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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.

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.

Introduction A comprehensive analysis of the immune cell infiltrate collected from pleural fluid and from biopsy specimens of malignant pleural mesothelioma (MPM) may contribute to understanding the immune-evasion mechanisms related to tumor progression, aiding in differential diagnosis and potential prognostic stratification. Until now such approach has not routinely been verified. Methods We enrolled 275 patients with an initial clinical diagnosis of pleural effusion. Specimens of pleural fluids and pleural biopsy samples used for the pathologic diagnosis and the immune phenotype analyses were blindly investigated by multiparametric flow cytometry. The results were analyzed using the Kruskal-Wallis test. The Kaplan-Meier and log-rank tests were used to correlate immune phenotype data with patients’ outcome. Results The cutoffs of intratumor T-regulatory (>1.1%) cells, M2-macrophages (>36%), granulocytic and monocytic myeloid-derived suppressor cells (MDSC; >5.1% and 4.2%, respectively), CD4 molecule–positive (CD4+) programmed death 1–positive (PD-1+) (>5.2%) and CD8+PD-1+ (6.4%) cells, CD4+ lymphocyte activating 3–positive (LAG-3+) (>2.8% ) and CD8+LAG-3+ (>2.8%) cells, CD4+ T cell immunoglobulin and mucin domain 3–positive (TIM-3+) (>2.5%), and CD8+TIM-3+ (>2.6%) cells discriminated MPM from pleuritis with 100% sensitivity and 89% specificity. The presence of intratumor MDSC contributed to the anergy of tumor-infiltrating lymphocytes. The immune phenotype of pleural fluid cells had no prognostic significance. By contrast, the intratumor T-regulatory and MDSC levels significantly correlated with progression-free and overall survival, the PD-1+/LAG-3+/TIM-3+ CD4+ tumor-infiltrating lymphocytes correlated with overall survival. Conclusions A clear immune signature of pleural fluids and tissues of MPM patients may contribute to better predict patients’ outcome.

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.

The research leading to these results received funding from the Obra Social “la Caixa” (to ME), the Cellex Foundation (to ME), the European Union's Horizon 2020 research and innovation programme under grant agreement No 727264 Epipharm (to ME), the 2015 Endowment fund for Research into Respiratory Health (“Fonds de Dotation Recherche en Santé Respiratoire”; to MD), the “Fondation ARC pour la recherche sur le cancer - Aide individuelle” (to MD), the Spanish Association Against Cancer (to VD), the Pla Estratègic de Recerca i Innovació en Salut (to SM), the Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional (PI16/01821 to LMM and PI15/02223, to IG-B), RETTICS grant (RD12/0036/0040 to IG-B), Fundación Merck Salud (to IG-B), Departamento de Salud, Gobierno de Navarra (074-2017, to IGB), the European Commission MSCA IMMUNOMARK-799818 (to IBara), the China Scholarship Council and Karolinska Institutet Fonder (to QX), and the Health and Science Departments of the Generalitat de Catalunya (to ME). We thank IGTP–HUGTP Biobank (PT13/0010/0009) University Hospital Ramon y Cajal-IRYCIS Biobank (PT13/0010/0002), members of the Spanish National Biobanks Network of the Instituto de Salud Carlos III, Andalusia Public Health System Biobank, the Biological Resource Center of the Hospices Civils de Lyon (Tissu-tumorothèque Est), and the Tumor Bank Network of Catalonia for their help with the collection of samples., Duruisseaux, M., Martínez-Cardús, A., Calleja-Cervantes, M.E., Moran, S., Castro de Moura, M. , Davalos, V., Piñeyro, D., Sanchez-Cespedes, M., Girard, N., Brevet, M.,Giroux-Leprieur, E., Dumenil, C. Pradotto, M., Bironzo, P., Capelletto, E., Novello, S., Cortot, A. , Copin, M.-C., Karachaliou, N.,Gonzalez-Cao, M. ,Peralta, S., Montuenga, L.M.,Gil-Bazo, I. ,Baraibar, I.,Lozano, M.D., Varela, M. ,Ruffinelli, J.C.,Palmero, R., Nadal, E.,Moran, T., Perez, L., Ramos, I., Xiao, Q.,Fernandez, A.F., Fraga, M.F.,Gut, M., Gut, I. ,Teixidó, C. , Vilariño, N., Prat, A., Reguart, N., Benito, A.,Garrido, P., Barragan, I., Emile, J.-F., Rosell, R., Brambilla, E., Esteller, M.

