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T-acute lymphoblastic leukemia (T-ALL) is characterized by several genetic alterations and poor prognosis in about 20-25% of patients. Notably, about 60% of T-ALL shows increased Notch1 activity, due to activating NOTCH1 mutations or alterations in the FBW7 gene, which confer to the cell a strong growth advantage. Therapeutic targeting of Notch signaling could be clinically relevant, especially for chemotherapy refractory patients. This study investigated the therapeutic efficacy of a novel anti-Notch1 monoclonal antibody by taking advantage of a collection of pediatric T-ALL engrafted systemically in NOD/SCID mice and genetically characterized with respect to NOTCH1/FBW7 mutations. Anti-Notch1 treatment greatly delayed engraftment of T-ALL cells bearing Notch1 mutations, including samples derived from poor responders or relapsed patients. Notably, the therapeutic efficacy of anti-Notch1 therapy was significantly enhanced in combination with dexamethasone. Anti-Notch1 treatment increased T-ALL cell apoptosis, decreased proliferation and caused strong inhibitory effects on Notch-target genes expression along with complex modulations of gene expression profiles involving cell metabolism. Serial transplantation experiments suggested that anti-Notch1 therapy could compromise leukemia-initiating cell functions. These results show therapeutic efficacy of Notch1 blockade for T-ALL, highlight the potential of combination with dexamethasone and identify surrogate biomarkers of the therapeutic response.

5-Aminolevulinic acid (5-ALA) fluorescence has been proved advantageous in glioma surgery. Conflicting results have been reported by few studies published in literature about intra-operative 5-ALA-induced fluorescence of medulloblastoma (MDB). The aim of this study is to verify if these conflicting results could be explained by intra-tumoral histological and phenotypic differences. In the present case of a 45-year-old patient affected by a cerebellar MDB, histological analysis of cell phenotype and 5-ALA and CD133 correlation were performed in multiple samples according to different fluorescence patterns. Intra-operatively, the tumor appeared unevenly fluorescent under blue-violet light. Histologically, 5-ALA-intense biopsies from inner areas were characterized by a significant amount of cancer cells, whereas 5-ALA faint regions from peripheral areas displayed normal cerebellar features, with MDB cells infiltrating healthy tissues. Presenting our findings, we show the correlation between different 5-ALA fluorescence patterns of medulloblastoma with specific histological and phenotypical features. Thus, we hypothesize that a distinct relationship between CD133 expression and fluorescence accumulation presented in our study could partially explain the divergent results published in literature.

SummaryThe Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.

5-aminolevulinic acid (5-ALA) introduction in the surgical management of Glioblastoma (GBM) enables the intra-operatively identification of cancer cells in the mass by means of fluorescence. Here, we analyzed the phenotype of GBM cells isolated from distinct tumour areas determined by 5-ALA (tumour core, 5-ALA intense and vague layers) and the potency of 5-ALA labelling in identifying GBM cells and cancer stem cells (CSCs) in the mass. 5-ALA identified distinct layers in the mass, with less differentiated cells residing in the core of the tumour. 5-ALA was able to stain up to 68.5% of CD133(+) cells in the 5-ALA intense layer and, although 5-ALA(+) cells retrieved from different tumour areas contained a similar proportion of CD133(+) cells (range 27.5-35.6%), those from the vague layer displayed the lowest ability to self-renew. In conclusion, our data demonstrate that a substantial amount of GBM cells and CSCs in the mass are able to avoid 5-ALA labelling and support the presence of heterogenic CSC populations in the GBM mass.

Background Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4. Methodology and principal findings 103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8(+) lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33(+)CD14(-)HLA-DR(-) were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33(+)CD14(+)HLA-DR(-) IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive. Conclusion In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme (GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of β-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of β-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133(+) fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy.

