of RTK signalling by genetic alterations results into an abnormal proliferation advantage and finally into a malignant transformation. c-Ros is an orphan RTK displaying transforming activity whose role has been established in the development of neuronal neoplasia. The aim of this study was to evaluate the involvement of c-Ros in the pathogenesis of chronic myelomonocytic leukemia (CMML) and to establish the effects of c-Ros activation. c-Ros expression was evaluated by RQ-PCR in 133 samples collected from 96 CMML patients at diagnosis (96 BM and 37 PB) and 60 healthy donors (30 PB and 30 BM). The protein amount and localization was analyzed by westen blot and immunofluorescence assay. In order to establish the effects of c-Ros activation we generated a chimeric receptor containing the extracellular domain derived from epidermal growth factor receptor (EGFR) and the transmembrane and cytoplasmic domains from c-ros (ER). The chimeric receptor was then transfected in NIH3T3 and HEK293T cells. Transfected and control cells were then stimulated with 100 nM EGF ligand and proliferation and apoptosis evaluated by incorporation of 3H timidine and MTT assay and by FACS for the detection of annexin V, respectively. We found that Ros is undetectable in healthy subjects but it is overexpressed in CMML (p

Eph receptors tyrosine kinases are involved in many key developmental processes. Ephs are overexpressed or mutated in solid tumours suggesting their possible role in oncogenesis. The majority of the genes and processes involved in CML progression are still unknown. The aim of this study was to investigate the role of EphA3 in CML progression and to explore the possibility to selectively target EphA3 with a monoclonal antibody. The expression of EphA3 and ligands were analyzed by RQ-PCR in 92 samples from 76 CML patients and in 38 healthy controls. 56 patients were in chronic phase (CP) enrolled in TOPS (Cortes, EHA, 2008) studies, 10 in accelerated phase (AP) and 10 in blast crisis (BC). In 5 patients and 10 healthy subjects EphA3 was studied also in enriched CD34+ population. Protein expression and localization were examined using Western Blot and immunofluorescence using specific antibodies. EphA3 expression in CD34+ cells were analyzed by FACS. The effects of EphA3 overexpression were studied by transfecting EphA3 plasmid in 293T e COS cells. In addition, sequencing of EphA3 TK domain was performed in 45 EphA3+ patients. BM, PB and CD34+ cells from CP CML patients expressed very low levels of EphA3, similar to normal controls with a median of 2−Δ ΔCt of 0,0018. By contrast, during the advanced phases of disease (AP and BC) we found high transcript levels (mean of 2−Δ ΔCt 43 and 67 respectively). Moreover, purified CD34+ CML cells presented significantly higher levels as compared to the unfractioned sample (p=0,001). RT-PCR showed no increase of the expression of the ligands Ephrin A2,A3,A4,A5 and B2, all of them able to bind to the receptor EphA3. WB and immunofluorescence confirmed the presence of phosphorylated protein in EphA3 in AP and BC CML cells. Western Blot and immunofluorescence confirmed the presence and the high level of phophorylation of EphA3 protein in AP and BC, but not in CP cells. EphA3 transfection into normal cells results in loss of adhesion, cell rounding and increased proliferation. Incubation of CML cells or transfected cells with a specific antibody able to block the EphA3 receptor induced a significant reduction of proliferation (31±4% of reduction in primary cells and 45±9% in transfected cells), it reduced colony growth (median value of 34,2 vs 76,5) and mainly, it changes the adhesion properties. The antibody did not induced any significant change of proliferation or colony growth in CP cells and normal controls. Finally, no kinase domain mutations were found in EphA3 overexpressing cells. In conclusion, our experimental data have shown that EphA3 was abnormally expressed in cells derived from advanced stage CML. The ectopic expression of EphA3, as demonstrated in our experimental system is responsible for increased proliferation and loss of cell adhesion. The inhibition of EphA3 induced by a specific antibody results in proliferation and colony growth inhibition. Although preliminary, our findings suggest that EphA3 may represent a potential candidate for a molecular therapy in advanced phase of chronic myeloid leukaemia. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

