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Background: ALL is a disease characterised by lack of differentiation and uncontrolled proliferation of lymphocyte precursor cells, driven by activation of oncogenes and inactivation of tumour suppressor genes. About 30% of B-cell ALL carries the Philadelphia chromosome (Ph), originated by the translocation t(9;22)(q34;q11), and thus express the fusion gene BCR-ABL. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the differentiation of B precursor cells, prevents apoptosis and also causes genetic instability. This ultimately leads to the acquisition of new mutations, resistance to BCR-ABL inhibitors and disease progression. In particular BCR-ABL stimulates DNA repair by non-homologous end-joining and homologous recombination repair mechanisms after various genotoxic stimuli, but a lot of deletions and abnormalities are also generated. Methods and patients: In order to identify whether some genetic lesions could contribute to genetic instability in Ph+ ALL, we analyzed the genome of 18 Ph+ ALL patients using high-resolution single nucleotide polymorphism (SNP) array. 250 ng of genomic DNA were processed on 250K NspI array according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH) and real-time quantitative polymerase chain reaction (PCR) were performed to validate our results. Results: We found a significant deletion on chromosome 22, cytoband q11.22, close to immunoglobulin lambda light chain genes, in the region 20913571-20931814, corresponding to the VpreB1 (CD179a, Pre-B-Lymphocyte gene 1) gene in about 30% of newly diagnosed Ph+ ALL patients. It is highly conserved among mammals and selectively expressed during the pre-B stage of B cells ontogenesis and together with IGLL1 encodes for the surrogate light chain of the pre-B cell receptor. In mice its monoallelic deletion is associated with an increase of lymphocytes in pre-B stage in bone marrow, while its complete absence causes the impossibility to generate mature B cells. Since Ph+ ALL cells are immunophenotypically blocked at the pre-B stage, we are investigating whether VpreB1 deletion could interfere with normal B cell development and thus could have a role in leukeamogenesis and disease progression. Conclusion: This study demonstrated that the use of high-resolution SNP array is a powerful technology to detect genetic anomalies not revealed by classical cytogenetics which may provide new insights into leukemogenesis.

Protein kinase C α has been reported to regulate cell cycle in several cell lines. Most of the reports describe a role for PKCα in G1/S transition but little is known about its possible involvement in G2/M progression. Our studies on the effects of PKC inhibitors, PKCα silencing and overexpression demonstrated a novel and positive role for PKCα in cyclin B1 regulation in human erythroleukemia cell line, K562. On the other hand, using PKC inhibitors and a PKCα inactive mutant, we could report that PKCα activity was not necessary for cyclin B1 regulation. Moreover, immunoprecipitation and immunocytochemistry experiments showed that these two proteins could physically interact each other and enter into the nuclei during G2/M progression. In order to better understand this mechanism, we investigated how PKCα could be attracted into the nuclei. We found a high increase of nuclear DAG during the G2/M phase. Then, using PMA and PLC inhibitors, we showed that PKCα translocation was due to the increase in nuclear DAG. Surprisingly, we saw the same effect on cyclin B1. Finally, in order to discover which PLC was involved, we silenced the nuclear localized PLCβ1 finding a decrease in PKCα and cyclin B1 nuclear amount. Taken together, our data demonstrated the existence of a novel DAG dependent mechanism linking PKCα and cyclin B1 which can regulate their entry into the nuclei during the G2/M phase of cell cycle.