Context An altered function of lymphocytes, DC and immature myeloid cells appears to be an hallmark of tumor-mediated immune suppression and the two inhibitory co-stimulatory receptors PDL-1 and CTLA4 might have a role in this context. Objective The aim of the present in vitro study was to assess whether PaCa cells cross-talk with normal mononuclear circulating cells (PBMC) causing them to acquire an immune­suppressive phenotype and to evaluate whether PDL1 and CTLA4 are involved. Methods PBMC from blood donors were cultured for 4 days in control (CTL) and in the PaCa cancer cell line Capan1 conditioned media (CM). Lymphocytes subsets (CD4+, CD8+, CD4+CD25+) and CD33+ immature myeloid cells subsets (CD14+/-; HLA-DR+/-) expressing or not PDL1 and/or CTLA4 were analyzed by flow cytometry. To assess immunosuppressive function, myeloid cells were FACS sorted and co-cultured with allogenic total T lymphocytes in 1:20 and 1:40 ratios. Total T lymphocytes proliferation was determined by 3H-thymidine uptake. Results Capan1 CM caused an expansion of CD4+CD25+ (P=0.01) and a reduction of CD33+CD14-HLA-DR+ (P=0.03) cells. In this latter cellular subset, CM caused also an increase of PDL1 (P=0.046) and a decrease of CTLA4 (P=0.05) positive cells. FACS sorted CTL and CM CD33+CD14-HLA-DR+ cells did not significantly affect the proliferation of allogenic total T lymphocytes both at 1:20 (P=0.54) or at 1:40 ratios (P=0.81). The CD33+CD14-HLA-DR+ PDL-1+ cells did not significantly modify allogenic T cells proliferation with respect to PDL- cells (P=0.11), while those cells which were CTLA4 negative caused a significant inhibition of T cell proliferation in comparison of CTLA4 positive cells (P=0.008). Conclusions PaCa-derived soluble factors induce the expansion of the inhibitory lymphocyte subset CD4+CD25+ and a reduction of the immature CD33+CD14-DR+ dendritic cells. The tumor associated reduced expression of the inhibitory molecule CTLA4 in this cell population was demonstrated to characterize an immunosuppressive phenotype and this study suggests to take care in the use of anti-CTLA4 therapies., JOP. Journal of the Pancreas, Vol 13, N° 5S (2012): September (Suppl.) - p. 548-650

Abstract 2818 Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder of early childhood characterized by excessive proliferation of granulocytic and myelomonocytic cells. So far, about 85–90% of patients harbor mutations in genes (RAS, PTPN11, CBL, NF1) involved in the RAS signaling pathway and 25% of cases show monosomy of chromosome 7. In 10–15% of patients with JMML no recurrent mutations have been identified. In JMML, evidence of mutated cells in the myeloid lineage is well known; however, to which extend cell populations of other hematopoietic lineages harbor the same mutation is not well understood. We calculated the mutated allele frequencies of PTPN11, KRAS and CBL mutations of 10 patients with JMML using amplicon ultra deep sequencing 454 Roche Technology. KRAS and PTPN11 mutated allele frequencies in the total bone marrow (BM) varied in a range from 37.40% to 51.58%, and no statistical difference was identified in the variants allele read counts of the latter two genes. The only patient analyzed with a CBL mutation showed a mutated allele frequencies of nearly 100% pointing to homozygosity of the mutation and suggesting loss of the non-mutated CBL allele through somatic uniparental disomy of chromosomal region 11q23. Assuming occurrence of PTPN11 and KRAS mutations in heterozygosity (presence once per diploid genome), a mutated allele frequency of approximately 50% would imply that all cells were mutated (100% of cells). Along this line, our analysis of mutation frequencies, largely below 50%, pointed to a mosaicism of mutated and non-mutated cells in the BM of JMML patients. To further analyze the spectrum of mutations in BM of JMML, we sorted different hematopoietic subpopulations from 6 patients carrying a PTPN11 mutation. We choose different combinations of surface markers: CD34 and CD38 for stem and progenitor cells, CD33, CD14, CD15 and CD16, CD11b for myeloid lineages at different stages of maturation, CD3 and CD19 for T and B cells, respectively. Analysis of sorted cells revealed high mutated allele frequencies for all maturation stages of the myeloid lineage with more than 90% heterozygous mutated cells. Presence of high mutated allele frequencies were also identified in hematopoietic stem and progenitors cells and in B-cell lineage. Interestingly, in the T-cell lineage, a low mutated allele frequency was detected ranging from 9.4% to 29,35%, this finding suggesting that about 20% to 60% of T cells in the BM harbor PTPN11 mutations. Furthermore, with the aim of detecting still unknown mutations, associated with JMML we performed whole exome sequencing of BM cells of 4 JMML patients lacking any of the currently known mutations. In conclusion all hematopoietic lineage cells carry PTPN11 mutations in different percentages, indicating a mosaic distribution of mutated and non-mutated cells in the whole BM of JMML patients. We studied the power function of a binomial test to understand the probability of detecting a real deviation of variant allele read count from the hypothetical 50%, as expected for 100% of mutated heterozygous alleles. Moreover we provided a sensitivity analysis of the possible p-values when the 50% hypothetical model is true. Also using this model, a scenario of mosaicism appeared in the various subpopulations of JMML BM. The presence of non-mutated apparently healthy cells in the BM of JMML patients may hold a promise for exploring therapies that could interfere only with the mutated population sparing healthy cells in the myeloid progenitor cells populations. Disclosures: No relevant conflicts of interest to declare.

