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Large HERC E3 ubiquitin ligase family members, HERC1 and HERC2, are staggeringly complex proteins that can intervene in a wide range of biological processes, such as cell proliferation, DNA repair, neurodevelopment, and inflammation. Therefore, mutations or dysregulation of large HERCs is associated with neurological disorders, DNA repair defects, and cancer. Though their role in solid tumors started to be investigated some years ago, our knowledge about HERCs in non-solid neoplasm is greatly lagging behind. Chronic Myeloid Leukemia (CML) is a model onco-hematological disorder because of its unique and unambiguous relation between genotype and phenotype due to a single genetic alteration. In the present study, we ascertained that the presence of the BCR-ABL fusion gene was inversely associated with the expression of the HERC1 and HERC2 genes. Upon the achievement of remission, both HERC1 and HERC2 mRNAs raised again to levels comparable to those of the healthy donors. Additionally, our survey unveiled that their gene expression is sensitive to different Tyrosine Kinases Inhibitors (TKIs) in a time-dependent fashion. Interestingly, for the first time, we also observed a differential HERC1 expression when the leukemic cell lines were induced to differentiate towards different lineages revealing that HERC1 protein expression is associated with the differentiation process in a lineage-specific manner. Taken together, our findings suggest that HERC1 might act as a novel potential player in blood cell differentiation. Overall, we believe that our results are beneficial to initiate exploring the role/s of large HERCs in non-solid neoplasms.

In aerobic organisms, oxygen is essential for efficient energy production, and it acts as the last acceptor of the mitochondrial electron transport chain and as regulator of gene expression. However, excessive oxygen can lead to production of deleterious reactive oxygen species. Therefore, the directed migration of single cells or cell clumps from hypoxic areas toward a region of optimal oxygen concentration, named aerotaxis, can be considered an adaptive mechanism that plays a major role in biological and pathological processes. One relevant example is the development of O2 gradients when tumors grow beyond their vascular supply, leading frequently to metastasis. In higher eukaryotic organisms, aerotaxis has only recently begun to be explored, but genetically amenable model organisms suitable to dissect this process remain an unmet need. In this regard, we sought to assess whether Dictyostelium cells, which are an established model for chemotaxis and other motility processes, could sense oxygen gradients and move directionally in their response. By assessing different physical parameters, our findings indicate that both growing and starving Dictyostelium cells under hypoxic conditions migrate directionally toward regions of higher O2 concentration. This migration is characterized by a specific pattern of cell arrangement. A thickened circular front of high cell density (corona) forms in the cell cluster and persistently moves following the oxygen gradient. Cells in the colony center, where hypoxia is more severe, are less motile and display a rounded shape. Aggregation-competent cells forming streams by chemotaxis, when confined under hypoxic conditions, undergo stream or aggregate fragmentation, giving rise to multiple small loose aggregates that coordinately move toward regions of higher O2 concentration. By testing a panel of mutants defective in chemotactic signaling, and a catalase-deficient strain, we found that the latter and the pkbR1null exhibited altered migration patterns. Our results suggest that in Dictyostelium, like in mammalian cells, an intracellular accumulation of hydrogen peroxide favors the migration toward optimal oxygen concentration. Furthermore, differently from chemotaxis, this oxygen-driven migration is a G protein-independent process.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