Iron overload is a critical issue for low risk myelodisplastic syndrome (MDS) patients with a long tranfusional history and often requires chelation therapy. Iron chelation is as an independent prognostic factor for survival in MDS but can also improve haemoglobin level in some cases with different drugs and modalities. Recently a once daily oral chelator Deferasirox became available for the treatment of secondary hemosiderosis also in MDS patients and it has been described an haemoglobin improvement in a many patients within a few months of treatment. Moreover it was demonstrated that the transcriptional factor NF-kB is abnormally activated in MDS blast cells. Its pathway can be mediated by a broad variety of stimuli, sometimes dependent by reactive oxygen species generated by iron overload but it is activated even in the absence of iron overload. Aim of our study was to compare the effects of the 3 commercially available iron chelators on NF-kB activity in MDS and to identify a possible mechanism responsible for the observed reduced transfusion requirement during iron chelation therapy. We collected 40 PB samples from MDS patients: 18 RA, 14 RAEB, and 8 s-AML. Thirty of them presented hepatic iron overload (measured by SQUID biomagnetic liver susceptometry) and serum ferritin levels over 5000 ng/ml. Ten samples were collected before starting transfusion therapy. MNC cells were incubated with 50 microM Deferasirox for 3 hrs. K562 and HL60 cells were analyzed as controls and were incubated with Deferasirox 50 mM, Deferiprone 0,3 mM and Deferioxamine 0,3 mM for 18 hours and with the DL-Dithiothreitol (DTT) 100 mM for 18 hours as control. NF-kB activity was evaluated using both EMSA and ELISA methods. Apoptosis was evaluated by FACS for the detection of annexin V. Deferasirox incubation induced a significant decrease of NF-kB activity both in HL60 and K562 cells lines (EMSA method). We detected an increased activation of NF-kB as compared to healthy subjects in 6 RA, 12 RAEB, in all the s-AML PB samples and in cell lines. No significant difference was detected in NF-kB activity by comparing patients with or without iron overload (p=0,5). The percentage of samples presenting NF-kB activity increases during disease progression being higher in RAEB and s-AML as compared to RA (p=0,003). Among patients with increased NF-kB (n=14) the incubation with Deferasirox induced a significant reduction of NF-kB activity (p=0,0002). The degree of NF-kB inhibition was not significantly different according to the level of iron overload. Apoptosis was not significantly triggered by incubation with any of the different chelators. HL60 and K562 cells incubation with the Deferiprone and Deferioxamine failed to induce a significant reduction of NF-kB activity. NF-kB is abnormally activated in MDS patients and this activity is not strictly related to iron overload being present in patients with normal serum ferritin levels and in cell lines. Deferasirox is the only commercially available iron chelator acting as a potent NF-kB inhibitor and this effect is not shared by Deferioxamine, Deferiprone or DTT. This behaviour suggests that it is independent from ROS scavenging that is a common feature of all the mentioned drugs. Our in vitro observation could be an explanation for the early improvement of hemoglobin levels observed in some patients under deferasirox chelation that seems not related to a sharp decrease of iron overload.