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.

Background aims Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34 + -derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. Methods The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34 + cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34 + cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. Results Gene expression analysis of in vitro -expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14 + subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo , we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. Conclusions Our data suggest that combining basal and expanded BM-derived CD34 + cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.

Ontogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4+ and single-positive CD8+ maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4+ and CD8+ T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.

Glioblastoma multiforme (GBM) are highly proliferative brain tumors characterized by a hypoxic microenvironment which controls GBM stem cell maintenance. Tumor hypoxia promotes also elevated glycolytic rate; thus, limiting glucose metabolism is a potential approach to inhibit tumor growth. Here we investigate the effects mediated by 2-deoxyglucose (2-DG), a glucose analogue, on primary GBM-derived cells maintained under hypoxia. Our results indicate that hypoxia protects GBM cells from the apoptotic effect elicited by 2-DG, which raises succinate dehydrogenase activity thus promoting succinate level decrease. As a consequence hypoxia inducible factor-1α (HIF-1α) degradation occurs and this induces GBM cells to acquire a neuronal committed phenotype. By adding succinate these effects are reverted, as succinate stabilizes HIF-1α and increases GBM stem cell fraction particularly under hypoxia, thus preserving the tumor stem cell niche. 2-DG inhibits anaerobic glycolysis altering GBM cell phenotype by forcing tumor cells into mitochondrial metabolism and by inducing differentiation.

Interleukin (IL)–23 is a proinflammatory cytokine belonging to the IL-12 superfamily. The antitumor activity of IL-23 is controversial, and it is unknown whether or not the cytokine can act directly on tumor cells. The aim of this study was to investigate the potential direct antitumor activity of IL-23 in pediatric B-acute lymphoblastic leukemia (B-ALL) cells and to unravel the molecular mechanisms involved. Here, we show, for the first time, that IL-23R is up-regulated in primary B-ALL cells, compared with normal early B lymphocytes, and that IL-23 dampens directly tumor growth in vitro and in vivo through the inhibition of tumor cell proliferation and induction of apoptosis. The latter finding is related to IL-23–induced up-regulation of miR15a expression and the consequent down-regulation of BCL-2 protein expression in pediatric B-ALL cells. This study demonstrates that IL-23 possesses antileukemic activity and unravels the underlying mechanisms. Thus, IL-23 may be a candidate novel drug for the treatment of B-ALL patients unresponsive to current therapeutic standards.