Simple Summary The pathological role/s of the HERC family members has recently been initiated to be explored in few solid tumors and the assessment of their transcript amount reveals that they might act as effective prognostic factors. However, evidence concerning the non-solid tumors, and especially myeloid related neoplasms, is currently lacking. In the present article for the first time we provide original data for a clear and well-defined association between the gene expression level of a giant HERC E3 ubiquitin ligase family member, HERC1, and some myeloid related disorders, namely Acute Myeloid Leukemia, Myeloproliferative neoplasms and Chronic Myeloid Leukemia. Furthermore, our findings unveil that the HERC1 protein physically interacts, likely forming a very large supramolecular complex, and it is a putative BCR-ABL1 tyrosine kinase substrate. We hope that this work will contribute to the advance of our understanding of the roles played by the giant HERCs in myeloid related neoplasms. Abstract HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that HERC1 gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm HERC1 level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells HERC1 mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of HERC1 in myeloid related blood disorders.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9, 22)(q34, q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Nandrolone decanoate (ND), an anabolic-androgenic steroid prohibited in collegiate and professional sports, is associated with detrimental cardiovascular effects through redox-dependent mechanisms. We previously observed that high-dose short-term ND administration (15 mg/kg for 2 weeks) did not induce left heart ventricular hypertrophy and, paradoxically, improved postischemic response, whereas chronic ND treatment (5 mg/kg twice a week for 10 weeks) significantly reduced the cardioprotective effect of postconditioning, with an increase in infarct size and a decrease in cardiac performance. We wanted to determine whether short-term ND administration could affect the oxidative redox status in animals exposed to acute restraint stress. Our hypothesis was that, depending on treatment schedule, ND may have a double-edged sword effect. Measurement of malondialdehyde and 4-hydroxynonenal, two oxidative stress markers, in rat plasma and left heart ventricular tissue, revealed that the levels of both markers were increased in animals exposed to restraint stress, whereas no increase in marker levels was noted in animals pretreated with ND, indicating a possible protective action of ND against stress-induced oxidative damage. Furthermore, isolation and identification of proteins extracted from the left heart ventricular tissue samples of rats pretreated or not with ND and exposed to acute stress showed a prevalent expression of enzymes involved in amino acid synthesis and energy metabolism. Among other proteins, peroxiredoxin 6 and alpha B-crystallin, both involved in the oxidative stress response, were predominantly expressed in the left heart ventricular tissues of the ND-pretreated rats. In conclusion, ND seems to reduce oxidative stress by inducing the expression of antioxidant proteins in the hearts of restraint-stressed animals, thus contributing to amelioration of postischemic heart performance.

// Valentina Rosso 1 , Jessica Petiti 1 , Enrico Bracco 2 , Roberto Pedrola 1 , Francesca Carnuccio 1 , Elisabetta Signorino 1 , Sonia Carturan 1 , Chiara Calabrese 1 , Giada Bot-Sartor 1 , Michela Ronconi 1 , Anna Serra 1 , Giuseppe Saglio 1 , Francesco Frassoni 3 , Daniela Cilloni 1 1 Department of Clinical and Biological Sciences, University of Turin, Turin, Italy 2 Department of Oncology, University of Turin, Turin, Italy 3 Department of Pediatric Hemato-Oncology and Stem Cell, Cellular Therapy Laboratory, Institute G. Gaslini, Genova, Italy Correspondence to: Daniela Cilloni, email: daniela.cilloni@unito.it Keywords: CALR, PNA, MPN, PCR clamping, diagnostic assay Received: September 14, 2016 Accepted: December 15, 2016 Published: December 23, 2016 ABSTRACT The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene ( CALR ) were detected in 56–88% of JAK2/MPL -negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs. We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.

// Valentina Monica 1 , Marco Lo Iacono 1 , Enrico Bracco 1 , Simone Busso 1 , Laura Di Blasio 2 , Luca Primo 2 , Barbara Peracino 3 , Mauro Papotti 1 , Giorgio Scagliotti 1 1 Department of Oncology, University of Torino, San Luigi Hospital, Orbassano, Torino, Italy 2 Department of Oncology, University of Torino, IRCCS Candiolo, Torino, Italy 3 Department of Clinical and Biological Sciences, University of Torino, San Luigi Hospital, Orbassano, Torino, Italy Correspondence to: Valentina Monica, email: valentina.monica@unito.it Keywords: pemetrexed, dasatinib, malignant pleural mesothelioma, cell lines, thymidylate synthase Received: August 17, 2015 Accepted: June 16, 2016 Published: July 06, 2016 ABSTRACT Background: Thymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated. Results: MPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling. Cell lines and Methods: In three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed. Conclusions: These data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.