Introduction Les anti-PD-1 permettent un gain de survie dans les cancers bronchiques non a petites cellules (CBNPC) metastatiques. Cependant, la majorite des patients ne tire pas de benefice de ces traitements. Nous avons evalue l’interet de l’analyse a haut debit du methylome de CBNPC dans la prediction de l’efficacite des anti-PD-1. Methodes Etude multicentrique (15 centres) internationale (France, Espagne, Italie) retrospective. Criteres d’inclusion : CBNPC metastatique traite par anti-PD-1et prelevement tumoral disponible. Une cohorte decouverte (n = 34) a permis d’evaluer la correlation entre caracteristiques epigenetiques des tumeurs evaluees par methylome a haut debit (Illumina 850 K array beadchip) et efficacite des anti-PD-1. Une signature epigenetique (EPIMMUNE) predictive de l’efficacite des anti-PD-1 a ete elaboree et confirmee dans une cohorte de validation (n = 47). Nous avons isole un site specifique de methylation associe au benefice clinique et confirme son interet predictif par une technique de pyrosequencage dans une troisieme cohorte independante (n = 61). La methode de Kaplan–Meier a ete utilisee pour estimer la survie sans progression (SSP) et la survie globale (SG), le test de log-rank pour comparer les groupes de patients. Un modele de COX multivarie a ete construit pour identifier les variables independamment associees avec la SSP et la SG. Resultats La signature EPIMMUNE+ etait associee a une SSP allongee (HR 0,010, IC95 % 3,29 × 10–0,0282 ; p = 0,0067) et une SG allongee (HR 0,080, IC95 % 0,017–0,373 ; p = 0,0012) avec les anti-PD-1 dans la cohorte decouverte ( Fig. 1 ). Ces resultats etaient confirmes en SSP dans la cohorte validation. EPIMMUNE+ n’etait pas associee a l’expression de PD-L1, a l’infiltrat CD8 ou a la charge mutationnelle. Les tumeurs EPIMMUNE–etaient enrichies en macrophages, en polynucleaires neutrophiles, en fibroblastes et en cellules endotheliales senescentes. Le status demethyle du gene FOXP1 etait associe a une SSP allongee (p = 0,0063) et une SG allongee (p = 0,0094) dans la cohorte decouverte et dans une deuxieme cohorte de validation. EPIMMUNE+ et la demethylation de FOXP1 n’avait pas de valeur pronostique dans le TCGA. Conclusion Les caracteristiques epigenetique des cellules tumorales et du microenvironnement conditionnent l’efficacite des anti-PD-1 dans les CBNPC. La demethylation de FOXP1 pourrait etre un biomarqueur predictif de l’efficacite des anti-PD1 utilisable en clinique, complementaire de PD-L1 ou de la charge mutationnelle. Etude soutenue par le FRSR.

Objectives Cisplatin-based chemotherapy is moderately active in malignant pleural mesothelioma (MPM) due to intrinsic drug resistance and to low immunogenicity of MPM cells. CAAT/enhancer binding protein (C/EBP)-β LIP is a pro-apoptotic and chemosensitizing transcription factor activated in response to endoplasmic reticulum (ER) stress. Materials and methods We investigated if LIP levels can predict the clinical response to cisplatin and survival of MPM patients receiving cisplatin-based chemotherapy. We studied the LIP-dependent mechanisms determining cisplatin-resistance and we identified pharmacological approaches targeting LIP, able to restore cisplatin sensitiveness, in patient-derived MPM cells and animal models. Results were analyzed by a one-way analysis of variance test. Results We found that LIP was degraded by constitutive ubiquitination in primary MPM cells derived from patients poorly responsive to cisplatin. LIP ubiquitination was directly correlated with cisplatin chemosensitivity and was associated with patients’ survival after chemotherapy. Overexpression of LIP restored cisplatin’s pro-apoptotic effect by activating CHOP/TRB3/caspase 3 axis and up-regulating calreticulin, that triggered MPM cell phagocytosis by dendritic cells and expanded autologous anti-tumor CD8+CD107+T-cytotoxic lymphocytes. Proteasome inhibitor carfilzomib and lysosome inhibitor chloroquine prevented LIP degradation. The triple combination of carfilzomib, chloroquine and cisplatin increased ER stress-triggered apoptosis and immunogenic cell death in patients’ samples, and reduced tumor growth in cisplatin-resistant MPM preclinical models. Conclusion The loss of LIP mediates cisplatin resistance, rendering LIP a possible predictor of cisplatin response in MPM patients. The association of proteasome and lysosome inhibitors reverses cisplatin resistance by restoring LIP levels and may represent a new adjuvant strategy in MPM treatment.

