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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.

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.

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.

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.

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

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