Acute myeloid leukemia is mainly characterized by a complex and dynamic genomic instability. Next-generation sequencing has significantly improved the ability of diagnostic research to molecularly characterize and stratify patients. This detailed outcome allowed the discovery of new therapeutic targets and predictive biomarkers, which led to develop novel compounds (e.g., IDH 1 and 2 inhibitors), nowadays commonly used for the treatment of adult relapsed or refractory AML. In this review we summarize the most relevant mutations affecting tumor suppressor genes that contribute to the onset and progression of AML pathology. Epigenetic modifications (TET2, IDH1 and IDH2, DNMT3A, ASXL1, WT1, EZH2), DNA repair dysregulation (TP53, NPM1), cell cycle inhibition and deficiency in differentiation (NPM1, CEBPA, TP53 and GATA2) as a consequence of somatic mutations come out as key elements in acute myeloid leukemia and may contribute to relapse and resistance to therapies. Moreover, spliceosomal machinery mutations identified in the last years, even if in a small cohort of acute myeloid leukemia patients, suggested a new opportunity to exploit therapeutically. Targeting these cellular markers will be the main challenge in the near future in an attempt to eradicate leukemia stem cells.

ATF2 is a transcription factor involved in stress and DNA damage. A correlation between ATF2 JNK-mediated activation and resistance to damaging agents has already been reported. The purpose of the present study was to investigate whether ATF2 may have a role in acquired resistance to cisplatin in non-small cell lung cancer (NSCLC). mRNA and protein analysis on matched cancer and corresponding normal tissues from surgically resected NSCLC have been performed. Furthermore, in NSCLC cell lines, ATF2 expression levels were evaluated and correlated to platinum (CDDP) resistance. Celastrol-mediated ATF2/cJUN activity was measured. High expression levels of both ATF2 transcript and proteins were observed in lung cancer specimens (p << 0.01, Log2 (FC) = +4.7). CDDP-resistant NSCLC cell lines expressed high levels of ATF2 protein. By contrast, Celastrol-mediated ATF2/cJUN functional inhibition restored the response to CDDP. Moreover, ATF2 protein activation correlates with worse outcome in advanced CDDP-treated patients. For the first time, it has been shown NSCLC ATF2 upregulation at both mRNA/protein levels in NSCLC. In addition, we reported that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Altogether, our results indicate a potential increase in CDDP sensitivity, on Celastrol-mediated ATF2/cJUN inhibition. These data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

Background: The TP73 and TP63 genes are members of the p53 tumor suppressor family and are expressed in different N -terminal isoforms either with proapoptotic (transactivation domain, TA) and antiapoptotic (N-terminally truncated, ΔN) function. Unlike p53, the role of p73 and p63 in tumor is controversial. It has been recently hypothesized that altered ΔN:TA expression ratio, rather than single isoform overexpression, plays a role in the pathogenesis of many diseases, including lung cancer. Methods: Isoform-specific, real-time polymerase chain reaction and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLCs) have been performed aiming to explore the expression levels of each p63 and p73 N -terminal isoforms and their ΔN:TA expression ratio. Results: For both p63 and p73, a N -terminal isoform-specific modulation that alter ΔN:TA isoform balance was identified. In particular, ΔNp63 isoform was significantly up-modulated, whereas TAp63 was slightly down-modulated in NSCLC specimens. Likewise, Δ2p73 and Δ2/3p73 were up-modulated, whereas ΔNp73 and ΔN′p73 isoforms were down-modulated. Moreover, a higher TAp63 and ΔN′p73 transcripts expression, detected in the normal tissue surrounding the tumors, correlates with poor patient outcome, representing independent prognostic factors for overall survival (ΔN′p73: p = 0.049, hazard ratio = 3.091, 95% confidence interval = 1.005–9.524 and TAp63: p = 0.001, hazard ratio = 8.091, 95% confidence interval = 2.254–29.05). Conclusion: Our findings suggest that p63 and p73 altered ΔN:TA expression ratio occurs in NSCLC likely contributing to the molecular pathogenesis of this tumor.