Although the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression are largely unknown. The aims of the study were to perform a genetic screening to identify new genes and pathways leading to CML progression and imatinib resistance and to provide a powerful tool allowing a wide screening of drug libraries. We developed a genetic model based on transgenic human p210 Bcr-Abl Drosophila melanogaster (Dm). We generated two different fly lines expressing either h-p210 wt or carrying the T315I mutation, in a tissue specific manner such as fly eyes or lymph gland, which represents the Dm hematopoietic system. Bcr-Abl expression results into a glazed phenotype of the eyes correlated with the amount of p210 protein. A wide modifier screening was performed using commercially available stocks of Dm carrying small and well characterized chromosome deletions. The resulting progeny was first screened using eye phenotype as read-out system. A first group of flies displayed a more aggressive phenotype since they lack one or more genes encoding for Bcr-Abl negative regulators while a second group displayed a mild phenotype most likely due to the absence of a gene encoding for a gene involved in the oncogenic signalling. Each deletion, responsible for any phenotype change in the progeny, was further analyzed by crossing Bcr-Abl flies with flies carrying the single deletion of each gene included in the identified region. Among the genes identified, PI3K loss of function results into a phenotype improvement thus supporting the tool effectiveness. As control, the cross between Bcr-Abl flies and the constitutively active form of PI3K results not only into a worse phenotype but also into an increased size of the eye, corresponding to an abnormal proliferation process. With this tool we have at now identified a list of genes responsible for a phenotype change including Fax, Dab and Pros which have been more extensively studied in human primary cells collected from patients at diagnosis enrolled in TOPS studies (Cortes, EHA, 2008) and during disease progression, so confirming their involvement in human disease. We found that these genes are downregulated during accelerated phases and blast crisis. Transfection of CML cells with Fax, Dab and Pros reduced proliferation and/or induced apoptosis. By contrast, loss of function of ENA, the CRKL hortologous in Dm did not induce any significant change of the phenotype. In addition, a drug screening was performed by feeding flies with drugs. We set up a rapid method for drug testing based on wt and T315I/Bcr-Abl phenotypes rescue induced by several TKs inhibitors or by combinations. In conclusion, Dm is a rapid genetic tool which allow the selection of a number of genes involved in CML progression and IM resistance. In addition, it allows to identify drugs or combination of drugs active on Bcr-Abl or T315I mutant form. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

Introduction Tyrosine kinases (TKs) play different roles either in physiological or in pathological cellular processes ranging from the regulation of intracellular signaling pathways leading for example to proliferation, differentiation and/or DNA syntheisis. The whole human genome encodes for approximately 95 TKs classified in two major families: transmembrane and non-transmembrane TKs. Transmembrane TKs are further splitted in two groups: Receptor Tyrosine Kinases (RTKs) and Non-Receptor Transmembrane Tyrosine Kinases. Despite in the last decade the role of RTKs has been deeply investigated very little is known about the second class of TKs, Non-Receptor Transmembrane Tyrosine Kinases. Recently, we began to investigate the roles played by these peculiar molecules in hematological tissues and preliminary data are herein presented and discussed. Material & Methods and Results STYK1 is a Non-Receptor Transmembrane TK characterized by a very short extracellular domain, of approximately 25 aminoacid residues followed by a single transmembrane alpha-helix sequence and a conserved cytoplasmic region harboring the catalytic domain, splitted in two halves separated by a short kinase insert sequence, peculiar of class III RTKs, VEGFR and FGFR family members. Indeed, sequence analysis alignment reveals the higher homology with PDGFR and FGFR members. Surprisingly, data mining searches, performed with the Ensembl tools (http://www.ensembl.org) and using blast algorithm with human STYK1 protein as query, revealed that this protein possesses orthologues either in vertebrate model organisms, both mammalian and non-mammalian or in invertebrate metazoan, such as Ciona. Furthermore, a bioinformatic analysis carried-out at CBS (http://www.cbs.dtu.dk/services/NetPhos/) revealed 4 tyrosine residues putatively phosphorylated. Interestingly one of the tyrosine residue displays high homology towards a consensus sequence for the p85 regulatory subunit of the PI3K. Since our interest aims to understand the roles played by STYK1 in hemopoietic cells, we first investigated its expression in different blood cells populations. Therefore the different blood cellular components were separated by immunoaffinity sorting and total mRNA was extracted. Afterwards c-DNA was synthesized and STYK1 mRNA expression studied by RT-PCR. The results reveal that STYK1 mRNA is expressed predominantly in mature lymphocytes. To study more in details its expression within lymphoid population we attempted to separate B-from T-lymphocytes and STYK1 mRNA expression evaluated in the two populations. Furthermore, we also investigated STYK1 expression in leukemic hematological disorders. Nevertheless STYK1 mRNA expression is typical of mature hemopoietic cells populations, such as lymphocytes, its expression resulted deregulated in patients affected by different kind of leukemia (i.e. CML, ALL, AML). Currently a constitutively active form of STYK1 has been generated and its signaling is under investigation. Conclusion The non-receptor transmembrane tyrosine kinase STYK1 represents a novel lymphocyte molecular marker whose expression results deregulated in patients affected by leukemia. Interestingly, due to its homology with class III RTKs like PDGFR, which are easily targeted by small molecules such as Imatinib mesylate, it may represents a novel druggable molecular target for leukemic cells.