Introduction. The proteasome inhibitor bortezomib is a new treatment option for patients with refractory or relapsed ALL, particularly when used in combination with conventional chemotherapy or other targeted agents. Indeed, a limited efficacy of bortezomib alone in ALL patients has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. CX-4945, a potent CK2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. Here, we have explored the cytotoxic effects of a bortezomib/CX-4945 combination in a panel of B- and T-ALL cell lines. Methods. B- (KOPN-8, NALM-6, RS4;11) and T- (MOLT-4, Jurkat, CEM-R) ALL cell lines were pretreated with bortezomib (Selleck Chemicals, Houston TX, USA), for six hours and then with CX-4945 (Selleck Chemicals, Houston TX, USA), for 16/24 hours. MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining and flow cytometric analysis. Protein expression was studied by western blot. Results. Cells were cultured in the presence of bortezomib or CX-4945, either alone or in combination at a fixed ratio. The combined treatment was more effective in reducing cell viability in all B-ALL cell lines and in MOLT-4 cells. Annexin V/PI staining was performed; interestingly, no synergism was detected if the two drugs were administered together since the beginning of treatment. In response to treatment with 2.5 nM bortezomib followed by 5 μM CX-4945, we detected an increase in apoptosis in B-ALL cell lines and in MOLT-4 cells after 24 h. Western blot analysis for the cleaved forms of caspase-3 and PARP, confirmed a higher apoptosis induction by the combined treatment. A reduction in anti-apoptotic Bcl2 concomitant with an increase in proapoptotic Bax, suggested that bortezomib/CX-4945 treatment caused a mitochondrial apoptosis. IRE1a and CHOP (established markers of ER stress/UPR-mediated apoptosis) levels increased in response to the combined treatment, in contrast the expression of GRP78/BIP (a marker of UPR activation) decreased, suggesting that a potential mechanism by which the drug combination induced cell death, involved ER stress induction by bortezomib and the inability to respond by adequate activation of the UPR signaling which was blocked by CX-4945. Conclusions. Here, we demonstrated that the proteasome inhibitor bortezomib and the CK2 inhibitor CX-4945 interact in a synergistic manner to induce apoptosis both in B- and in T-ALL cell lines. Drug cytotoxicity was associated with modulation of the ER stress/UPR signaling pathway. Importantly, the synergism was observed only when bortezomib treatment preceded CX-4945 administration. Therefore, our findings support clinical application of bortezomib in combination with CX- 4945 in B- and T-ALL treatment.

Background: The Bcr-Abl kinase expressed in ALL drives malignant transformation of human pre-B cells and promote aberrant alternative splicing of genes involved in B-cell signaling and differentiation. The fact that Ikaros (Ik) transcription factor functions as a critical regulator of normal lymphocyte development and the observation of rapid development of leukaemia in mice expressing non-DNA binding isoforms, prompted our study to investigate whether normal Ik expression and function might be altered in Ph+ ALL. Patients and methods: Bone marrow and peripheral blood samples from 47 adult patients (median age 55 yrs, 18-76) with Ph+ ALL were collected after informed consent. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning, nucleotide sequencing and western blot analyses were performed to identify the specific Ik isoforms. Results: In 43/47 (90%) patients the Ik6 isoform, lacking all N-terminal zinc-fingers responsible for DNA-binding, was detected and in 23 of them (54%) it was the predominant isoform. Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patients expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In the patients who seemed to express by RT-PCR only full-length Ik isoforms cloning and subsequent sequencing revealed clones harboring different transcripts. We detected an in frame deletion of 10 aa (ΔKSSMPQKFLG) at the end of ex6 due to the selection of an alternative donor splice site. The consequences were impaired activation of transcription and impaired ability to form dimers. We also observed isoforms with a 60-bp insertion (TYGADDFRDFHAIIPKSFSR) immediately upstream of exon 4 at the exon 2/exon 4 junction either alone or together with the in-frame deletion. Also in this in case the aberration is due to the selection of alternative splice site which determines the skipping of exon 3 and the insertion of a sequence inside the intron 3-4 which starts with an alternative acceptor splice site. Aberrant Ik isoforms had a cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. Conclusions: Our data demonstrated that aberrant splicing of Ikaros represents a frequent feature in Ph+ ALL patients and that dominant negative Ik6 splice variant correlates with BCR-ABL mRNA levels, disease progression and relapse.

Expression of dCK and dGK in T-ALL cell lines. Western blotting analyses for the expression of dCK and dGK proteins in T-ALL cell lines. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.

Specific effects of nelarabine on PI3K/AKT and MEK/ERK1/2 pathways. Western blotting analyses for the expression of p-AKT and p-ERK in resistant T-ALL cell lines treated with the specific inhibitors LY294002 (PI3K inhibitor), CCI-779 (mTOR allosteric inhibitor) or trametinib (MEK1/2 inhibitor) alone or in combination with nelarabine. Thirty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. CTRL: untreated cells; Nela and N: nelarabine at 10 μM; LY: LY294002 at 10 μM; CCI: CCI-779 at 100 nM; Tram: trametinib at 1 μM. Cells were treated for 48 h.

The combination of nelarabine and ZSTK-474 is synergistic in CEM-R cells, which overexpress P-gp. Cell viability assay of CEM-R cell line treated for 48Â h with increasing concentrations of nelarabine alone or combined with the pan PI3K p110 inhibitor ZSTK-474. One representative of two different experiments is shown.

Bcl2 expression in T-ALL cell lines. Western blotting analyses for the basal expression of Bcl2 in untreated T-ALL cell lines. Twenty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. Densitometric analysis was performed using a Chemidoc 810 Imager with the appropriate software (UVP, Upland, CA, USA), and for each cell line, Bcl2 protein expression is indicated relatively to β-actin expression.