Meningioma 1 (MN1) gene overexpression has been reported in acute myeloid leukaemia (AML) patients and identified as a negative prognostic factor. In order to characterize patients presenting gene overexpression and to verify if MN1 transcript could be a useful marker for minimal residual disease detection, MN1 was quantified in 136 AML patients with different cytogenetic risk and in 50 normal controls. In 20 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment and in 8 patients with NPM1 mutation, we performed a simultaneous analysis of MN1 and the fusion-gene transcript or NPM1 mutation during follow-up. Sequential MN1 and WT1 analysis was also performed in 13 AML patients lacking other molecular markers. The data obtained show that normal cells consistently express low levels of MN1 transcript. In contrast, high levels of MN1 expression are present in 47% of patients with normal karyotype and in all cases with inv(16). MN1 levels during follow-up were found to follow the pattern of other molecular markers (fusion gene transcripts, NPM1 and WT1). Increased MN1 expression in the BM during follow up was always found to be predictive of an impending hematological relapse.

BACKGROUND The objective of this study was to evaluate the ability of the clinically available histone deacetylase (HDAC) inhibitor valproate to enhance the cytotoxicity of the Bcr-Abl inhibitor imatinib in imatinib-resistant cell lines. METHODS Interactions between imatinib, and valproate have been examined in imatinib-sensitive and -resistant chronic myeloid leukemia (CML)cell lines (K562, KCL-22, CML-T1) and in bone marrow mononuclear cells (MNCs) derived from imatinib-resistant CML patients. RESULTS In imatinib-sensitive cell lines, cotreatment with imatinib 0.5 μM and valproate 5 μM for 48 hours potently enhanced imatinib-induced growth arrest and apoptosis. In resistant cell lines and in primary MNCs derived from imatinib-resistant patients, valproate restored sensitivity to the cytotoxic effects of imatinib. Coexposure of cells to valproate and imatinib was associated with repression of several genes involved in Bcr-Abl transformation. In particular, the combination valproate–imatinib downregulated the expression of Bcr-Abl and the antiapoptotic protein Bcl-2, which is particularly overexpressed in imatinib-resistant clones. CONCLUSIONS Data from this study suggested that administration of the clinically available HDAC inhibitor valproate may be a powerful strategy to enhance cytotoxic effects of imatinib in those patient resistant to imatinib or in which complete cytogenetic remission has been not reached. Cancer 2006. © 2006 American Cancer Society.

Imatinib represents at present the most attractive therapy for BCR-ABL positive leukemias, even though a percentage of CML patients develop resistance to this compound. For these resistant patients a therapeutic approach based on a combination of drugs is more likely to be effective. In the last years, constitutive NF-kappaB/Rel activity has been demonstrated in several hematological malignancies. As a result, NFkB/Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-kappaB activation pathway offers a selective target to address tailored therapies. In the current study, we show that the IKK inhibitor PS1145 is able to inhibit the proliferation of CML cell lines and primary BM cells. Moreover, the addition of Imatinib increases the effects of PS1145 in resistant cell lines and BM cells from resistant patients, with a further increase of apoptosis and inhibition of proliferation and colony growth. Our data provide the rational for a new therapeutic approach, which combines Imatinib and the IKK inhibitor PS1145 in CML resistant patients.

To the Editor: Many anti‐cancer vaccination strategies have been tested in mice and humans in the attempt to eradicate leukemia cells 1. The vast majority of clinical trials are based on peptide vaccination which allows the induction of cellular response to specific tumor associated antigens 2. WT1(Wilms tumor‐1) gene is located on chromosome 11p13 and encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation. WT1 was originally described as a tumor suppressor gene although many evidences demonstrated that it plays an oncogenic function in the setting of leukemia. WT1 protein represents an optimal tumor antigen since it is highly expressed in acute leukemias, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms 3. By contrast, it is expressed at very low levels in normal hematopoietic progenitors. Expression of the WT1 protein is restricted to a limited set of tissues, including the gonads, uterus, kidney, and spleen.

Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL T315I mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation. The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors. We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm 2 (SS5) or 15 dyne/cm 2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45±5.5 and 30±1.5 cells per field, respectively ( P 2 and also when SS of 5 and 15 dyne/cm 2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9±7 and 18±5 ng/10 6 cells for 16 hours, respectively ( P 6 cells for 16 hours, respectively ( P CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRα or PDGFRβ increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRα inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB ( P

The detection of somatic mutations in a tumor represents a valuable tool for tumor characterization and provides the clinicians with information for setting up the most appropriate therapy. KRAS mutations in codons 12 and 13 are important biomarkers routinely analyzed in the clinic for the management of anti-EGFR treatment in colorectal carcinoma (CRC). Here we report a sensitive and inexpensive assay for KRAS mutations based on a PNA-mediated PCR clamping. The assay displays very high sensitivity (0.7%) and specificity (96.7%) when compared to traditional sequencing (SS) and pyrosequencing (PS), two of the most commonly and routinely used methods employed today by diagnostic laboratories. Furthermore, the PNA assay requires only basic and low-cost laboratory equipment, in contrast with all the most recent PCR-based technologies, which are highly sensitive but also much more expensive. Finally, despite the PNA assay does not allow for the definition of specific mutations, it is the cheapest and easiest screening method to firstly stratify wild-type and mutated patients, information that is strictly necessary to clinicians for the management of CRC and anti-EGFR treatment.

Background Mutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative Myeloproliferative Neoplasms (MPN) patients and its detection is now a cornerstone in the diagnostic algorithm. Methods We developed 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 JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence. Results Using this assay we found a percentage of mutated CD34+ cells ranging from 40% to 100% in Polycythemia Vera patients, from 15% to 80% in Essential Thrombocythemia and from 25% to 100% in Primary Myelofibrosis. This method allows to distinguish, with a high degree of specificity, at single cell level, between CD34+ progenitor stem cells harbouring the mutated or the wild type form of JAK2 in NPM patients. Conclusions This method allows to identify multiple gene abnormalities which will be of paramount relevance to understand the pathophysiology and the evolution of any type of cancer.

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.

Background Aurora kinase A (AURKA) is a member of serine/threonine kinase family. Several kinases belonging to this family are activated in the G2/M phase of the cell cycle being involved in mitotic chromosomal segregation. AURKA overexpression is significantly associated with neoplastic transformation in several tumors and deregulated Aurora Kinases expression leads to chromosome instability, thus contributing to cancer progression. The purpose of the present study was to investigate the expression of AURKA in non small cell lung cancer (NSCLC) specimens and to correlate its mRNA or protein expression with patients' clinico-pathological features. Materials and methods Quantitative real-time PCR and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLC) have been performed aiming to explore the expression levels of AURKA gene. Results AURKA expression was significantly up-modulated in tumor samples compared to matched lung tissue (p < 0.01, mean log2(FC) = 1.5). Moreover, AURKA was principally up-modulated in moderately and poorly differentiated lung cancers (p < 0.01), as well as in squamous and adenocarcinomas compared to the non-invasive bronchioloalveolar histotype (p = 0.029). No correlation with survival was observed. Conclusion These results indicate that in NSCLC AURKA over-expression is restricted to specific subtypes and poorly differentiated tumors.

Background Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-κB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes.Design and Methods We evaluated deferasirox activity on nuclear factor-κB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 μM deferasirox for 18h.Results Nuclear factor-κB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate.Conclusions Nuclear factor-κB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

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.

c-Src is a tyrosine kinase involved in tumor proliferation, migration, and angiogenesis and has been shown to modulate the cytotoxicity following cisplatin-induced DNA damages. c-Src is frequently activated in non–small cell lung cancer (NSCLC) tissues and cell lines, but no preclinical data regarding the effects of the novel potent Src inhibitor, dasatinib (BMS-354825), in the modulation of cisplatin resistance are currently available. The present study reports that treatment with dasatinib completely abrogated Src phosphorylation in the majority of the NSCLC cell lines tested (n = 7), with modest effects on cell proliferation and survival. In five cell lines, a higher cytotoxicity was observed delivering cisplatin in combination with dasatinib: the most evident effects were found in the squamous H520 cells due to the effective block of cisplatin-induced Src phosphorylation. Moreover, dasatinib treatment significantly blocked cisplatin-induced transcription of a panel of DNA repair and synthesis genes. In addition, a real-time PCR analysis done on tumor and matched normal lung specimens from 44 surgically resected NSCLC patients showed that Src transcripts are significantly upregulated in 23% of cases. In conclusion, Src-directed therapeutic strategies could interfere with cisplatin resistance, possibly allowing to reduce cisplatin doses, thus improving its efficacy. The data of this study support further clinical studies aimed to evaluate the efficacy of Src-inhibiting agents in combination with cisplatin in the treatment of NSCLC. [Mol Cancer Ther 2009;8(11):3066–74]