The transferrin receptor 2 gene (TFR2) encodes for a transmembrane protein that plays a pivotal role in iron metabolism, since TFR2 mutations are responsible of type 3 Hereditary Hemochromatosis (HH). Together with other HH proteins (Hfe and Rgmc) Tfr2 participates to modulation of Hepcidin (Hepc) production, a small peptide able to regulate body iron availability through its negative effect on cellular iron exporter Ferroportin (Fpn1). Tfr2 protein is a member of the transferrin receptor family, showing moderate homology to transferrin receptor (Tfrc). It is able to bind transferrin even thought at lower affinity compared to TFRC. Two alternative transcripts of TFR2 have been reported, named alfa and beta. TFR2 alfa mRNA is highly expressed in the hepatocytes while TFR2 beta has a low ubiquitous expression (Kawabata et al, 1999). The beta isoform is identical to alfa but it lacks the transmembrane and cytoplasmic domains; it has been considered an alternative splicing form and its role is still poorly understood. Nevertheless, clinical observations support the hypothesis that TFR2 beta has an important functional role since HFE3 patients with mutations not compromising its production have a milder phenotype compared to those unable to synthesize both alfa and beta TFR2 isoforms (Roetto et al,2002; Le Gac et al, 2004). In order to elucidate whether TFR2 beta was differentially expressed and produced during life span its mRNA and translated protein were analyzed in C57BL/6 mice during their growth and adult age. Since Tfr2 beta protein has never been demonstrated to exist before, its cDNA has been cloned in an expression vector and transfected in HEK293T cells. Cells lysates have been electhrophoresed, transferred onto nitrocellulose membrane, decorated with Tfr2 Ab (Santa Cruz Biotechnoloy, Inc) and visualized on Western Blot. A 65 kDa band has been obtained from the transfection, consistent with the aminoacid sequence predicted molecular weight, and used as a positive control for further in vivo experiments. C57BL/6 mice livers from seven different ages, starting from birth until adult age (post natal days 0, 1, 3, 7, 14, 28, 40 and 60) have been analyzed in WB utilizing Tfr2 antibody. At least three mice per point have been analyzed. One band of a molecular weight of approximately 95 KDa has been detected during whole animal life, quantitatively increasing during life time, corresponding to Tfr2 alfa. Meanwhile, the beta isoform appeared to be consistently produced until the 7th day after birth and disappears starting from day 14. To evaluate the possible consequences of this result on iron metabolism, HH proteins (Hfe, Fpn1, Rgmc) mRNA expression pattern has been analyzed in real time PCR at the same points, as well as Hepcidin (Hepc) transcript, in order to evaluate whether TFR2 beta production could in some way influence Hepc modulation. HEPC transcription resulted to be significantly downmodulated from day 0 after birth until day 14 as compared to later stages in mice life (adulthood). HFE transcription resulted to be lower in young animals compared to adults while FPN1 increased significantly between day 3 and 7, according to HEPC decreasing. On the whole, these results suggest a functional role for the Tfr2 beta isoform in iron sensing, since this isoform resulted to be selectively produced during animal growth. At the same life period Hepc transcription resulted to be significantly downmodulated, allowing in this way increased iron absorption. These data are consistent with an higher iron requirement necessary for massive animal body growth.