Le traslocazioni di MLL, presenti in oltre il 20% delle AML pediatriche, si associano a prognosi sfavorevole. Pertanto, continui sforzi sono necessari per identificare nuove terapie efficaci per questi pazienti. EPZ- 5676 è un inibitore di DOT1L, metiltranferasi con attività leucemogenica in presenza di riarrangiamenti di MLL (MLL-r). Sorafenib è un inibitore di tirosino-chinasi, tra cui FLT3, frequentemente utilizzato nel trattamento dei pazienti MLL-r. Scopo dello studio è la valutazione degli effetti citotossici e biologici della combinazione EPZ-5676 e Sorafenib. Sono state utilizzate linee cellulari con o senza MLL-r, e cellule primarie ottenute da pazienti AML pediatrici con MLL-r. Sono stati valutati gli effetti citotossici e biologici dei singoli farmaci e della loro combinazione mediante analisi citofluorimetriche, Western Blotting e RQ-PCR. EPZ-5676 inibisce DOT1L inducendo demetilazione del target H3k79 in tutte le cellule, ma la proliferazione cellulare è significativamente inibita solo nelle cellule MOLM13 e nOMO1 (MLL-r) e OCI-AML3 (non MLL-r). Ciò si associa ad induzione dell’apoptosi o effetti citostatici. EPZ-5676 riduce l’espressione dei geni HOXA9, MEIS1 e FLT3 in tutte le cellule; STAT5 e c-Myc nelle cellule MLL-r. Inoltre, EPZ-5676 modula parzialmente le vie di sopravvivenza PI3k/Akt e MAPk. La combinazione EPZ-5676 e Sorafenib risulta sinergica nelle linee cellulari (anche in assenza di MLL-r) e nelle cellule primarie, in cui induce un significativo incremento dell’apoptosi. In conclusione, gli effetti di EPZ-5676 non sembrano dipendere esclusivamente dalla presenza di MLL-r. Inoltre, EPZ-5676 è sinergico con Sorafenib, suggerendo un nuovo approccio terapeutico per il trattamento dei pazienti pediatrici affetti da AML.

Background: About 20% of the childhood AML present MLL gene translocations, that are associated with a very poor prognosis. That is why there is a strong interest in developing novel therapeutic strategies for these patients. As DOT1L is responsible for H3K79 methylation and is associated with MLL-r leukemogenesis, DOT1L inhibitors entered in clinical trials as promising treatments. MLL-r AML also showed an overexpression of FLT3 receptor tyrosine kinase. Therefore, FLT3 inhibitors, such as the multi-tyrosine kinase inhibitor Sorafenib, demonstrated encouraging efficacy in AML. Aims: To investigate the efficacy of a combination treatment using DOT1L inhibitor EPZ-5676 and Sorafenib to treat MLL-r AML. Methods: MLL-r (MOLM13, NOMO1, THP1) and non MLL-r (OCI-AML3, HL60 and U937 harboring CALM-AF10 translocation) AML cell lines, and MLL-r primary samples from pediatric AML patients were used. Flow cytometry analyses were performed to assess absolute cell counting and apoptosis (AnnexinV FITC/PI staining). Protein expression and H3K79 methylation were quantified by Western blot. mRNA expression was studied by quantitative Real-Time PCR (Q-PCR). Results: Firstly, the specific effects of DOT1L inhibition were examined in AML cell lines treated with increasing concentrations of EPZ-5676, up to 18 days. Cell growth was significantly inhibited after at least 8 days of treatment in MOLM13 and NOMO1 cells, but time-dependent apoptosis occurred only in MOLM13, thus suggesting that DOT1L inhibition alone might not be able to induce cytotoxicity. Whereas no effects were observed in HL60, U937 (non MLL-r), or THP1 (MLL-r) cells, both cell growth impairment and apoptosis were detected in non MLL-r OCI-AML3 cells, so that the impact of DOT1L inhibition could not exclusively rely on MLL rearrangements. Repression of DOT1L occurred since the 4th day of treatment, as demonstrated by the complete loss of H3K79me2. To further explore the consequence of this phenomenon, both MLL targets and key component of signaling pathways involved in AML survival (i.e. PI3K, FLT3 and MAPK) were investigated in cells treated with 1 uM EPZ-5676, up to 28 days. Gene expression of HOXA9, MEIS1, FLT3 and CDK6 protein (MLL target) decreased in all cell lines, whereas STAT5 and c-Myc mRNAs, along with STAT5 protein expression, were downregulated only in MLL-r cells. Furthermore, in MOLM13 and NOMO1 cells p-Erk was strongly reduced. Finally, p-Akt slightly decreased in nearly every case, whereas a strong induction in p-S6RP was observed only in U937 cells after prolonged treatment, suggesting the possible involvement of PI3K pathway in drug resistance mechanisms. To increase the benefit of DOT1L inhibition, both AML cell lines and MLL-r primary samples were pretreated with increasing concentration of EPZ- 5676 for 4/8 days, following 24/48h treatment with Sorafenib. This combination resulted in a synergistic effect in nearly all cases. Importantly, EPZ-5676 pretreatment increased Sorafenib-induced cell growth inhibition in EPZ-5676 refractory cells HL60, U937 and THP1. Moreover, in primary AML samples both EPZ-5676 and Sorafenib showed a limited effect as single agent, whereas their combination induced a drastic increase of apoptosis. Summary/Conclusions: These results demonstrated that the single administration of EPZ-5676 has a limited antileukemic activity, which is not restricted to MLL-r AMLs. However, the combination of EPZ-5676 with Sorafenib revealed a synergistic effect in both MLL-r and non MLL-r AMLs, paving the way to innovative and more effective treatments for pediatric AML patients.