Fluctuating levels of estradiol and progesterone during the estrous cycle may induce structural changes in several brain nuclei including the hippocampus, where some neurons express estrogen receptors. Nitric oxide plays a wide range of functions in the nervous system generally by acting as a neurotransmitter-like molecule. It has been demonstrated that long-term treatments with estradiol in ovariectomized females and with testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in rat hypothalamus, whereas changes in nNOS immunoreactivity or in associated NADPH-diaphorase activity were observed both in hypothalamus and in amygdala during different phases of estrous cycle. Estradiol could induce nNOS expression in several brain regions in rodents. Therefore, to clarify if the hippocampal NO producing system is a target for gonadal hormones in physiological conditions, we have investigated the effects of estrous cycle in the expression of nNOS immunoreactivity on two-month-old intact female mice. Immunoreactive cells were observed in all hippocampal subregions: the higher number was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate gyrus. The number of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during proestrus and metaestrus, and these variations were statistically significant in CA1, CA2 and CA3 regions. These results suggest that the nitrinergic system is a target for estrogen action in the hippocampus, and that this action may take place in physiological conditions according to the short-term variations of gonadal hormones during the estrous cycle.

Nitric oxide (NO) is a gaseous neurotransmitter that plays an important role in the regulation of sexual behavior in rodents. NO is produced, within the central nervous system, by the enzyme neural NO synthase (nNOS) whose expression is influenced by gonadal hormones. In previous studies, we demonstrated that part of the nitrergic hypothalamic and limbic system is influenced, in physiological conditions, by the hormonal fluctuations during the estrous cycle, but we were unable to distinguish among the role played by progesterone (P) or estradiol (E 2 ) in inducing these changes. In the present study, we investigated the effects of E 2 and P (alone or together) on the nitrergic system of gonadectomized female mice, following a timing of administration that emulates the different phases of estrous cycle. In parallel, we tested the influence of the two hormones on sexual behavior, confirming that P works in synergistic fashion with E 2 to facilitate female receptivity. The quantitative analysis of nNOS-ir system demonstrated a statistically significant variation in the number of positive cells only in those part of the limbic–hypothalamic nitrergic system that are affected in cycling females, i.e. the bed nucleus of the stria terminalis, the arcuate nucleus and the medial preoptic area, with the highest number of positive neurons observed in E 2 + P group. The variable effects of E 2 and P may depend on the different distribution of their receptors within the analyzed nuclei, but the relationships among variations of estrogen and progesterone levels and in vivo modulation of nNOS expression remain unknown and needed further investigations.

Core-binding factor (CBF) leukemias are characterized by a high degree of sensitivity to high-dose cytarabine (ARA-C) treatment and by a relatively favorable prognosis compared with most other forms of adult acute myeloid leukemia (AML). The molecular basis of the response to chemotherapy is still being analyzed. The 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, and when it is abnormally expressed, it confers factor-independent growth to hematopoietic cells. In this study, we analyzed the expression levels of PR3 in 113 AML patients. PR3 is highly expressed in AML, mainly in CBF leukemias in which PR3 is not only expressed, but also abnormally localized within the nuclear compartment. Nuclear PR3 results in cleavage of nuclear factor (NF)-kappaB p65 into an inactive p56 subunit lacking any transcriptional activity. The nuclear localization of PR3 is responsible for increased proliferation, apoptosis arrest and increased sensitivity to high-dose ARA-C. This study provides a new molecular mechanism that is responsible for NF-kappaB inactivation and increased sensitivity to chemotherapy in CBF leukemias.Leukemia advance online publication, 14 January 2010; doi:10.1038/leu.2009.207.