The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. In Chronic Myeloid Leukemia the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO in Bcr-Abl induced apoptotic arrest and cell growth, the consequence of imatinib (IM) treatment on FoxO activity and the alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 20 CML patients at diagnosis and during IM treatment and from 20 healthy donors. The expression of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by Bcr-Abl. We have previously described the absence of Spred1 is CML cells and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells and BV173 Ph+ cell line were incubated with 1 μM IM, 5 μM of the PI3K inhibitor LY294002 and 20 μM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation, and with the combination of IM plus PS1145 and LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in CML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in Ph+ CML cells and it enters the nucleous during IM treatment. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in CML cells is 6.6±0.8 in the nucleous and 16.7±1.1 in the cytoplasm. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears during therapy or after IM incubation. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,001±0,09) and is upregulated during remission (mean value 2−ΔΔCt= 1,2±1,8) or after IM incubation (mean value = 0,7±0,9) or LY294002(1±0,7). Exposure to IM, LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±5, 17 ±3 and 12±2 respectively. Interestingly, the association of PS1145 and IM or PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 23±4 and 24±4 respectively, suggesting that both pathways are implicated in FoxO inactivation. These observations suggest that FoxO inactivation may be crucial for Bcr-Abl induced proliferation and apoptosis arrest. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. Nevertheless, we demonstrated that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of IM plus an IKK inhibitor.

INTRODUCTION The mammalian target of rapamycin (mTOR) is a serine/threonine-specific protein kinase, downstream of the phoshatidylinositol 3-kinase (P13K)/AKT pathway. It controls many aspects of cellular physiology, including transcription, translation, cell size, cytosckeletal organization, autophagy and progression from the G1 to S phase of the cell cycle. Constitutive activation of the mTOR related downstream effectors including P13K, AKT, p70S6K and 4EPB1 was found in numerous malignancies. Rapamycin and its analogues are mTOR inhibitor currently being tested in solid and hematological tumours. Previous studies demonstrated that rapamycin have preclinical potential as therapy for multiple myeloma, especially when associated with other drugs. We first determined the frequency of the activation status of AKT/mTOR/p70S6K pathway in multiple myeloma patients and correlated its activation with FISH analysis and clinical data. METHODS Immunohistochemical analysis with phospo-AKT (Ser 473), phospho-mTOR (Ser2448) and phosphor-p70S6K (Thr389) (Cell Signaling Technology) was performed on a series of 44 multiple myeloma bone marrow sections. Slides were scored by two independent observers and staining was defined as weakly positive (>10–30, +), moderately positive (>30–70, ++) and strongly positive (>70, +++). FISH analysis was performed on CD138 purified plasma cells of 31 myeloma patients with specific probes for the detection of the t (4;14)(p16.3;q32) and del 13q14 (Vysis). Clinical data of all patients including age, presence of bone lesion, isotype, serum beta2-microglobulin, C-reactive protein, haemoglobin level and albumin were evaluated and Fisher exact test was used to compare the expression of p-mTOR with p-AKT and p-p70S6K signaling protein and clinical data. RESULTS p-mTOR and p-AKT were expressed respectively in 26 of 44 (59.9%) and 23 of 44 (52.3%) patients, with a predominance of cytoplasmic staining pattern. p-mTOR immunoreactivity was strongly, moderately and weakly positive in 36%, 36% and 28% of the samples, respectively. p-p70S6S was detected in 26 patients (59.9%) with a predominantly nuclear staining pattern. Of the 26 myelomas stained positive for p-mTOR, 22 expressed p-AKT and all of them demonstrated p-p70S6S positivity. p-mTOR expression significantly correlated with both p-AKT (p CONCLUSION mTOR signaling protein are activated in a fraction of multiple myeloma cases and may contribute to tumour cell proliferation and survival of myeloma cells. Our results also suggest that the AKT/mTOR/p70S6K pathway may have prognostic implications and may provide novel therapeutical targets in this subset of myeloma patients.