Glioblastoma multiforme (GBM) are highly proliferative tumors currently treated by surgical removal, followed by radiotherapy and chemotherapy, which are counteracted by intratumoral hypoxia. Here we exploited image guided surgery to sample multiple intratumoral areas to define potential cellular heterogeneity in correlation to the oxygen tension gradient within the GBM mass. Our results indicate that more immature cells are localized in the inner core and in the intermediate layer of the tumor mass, whereas more committed cells, expressing glial fibrillary acidic protein and β-III-tubulin, are distributed along the peripheral and neo-vascularized area, where Smad1/5/8 and Stat3 result to be activated. Moreover, GBM stem cells, identified with the stem cell marker CD133, express high level of DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) known to be involved in chemotherapy resistance and highly expressed in the inner core of the tumor mass. Importantly, these cells and, particularly, CD133+ cells result to be resistant to temozolomide (TMZ), the most used oral alkylating agent for the treatment of GBM, which specifically causes apoptosis only in GBM cells derived from the peripheral layer of the tumor mass. These results indicate a correlation between the intratumoral hypoxic gradient, the tumor cell phenotype, and the tumor resistance to chemotherapy leading to a novel concentric model of tumor stem cell niche, which may be useful to define the real localization of the chemoresistant GBM tumor cells in order to design more effective treatment strategies.

The side population (SP), recently identified in several normal tissues and in a variety of tumors based on its ability to extrude some fluorescent dyes, may comprise cells endowed with stem cell features. In this study, we investigated the presence of SP in epithelial ovarian cancer and found it in 9 of 27 primary tumor samples analyzed, as well as in 4 of 6 cultures from xenotransplants. SP cells from one xenograft bearing a large SP fraction were characterized in detail. SP cells had higher proliferation rates, were much less apoptotic compared with non-SP cells, and generated tumors more rapidly than non-SP cells. We also investigated the effects of IFN-α, a cytokine that has widely been used to treat solid tumors, on epithelial ovarian cancer cells and observed that IFN-α exerted marked antiproliferative and proapoptotic effects on primary cultures containing high numbers of SP cells. In vitro, IFN-α treatment invariably caused a dramatic reduction in SP size in tumor cell lines of different origins; moreover, IFN-α treatment of purified SP cells was associated with a distinctive change in their transcriptional profile. Gene therapy with human IFN-α resulted in regression of established tumors bearing a large SP fraction, which was not observed when tumors bearing low SP levels were treated. These findings could have relevant clinical implications because they imply that tumors bearing large SP numbers, albeit rare, could be sensitive to IFN-α treatment. [Cancer Res 2008;68(14):5658–68]

Epstein-Barr virus (EBV)-seronegative transplant recipients are at high risk of developing EBV-associated post-transplant lymphoproliferative disorder (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. So far, efforts to activate CD8+ EBV-specific cytotoxic T lymphocytes (CTL) endowed with high specific cytotoxicity from EBV-seronegative children have failed. We compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on lymphoblastoid cell lines (LCL) stimulation potentially enhanced by either LCL presentation through dendritic cells, or selection of IFN-gamma+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, according to the standard protocol for reactivation of EBV-specific CTL. We found that only specific LCL stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children. The lines thus activated, which included specificities toward EBV latent and lytic proteins, showed high percentage CD8+ T cells, with10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7 T-cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, it is feasible to activate EBV-specific CD8+ CTL with suitable characteristics for in vivo employment from EBV-seronegative children.