The Wilms' tumor gene WT1 is a reliable marker for minimal residual disease assessment in acute leukemia patients. The study was designed to demonstrate the potential use of WT1 to establish quality of remission in acute leukemia patients for early identification of patients at high risk of relapse. A prospective study based on a quantitative Real-Time PCR (TaqMan) assay in 562 peripheral blood samples collected from 82 acute leukemia patients at diagnosis and during follow-up was established. The evaluation of WT1 in peripheral blood samples after induction chemotherapy can distinguish the continuous complete remission patients from those who obtain only an "apparent" complete remission and who could relapse within a few months. WT1 helps identify patients at high risk of relapse soon after induction chemotherapy allowing post-induction therapy in high risk patients to be intensified.

Mutations in nucleophosmin (NPM) exon 12 and the resulting delocalization of NPM into the cytoplasm are the most specific and frequent cellular events in acute myeloid leukemia patients (AML) with normal karyotype. Cytoplasmatic NPM (NPMc+) is associated with responsiveness to chemotherapy and better prognosis. The activation of nuclear factor-kappaB (NF-kappaB) has been demonstrated to occur in a subset of AML patients and is thought to induce resistance to many chemotherapeutical agents. In this study, we demonstrate the increased in vitro sensitivity of NPMc+ cells to chemotherapeutical agents and their reduced NF-kappaB activity. Furthermore, we provide evidence of the interaction between NPMc+ and NF-kappaB in the cytoplasm, resulting in the sequestration and inactivation of NF-kappaB. The cytosolic localization and consequent inactivation of NF-kappaB justifies the reduced NF-kappaB DNA-binding activity observed in NPMc+ patients. These data, taken together, may provide a possible explanation for the increased rate of chemosensitivity observed among the NPMc+ patients.

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.

CD7/CD56-positive acute myeloid leukemias are characterized by constitutive phosphorylation of the NF-kB subunit p65 at Ser536

Recent advances in molecular genetics have increased knowledge regarding the mechanisms leading to myelodysplastic syndrome (MDS), secondary acute myeloid leukemia (AML), and therapy-induced MDS. Many genetic defects underlying MDS and AML have been identified thereby allowing the development of new molecular-targeted therapies. Several new classes of drugs have shown promise in early clinical trials and may probably alter the standard of care of these patients in the near future. Among these new drugs are farnesyltransferase inhibitors and receptor tyrosine kinase inhibitors including FLT3 and VEGF inhibitors. These agents have been tested in patients with solid tumors and hematologic malignancies such as AML and MDS. Most of the studies in MDS are still in early stages of development. The DNA hypomethylating compounds azacytidine and decitabine may reduce hypermethylation and induce re-expression of key tumor suppressor genes in MDS. Biochemical compounds with histone deacetylase inhibitory activity, such as valproic acid (VPA), have been tested as antineoplastic agents. Finally, new vaccination strategies are developing in MDS patients based on the identification of MDS-associated antigens. Future therapies will attempt to resolve cytopenias in MDS, eliminate malignant clones, and allow differentiation by attacking specific mechanisms of the disease.

NF-kB is a transcription factor that mediates antiapoptotic signals in several cancer cell lines. Here we have demonstrated that the cytotoxic drug, Etoposide, activates NF-kB in K562, a chronic myeloid leukemia blast crisis cell line. Treatment with the NF-kB inhibitors MG-132, Bay11-7082, and Resveratrol impedes Etoposide-induced NF-kB activation, rendering K562 sensitive to Etoposide-induced apoptosis. Stable expression of mutant form of IkB-alpha, which retains NF-kB inactive in the cytoplasm of cells, confirmed the data obtained with molecular inhibitors. Both inhibitors and stable expression of SR-IkB are associated with down-modulation of the antiapoptotic protein Bcl-xL, suggesting that the survival pathway activated by Etoposide involves NF-kB-mediated Bcl-xL expression.