The introduction of novel chemotherapy protocols has improved the outcome of T-cell acute lymphoblastic leukemia (T-ALL) patients, but refractory and/or relapsing disease remains a major concern. In this context, a major contribution was provided by the introduction of the nucleoside analogs and in particular nelarabine, approved for salvage treatment of refractory/relapsed TALL patients. Aims: A serious nelarabine drawback is that it induces a dose-dependent neurotoxicity. To improve nelarabine efficacy, it is essential to study its molecular targets, testing selective inhibitors of such targets, to be administered in combination with nelarabine, allowing for a lower dosage of the chemotherapeutic. Methods: Human T-ALL cell lines (HBP-ALL, DND41, Jurkat, MOLT-4, MOLT16, CEM, CEM-R drug-resistant, ALL-SIL, Loucy, HSB2, Peer, PF382, P12-ICHIKAWA) and primary T-ALL refractory/relapsed lymphoblasts from patients were incubated with increasing concentrations of nelarabine alone or combinated with PI3K/mTOR inhibitors for cell viability assays and absolute cell counting by flow cytometry. Flow cytometry was also performed to analyze apoptosis and for phenotyping analyses. Protein expression was evaluated by Western Blot. ENT1/ENT2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: We examined cell viability of cell lines and primary T-ALL refractory/relapsed cells. Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC5015μM). The most resistant cell line (IC50>300μM) was Loucy, which is representative of ETP-ALL, a T-ALL subtype with a very poor prognosis. Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48h treatment with 2-5 μM nelarabine, as demonstrated by flow cytometric analyses of stained with AnnexinV FITC/PI cells and western blot analyses of caspases 8, 9, 3, and PARP. In contrast, resistant T-ALL cells were not perturbated. It has been reported that the levels of expression of ENT 1/2 nucleoside transporters were related to in vitro nelarabine sensitivity of T-ALL cell lines and primary samples. The expression of ENT1/2 transporters could be also dependent on interactions between leukemic cells and tumor microenvironment. No significant differences in ENT1/2 mRNA levels between samples sensitive or resistant to nelarabine were seen. Modulation of ENT1/2 gene expression was not related to nelarabine treatment, even if in some cases the co-culture with human stromal cells HS-5, which mimic the bone marrow microenvironment, partially supported cell survival. Upregulated phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation, survival, and drugresistance. We analyzed the effects of nelarabine on the phosphorylation status of key components of the PI3K/mTOR pathway in T-ALL settings. Sensitive TALL cells showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein (S6RP). In contrast, resistant cells treated with nelarabine alone, showed a hyperactivation of PI3K/mTOR and MEK/ERK signaling pathways. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing cell viability and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR signaling. Summary/Conclusions: Nelarabine combined with PI3K inhibitors efficiently reduced cell viability and induced apoptosis in T-ALL settings, allowing for a lower dosage of nelarabine and therefore, synergizing with conventional therapies in relapsed/refractory T-ALL patients with upregulation of PI3K signaling.

DNA damage and ROS production. A. Flow cytometric analysis of γH2AX in MOLT-4 and Jurkat sensitive to nelarabine cells (upper panel). Percentages of γH2AX are shown in the table (lower panel). B. Reactive oxygen species (ROS) production was assessed in MOLT-4 and Jurkat sensitive T-ALL cell lines after 1 and 24 h treatment with nelarabine. Fluorescence values (DCF) were reported as the percentage of intracellular ROS in respect to controls. ROS increased significantly in both 1- and 24-h treatment compared to controls in MOLT-4 and Jurkat cells. Statistical analyses were performed with the Dunnett’s multiple comparison test.