Purpose Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) –related malignancy expressing EBV antigens that are possible targets of cell therapy, including latent membrane protein 2 (LMP2). We conducted a clinical trial of EBV-targeted cell therapy with autologous virus-specific cytotoxic T lymphocytes (CTLs) for NPC refractory to conventional treatments. Patients and Methods Ten patients with EBV-related stage IV NPC in progression after conventional radiotherapy and chemotherapy received intravenously autologous EBV-specific CTLs reactivated and expanded ex vivo from peripheral blood lymphocytes through stimulation with EBV-transformed autologous B-lymphoblastoid cell lines (LCL). Toxicity, specific cellular immune responses, and clinical tumor responses were evaluated. Results EBV-specific CTLs could be generated in all patients and were predominantly CD3+/CD8+ T lymphocytes displaying specific killing of autologous EBV-LCL, autologous NPC cells as well as autologous targets bearing the EBV antigen LMP2. Patients received two to 23 infusions of EBV-specific CTLs that were well tolerated with the exception of grade 1 to 2 inflammatory reactions at the tumor site in two cases. Control of disease progression was obtained in six of 10 patients (two with partial response and four with stable disease). Analysis of interferon-γ–producing cells demonstrated an increased frequency of EBV-specific immunity, with appearance of LMP2-specific responses in four patients, of whom three had clinical benefit. Conclusion Cell therapy with EBV-targeted autologous CTLs is safe, induces LMP-2-specific immunologic responses, and is associated with objective responses and control of disease progression in patients with stage IV NPC resistant to conventional treatments.

Introduction MLL- rearranged acute myeloid leukemia (AML) remains a type of leukemia difficult to treat, for which alternative and more adequate treatment options are still urgently needed. The zinc finger protein 521 (ZNF521/EHZF) is a protein with multiple zinc finger domains that plays an essential role in the homeostasis of the hematopoietic stem/progenitor cell compartment. ZNF521 mRNA is highly expressed in hematopoietic stem and progenitor cells and its levels rapidly decrease during cell differentiation. Gene expression studies demonstrated that ZNF521 transcripts are abundantly expressed in MLL-rearranged AML. However, the functional significance of this up-regulation in AML with MLLrearrangements remains largely unknown. Methods and results Quantitative RT-PCR analysis (qRT-PCR) confirmed that ZNF521 expression was significantly up regulated in a cohort of MLL-rearranged pediatric AML patients (n=80) compared with other AML subtypes (n=30) together with healthy bone marrow samples (n=7, p Conclusions Collectively, these results demonstrate that ZNF521 plays an important role in MLL-AF9-induced AML leukemia wherein it maintains a block of differentiation. Thus, our findings make ZNF521 a novel attractive target for therapeutic intervention in MLL-rearranged AML. However, further studies on the function of ZNF521 in MLL-rearranged AML and on the possibility to block it are required. Disclosures: No relevant conflicts of interest to declare.

Correction to: Cell Death and Disease (2013) 4, e500; doi:10.1038/cddis.2013.32; published online 21 February 2013 Since the publication of this article the authors have noticed that the Authors Contribution section was omitted. The error has now been rectified. The article with the Authors Contribution appears online together with this corrigendum.