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.

Introduction K-Ras mutations, occurring prevalently within codons 12/13, are detected in different cancer types including colorectal carcinoma (CRC) and might be responsible for resistance to anti-EGFR monoclonal antibodies (Cetuximab®) treatment. A sensitive method for K-Ras mutations detection is therefore crucial issue for choosing the most appropriate therapy. Currently, Sanger sequencing (SS) is accepted as gold standard method for mutational analysis but it is time-consuming and cost-ineffective. In addition, since in tumor analysis mutated clone may be present at different percentage, the SS sensitivity limit of 15-20% is crucial. Pyrosequencing (PS) has 5% detection limit, but still cost-ineffective, not largely available and shown a grey area (5-10%) in which genotype remains undeterminable. Therefore, a more sensitive method for detecting k-Ras mutations represents still a challenge. In this study we developed a novel, sensitive, friendly to use and very cheap assay for K-Ras mutations in CRC, based on PNA-directed PCR-clamping. Methods PNAs are synthetic molecules acting as potent DNA mimic in terms of sequence specific hybridization. As consequence, PNA/DNA heteroduplex is thermodynamically more stable than DNA/DNA counterpart but PNA cannot be extended by polymerase to function as a primer. Based on this, we designed a PNA molecule and one of the PCR primers perfectly matching the WT K-Ras sequence (encompassing codons 12/13), thus leading to a direct competition towards complementary template genomic DNA. In WT samples, perfect matching occurs because PNA-template hybridization is favoured and DNA amplification suppressed. By contrast, in case of mutated K-ras a single base pair mismatch will strongly impair the PNA-template stability favouring PCR amplification. Sixty formalin-fixed paraffin-embedded specimens from metastatic CRC were analyzed for K-Ras mutations by using both SS and PS and results and compared with PNA-directed PCR clamping method. Sensitivity was tested performing serial dilutions with plasmids carrying either wt or mutated sequences. Results PNA-methods displayed higher sensitivity compared to both SS and PS, allowing mutations detection up to 98.5%. The results showed a concordance of 63.3% among the three methods, while the concordance between PS and PNA-method resulted significantly increased. 47 samples were found to be mutated by PNA-PCR clamping, while 30 and 26 by PS and SS, respectively. Major discrepancy (13 out of 14) regards the detection of mutations by PNA-methods, otherwise unidentified either by SS or PS. Four samples, previously fallen into grey area, were classified as mutated by PNA-method. PNA-method failed to detect mutations just in 1 out of 30 cases, thus displaying a specificity of 96.66%. Correlation between clinical outcome after Cetuximab treatment and genotyping by PNA-method, which will confirm the clinical usefulness of the new method, is ongoing. Conclusion We developed an innovative method, able to detect K-Ras mutations in tumor paraffin-embedded specimens with very high sensitivity (98.5%). This is a cheap, easy to apply, quick and sensitive technique that could represent a novel tool, useful in clinical practice to drive therapeutic decisions.

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. Using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we identified Dab1 and Dab2, the homologs of Dm Disabled (Dab), as genes involved in CML progression. In Dm the Dab loss of function induced a worsening of the hBcr-Abl eye phenotype and an even stronger phenotype was obtained using Dab RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Dab is an adaptor protein acting downstream of many receptor tyrosine kinases (RTK). One of the human homolog of Dab, Dab1 is a large common fragile site gene involved in neural migration, and the other homolog Dab2 encodes an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many cancers 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) distributed as follows: 55 patients at diagnosis (19 enrolled in TOPS study), 9 chronic phase (CP), 7 accelerated phase (AP) and 11 blast crisis (BC). 21 healthy donors (10 PB and 11 BM) were analyzed as control. In 18 patients, genes expression was analyzed during remission as well. Protein expression was evaluated by Western Blot (WB) and Immunofluorescence (IF). In addition, K562 cells were transfected with Dab plasmids to evaluate the effects on cell proliferation. We found that in CML patients Dab1/2 expression was significantly decreased both in BM and PB (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 251.

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

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.

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.