The Wilms’ tumor gene is overexpressed in many types of haematological malignancies including acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML) and Ph negative myeloproliferative disorders. WT1 holds great promise for immunotherapy of leukaemia since it is expressed at high levels in blast cells but not in normal tissues, it is involved in the maintenance of malignant phenotype and it is spontaneously immunogenic. Many clinical trials using MHC class I-restricted WT1 peptide, have been recently performed in patients affected by AML, MDS, lung and breast cancer with satisfactory clinical results and without relevant toxicity. The first aim of the study was to analyze the natural immunity against WT1 in different types of haematological malignancies and the second was to set up a vaccination approach in the mouse model using the WT1 full length protein and to test the safety and efficacy of this vaccine. Using a dot blot technique, we analyzed for the presence of WT1 specific antibodies 107 patients including: 18 AML, 20 MDS, 22 Multiple Myeloma, 14 CML, 12 Idiopatic Myelofibrosis (IM), 6 CMML, and 15 ALL and 30 healthy subjects as control. For the second aim, we purified the WT1 full length protein. Complete coding sequence was cloned in a pGEX bacterial expression vector for the production of the fusion protein GST-WT1 that was subsequently purified by glutathione conjugated beads. Twenty C57BL7/6 mice were immunized with 50 μg of purified WT1 protein and 50 μL of Freund adjuvant every 15 days, for a total of 3 immunizations, 20 mice were injected with GST and adjuvant and 20 with PBS alone as control. 2 weeks after the last administration, 10 vaccinated mice and 10 controls have been injected with 200.000 TRAMP-C2 cells. Ten mice for each group were sacrificed and lymphocytes, cultured in presence of IL-2 to perform a cytotoxicity assay. Antibodies against WT1 have been evaluated. Toxicity on normal tissues has been rouled out using the histochemical analysis of different tissues. Results: Dot blot analysis demonstrated the presence of significant levels of WT1 IgG antibodies in 81% and IgM in 76% of the patients but not in normal subjects so demonstrating that WT1 is highly immunogenic. Particularly 88% of AML, 85% of MDS, 63% of MM, 100% of CML, 83% of IF and 66% of CMML presented high levels of antibodies. After mice vaccination, no organ toxicity has been observed. Importantly, immunized mice showed high levels of both, IgM and IgG antibodies with a mean value of luminescence of 690000 in WT1 injected mice vs 7500 in control mice and high levels of CTLs against WT1 as compared to mice injected with PBS or GST alone. Importantly, in control mice the mean size of the tumor was 1,6±0,4 cm, by contrast in vaccinated mice the tumor was unable to develop. Data obtained from this study clearly demonstrated that WT1 is immunogenic. The full length protein vaccination approach is able to elicit both, umoral and cytotoxic response and it results in a significant antitumor effect in vivo. Although further studies are required to compare the efficacy of this approach to the peptide based vaccine, WT1 full length protein could represent a valid vaccination strategy allowing to overcome the HLA restriction limit of the peptide approach.

Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They are implicated in negative feedback interactions that regulate intracellular pathways. The repressive function of Spred proteins targets several TK receptors so resulting in a variety of biological effects. Spred proteins, after growth factor stimulation, translocate to the plasma membrane, become tyrosine phosphorilated and interact with components of the Ras/MAPK and Ras/Raf/Erk pathways. Abnormal activation of Ras/MAPK pathway has been demonstrated in many haematological disorders including AML. In order to assess whether the activation of this pathway, leading to the disruption of many biological process, could be ascribed not only to the constitutive activation of TKs but also to defective signalling inhibition, we studied the function of Spred1 in AML. Using a Real Time PCR we studied the expression level of Spred1 in 80 AML samples (15 PB and 65 BM), 26 normal controls (16 PB and 10 BM) and 12 CD34+ samples sorted from AML patients and 12 from healthy subjects. We found that Spred1 transcript amount is significant reduced in AML samples when compare to normal controls (2−DeltaDeltaCt =0,084 vs 2,4 p=0,0000001) (range in AML 0,3–0,003). This difference is even more sound in AML CD34+ cells where the Spred1 transcript is 3 logs lower compared to normal CD34+ (2−DeltaDeltaCt =0,001 vs 2,27 p=0,0000001). Western blot using an antibody against Spred1 demonstrated the reduction or the absence of Spred1 protein in AML cells. Sequence analysis allowed to exclude the presence of mutations in the coding and promoter regions. In order to better understand the mechanism leading to the abrogation of Spred1 we analyzed the factors responsible for Spred1 transcription. We demonstrated that the transcription factor WT1 binds to and activates the promoter region of Spred1. WT1 KTS- is the isoform with transcriptional activity, while the isoform KTS+ is mainly localized at the cytoplasmatic level as confirmed by immunofluorescence analysis. Immunofluorescence assay and western blot carried out in NIH3T3 cells transfected with plasmids containing KTS+ and KTS- isoforms demonstrated that Spred1 protein is present at high levels in WT1 KTS- cells but not in KTS+ transfected cells. Finally we demonstrated, using capillary electrophoresis, that in AML patients the WT1 KTS- isoform was reduced or completely absent (ratio KTS+/KTS- ranging from 2 to 9,6 compared to a normal ratio ranging from 1, to 1,4 in cells from healthy subjects. Finally, in patients who achieved a complete remission after chemotherapy, WT1 KTS+/KTS- ratio returned within the normal range and Spred1 transcript and protein were significantly upregulated. This study clearly demonstrates that WT1 transcription activity is altered in AML cells in which WT1 may probably play a role of oncogene rather than of tumor suppressor gene. This leads to the absence of Spred1 transcript and protein which is a physiological inhibitor of RTK mediated signalling.

Mutations in NPM1 exon 12 and the resulting shift of NPM1 into the cytoplasm are the most specific and frequent events in acute myeloid leukaemia patients with normal karyotype. Cytoplasmatic NPM is associated with responsiveness to induction chemotherapy, although its role in predicting outcome after remission remains to be defined. The aim of the study was to identify the molecular mechanisms responsible for chemosensitivity in leukemic cells carrying the mutation of NPM1. NF-kB is a transcription factor involved in many intracellular pathways including apoptosis. NF-kB is a heterodimer of p50 and p65 subunits sequestered in the cytoplasm in its inactive form through interaction with inhibitory IKB proteins and activated in the nucleous after degradation of IKB. The activation of NF-kB is responsible for chemoresistance to different drugs including anthracyclines. Based on this assumption, we analyzed the possible cytoplasmatic interaction between the mutated form of NPM1 (NPM+) and NF-kB. The NF-kB DNA binding activity was analyzed in 20 BM samples collected from AML patients carrying the NPM1 mutations and 30 NPM1 wild type samples (NPM1−) using an ELISA method. Immunofluorescence analysis using NPM1 and p65 antibodies was performed to identify the localization of both proteins. Western blot for p65 and NPM1 was used to confirm the protein amount and localization. Finally, co-immunoprecipitation assay was perform to study the interaction of the two proteins. We found a significant lower DNA binding activity in NPM1 mutated cells when compared to the wild type NPM cells (p=0,001). Immunofluorescence analysis confirmed the cytoplasmatic localization of NPM protein in mutated samples and the nuclear localization in wild type samples. In addition, immunofluoresence analysis performed with a monoclonal antibody against p65 subunit shows different pattern of NF-kB localization in NPM1+ when compared to NPM1−. In particular, in NPM1+ cells NF-kB is mainly localized in the cytoplasm in the inactive form and in NPM− cells is mainly nuclear localized. These data were confirmed by Western blot carried out with the same monoclonal antibody against p65 in the nuclear and cytosolic extracts. The interaction of NPM1 and NF-kB was further investigated and demonstrated by co-immunoprecipitation studies. In conclusion, we demonstrated that p65 and NPM1 interact with each other within the cytoplasm and this interaction results in the sequestration of NF-kB within the cytoplasm. The cytosolic localization of the inactive form of NF-kB explains the reduced NF-kB DNA binding activity observed in NPM1+ patients. These data may provide a possible explanation for the chemosensitivity observed in NPM+ patients. Furthermore, since NF-kB is involved in the transcription of many genes which regulate proliferation and differentiation processes, the disruption of NF-kB function may represent one of the mechanism of leukemogenesis induced by NPM1 mutated proteins.