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.

Alternative splicing plays a key role for generating protein diversity and contribute to essential biological processes among them differentiation, cell cycle regulation and apoptosis. Alterations in alternative splicing was identified in several human diseases, including cancer. Bcl-x is a Bcl-2 family member which is alternatively spliced in two distinct transcripts generating two different proteins: Bcl-xL (Long) and Bcl-xS (Short), with antagonistic functions. The long isoform inhibits apoptosis, in contrast the short one activates it. Bcl-xL is highly expressed in different types of cancer. CML is a myeloproliferative disorder characterized by a reciprocal chromosomal translocation t(9;22), that generate Bcr-Abl chimeric protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cell proliferation, alteration in cell-cell and cell-matrix adhesion and resistance to apoptosis. It has been reported that in CML Bcl-x isoforms ratio is altered in favor of the anti-apoptotic one (Bcl-xL).We investigated the involvement of Bcr-Abl in regulating splicing events affecting Bcl-x pre-mRNA occurring in CML. By proteomic approach, based on strategy using GST-Pull Down assay, we attempted to gain insight into functional interactions occurring among the oncogenic kinase Bcr-Abl and RNA-binding proteins, identifying physical interactions among the adapter protein Nck-beta, the spliceosome ribonucleoprotein hnRNPA1 and Sam68 which are assembled to form a functional quaternary complex. These interactions were further confirmed by immunofluorescence staining showing co-localization of the proteins within peculiar nuclear splicing regions (nuclear speackles). Using RNA Pull Down assays we detected that the quaternary complex was able to specifically bind BCL-xL mRNA. Using several specific tyrosine kinase inhibitor such as BMS-354825, AMN-107 or STI-571, we identified a key role of BCR-ABL phosphorylation on integrity of the multimeric complex. Furthermore, STI-571 impairs the interaction between quaternary complex and Bcl-xL mRNA. As observed also for other spliced genes like MCL1, PP1γ; PASG, VRK and CENT1. The Bcr-Abl mediated Bcl-x splicing was also confirmed by quantitative PCR and immunofluorescence analysis. Bcr-Abl leads to increased Bcl-xL mRNA expression as proved either by Bcr-Abl ectopic overexpression or by a Bcl-x minigene assay. In conclusion, these data indicate that phosphorilated Bcr-Abl is part of a complex affecting Bcl-x alternative splicing, increasing the anti-apoptotic isoform in respect to the pro-apoptotic one. These data represent the first original report of a direct involvement of Bcr-Abl in splicing events, thus opening new perspectives in CML therapeutic treatment field. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2011-4771

Despite the role of Bcr-Abl in the pathogenesis of CML is well established, the mechanisms responsible for CML progression remain 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 the genetic screening using our set-up model of human p210 Bcr-Abl (hBcr-Abl) transgenic Drosophila melanogaster (Dm) in which the tissue specific expression of hBcr-Abl is able to induce a severe eye phenotype or the formation of melanotic tumors, when expressed into the fly lymph gland, the Dm hematopoietic system. The whole fly genome containing 14.000 genes was analyzed using 278 fly stocks carrying well characterized chromosome deletions. The resulting progeny was screened using eye and lymph gland as first and second read-out system respectively. Data obtained from both screens were analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified genes were validated analyzing either BM or PB samples from CML patients and healthy donors. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screen 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. 42% of the 4000 Dm genes mapping in these regions displayed a known human counterpart. Gene Ontology profiles of these genes included oncogenes, tumor suppressors and genes involved in the regulation of transcription, signal transduction, proliferation, differentiation, apoptosis and splicing. Moreover, a computational comparison of our results in fly with gene expression signatures of CML from Microarray data, showed only a partial overlap. Interestingly, the 72% of identified genes was not known to be involved in human leukaemia. However, further confirmation of our findings comes from the validation in human samples in which 1250 genes were found to be associated with CML; among these genes, we found not only an alteration of their expression profiles in CML patients compared 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 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 points to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression. 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 246.

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.

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 2171 Poster Board II-148 Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks. Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood. The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively. CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L). The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis. Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors. Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA. 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 in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. 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, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. 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 pre-mRNA splicing scenario. Disclosures: No relevant conflicts of interest to declare.

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

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

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