HS-5 stromal cells protect MOLT-4 cells from nelarabine cytotoxicity at least in part through CXCL12/CXCR4 interactions. A. MTT assays of MOLT-4 cells growing alone or in co-culture system with HS-5 cells and treated with nelarabine (2 μM for 48 h) in a Transwell@ system. Three hundred seventy-five thousand MOLT-4 cells were seeded in the upper chamber of the Transwell@ system. Results are the mean of three different experiments ± SD. B. Western blot analysis for p-ERK, p-CXCR4, and their total forms in MOLT-4 cells grown either in the absence or the presence of HS-5 stromal cells in a Transwell@ system. Nelarabine treatment (2 μM) was for 24 h. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.

Introduction: Chk1 and Chk2 are serine/threonine kinases that are involved in cell cycle control and DNA repair pathways. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1-negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5-2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of baseline genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. Moreover, in all sensitive cell lines Chk1/Chk2 inhibition increased number of γH2AX foci (western blot and immunofluorescence analysis) and phosphorylation of Chk1 (ser317 and ser345) representative of induced DNA damage. Functionally, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15). The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients with 36% of them having IC50 within 100-500 nM. By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells. Noteworthy, in mice transplanted with T-lymphoid leukemia, PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts after 24 hrs to elucidate the mechanisms of action of PF-0477736. Results showed a differential expression (p < 0.05) of genes involved in chromatin modification (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in leukemia cells. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 provided by Pfizer.

Background: Detection of residual leukemic cells by measuring BCR-ABL1 transcript level with assays based on quantitative polymerase chain reaction (qPCR) is necessary in the monitoring of minimal residual disease in patients with BCR-ABL1 positive ALL. Aim: To investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared results obtained by conventional qPCR with those obtained by the nanofluidic approach. Patients and methods: We analyzed 60 BCR-ABL1 positive ALL samples expressing p190 (42) or p210 (18) isoform, first by TaqMan absolute qPCR (Applied Biosystems 7900HT Fast Real-Time PCR System). BCR-ABL1 target gene and ABL1 control gene copies were derived by the interpolation of cycle threshold values to the appropriate standard curve obtained using serial plasmid dilutions containing known BCR-ABL1 or ABL1 gene copies. Results: Samples resulted in complete (22) or major (38) molecular response (BCR-ABL1/ABL1 ratio ≪ 0.001 or < 0.1, respectively; 3,8 BCR-ABL1 mean copy number copies). Subsequently we reanalyzed the samples using the 12.765 Digital array (Fluidigm) and the integrated BioMark Digital PCR Analysis software (Fluidigm). The nanofluidic biochip consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR. As fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Sensitivity and reproducibility of the assay were assessed using six serial dilutions of plasmids (Ipsogen) expressing known BCR-ABL1 p190 transcript copy number (10000; 1000; 100; 50; 10; 1 copies). A 2 µl volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 ALL samples: digital array resulted positive in 21 of major molecular response samples (2,13 BCR-ABL1 mean copy number copies) and in any complete molecular response samples. In conclusion, the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Background. Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens. Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory T-ALL or T-LBL. Methods. After obtaining an informed consent, 16 patients (median age 33 years, range 19-45, M/F = 13/3) affected by T-ALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1-3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing. Results. Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8-54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively. Conclusions. N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored. Acknowledgments. European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Low RPKM estimates (0.01

Introduction: Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at at 2q33.3-q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3) and at 12p13 (ETV6). Deletions also affected genes involved in cell regulations as CDKN2A (9p21) and RB1 (13q14.2). Most frequent gains affected the TP73 gene at 1p36.3, the oncogenes MYC and PVT1 at 8q24 (4.33 Mb), PRAME at the 22q11.22. Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Patients with more than 10 CNAs were associated with a worse prognosis. Conclusions: These data demonstrate that different cooperating events may be involved in the generation of APL. These novel findings may be used to stratify patients according to genomic changes and to facilitate the screening for novel therapeutic targets. Supported by: AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo.

Background. Approximately 30% of adult ALL patients are characterized by the presence of the Philadelphia (Ph) chromosome, which derives from a reciprocal translocation t(9;22)(q34;q11) and results in a chimeric BCR-ABL oncogene. The prognosis of this subset of patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs.

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.