Abstract 1459 Microvesicles (MVs) are nano-sized lipid bodies (100–1000 nm in diameter) that are released by cells, both in vitro and in vivo. MVs are secreted in the extracellular space and as carriers of proteins, mRNA and miRNA represent vectors from donor to target cells involved in intercellular communication. MVs modulate the functional state of receiving cells through fusion with the target cell. In leukemia MVs were suggested to modulate the hematopoietic niche. In this study, we investigated the transcriptome of MVs released from leukemic cell lines. In particular, we analyzed K562, a BCR-ABL positive human erythromyeloblastoid leukemia cell line, and we collected RNA both from cells and MVs released in culture. Since many different methods have been described for microvesicles isolation and description of MV populations are often ambiguous, an accurate protocol has been developed in order to select a defined MVs population. In detail, for MVs isolation cell culture medium was centrifuged at 2500g for 15 minutes. These centrifugations allowed to delete cells and bigger bodies. Then supernatant was filtered by means of a 1.2um filter, in order to keep only vesicles of defined physical measure and to eliminate residual bigger vesicles, such as apoptotic bodies (>1000nm). The filtration allowed an accurate selection of a well defined MVs population. The filtered medium was then centrifuged at 18000g for 1h at 4°C. MVs were resuspended in Trizol for RNA isolation. Also RNA of cells, from which MVs have been released, was extracted using the Trizol method, according to manufacturer instructions. Cell line RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by means of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). To check MVs RNA integrity we determined the ratio of 5' amplicons to 3' amplicons of housekeeping transcripts by Real Time PCR. We screened the presence of several housekeeping transcripts in MVs and selected GAPDH as reference. The GAPDH 5':3' amplicon ratio should equal 1 to ensure MVs RNA quality. Afterwards, reverse transcription–polymerase chain reaction (RT-PCR) amplification was performed. cDNA was synthesized from 1mg of total RNA. We analyzed the gene expression profile of K562 cell line and MVs, released from these cells, using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Gene expression data have been compared between cells and MVs. Ninety-one percent of probe sets showed a similar expression (fold-change lower than 1.5) between cell line and MVs. Thirteen percent of these probes were recognized as present call probe sets in both cell line and MVs. Analysis of probe sets using DAVID Functional Annotation Bioinformatics Microarray Analysis revealed a conservation of MVs gene transcripts involved in pathways of cell function such as RNA processing, protein translation, aminoacid metabolism and cell respiration. Remarkably, in MVs we observed a high presence of gene transcripts coding for protein belonging to the Chronic Myeloid Leukemia pathway that are expressed downstream of the BCR-ABL tyrosin kinase fusion protein. The maintenance of this pathway in MVs highlights the intrinsic peculiarity of BCR-ABL positive K562 cells apparently also conserved in MVs mRNA outfit representing a hallmark of the parental cell from which they have been released. Furthermore, 3.8% of the probe sets resulted to be up-regulated in MVs compare to the cell line (fold-change higher than 1.5). In MVs, we observed a higher expression of genes belonging to cell communication pathways, adhesion and migration processes, membrane and ionic channels signals. In conclusion, we isolated MVs released by K562 leukemic cells using an accurate selection of the MV population based on physical measures and for the first time a whole transcriptome gene expression analysis has been performed comparing K562 cells and MVs. Moreover, we identified an enrichment of transcripts coding for proteins involved in several essential pathways in the MVs supporting the hypothesis of a functional selection from the parental cell transcriptome and underlining the relevant role of MVs as vehicles of messages to target cells. Disclosures: No relevant conflicts of interest to declare.

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.

Abstract 933 Acute lymphoblastic leukaemia (ALL) genesis, development and maintenance, has been shown to involve a number of aspects. On the one hand along the line of the somatic mutation theory of cancer there are accumulating genetic aberration, on the other hand there are cytokines and growth factors that contribute to initiate and maintain the disease. The bone marrow microenvironment plays an important role in regulating the hematopoietic stem cell niche modulating normal haematopoiesis but also affecting leukaemogenic transformation. In the bone marrow microenvironment leukaemia cells receive signals that protect them and promote their survival or proliferation. Horizontal transfer of cellular signals mediated by circulating microvesicles (cMVs) is an emerging mechanism of cell to cell interaction, also in the haematopoietic stem cell niche. These small plasma bodies can be isolated from peripheral blood and are characterised by varying sizes (exosomes of 50 –100 nm diameter and microparticles of 100–1000 nm), and MVs have been shown to transfer cellular messages from donor to target cells where they can modulate protein synthesis. In this study we investigated MVs that were recovered from peripheral blood (PB) and the type of signal that could be transferred by MVs in paediatric ALL. Initially we characterised MVs isolated from the supernatant of three cell lines (ALL MHH CALL4, LAM ME-1, osteosarcoma MG-63). We then isolated MVs from plasma of 28 ALL patients at diagnosis before the onset of treatment and of 5 control samples. Plasma free of platelets obtained from peripheral blood (in EDTA) was subjected to a centrifugation step (16000 g, at 4°C for 1 hour). After recovery of the pellet MVs were stained by cytoplasmic dye carboxyfluorescein diacetate, succinimidyl ester (CFSE) and by monoclonal antibodies specific for CD markers of patients leukaemia blasts and cell lines. MVs were analysed by flow cytometry to investigate at cMVs surface antigen marker expression and physical parameters. We focused our analysis on a particle population of 150–400 nm in diameter. MVs from cell lines showed surface antigen specific for the respective cell lineage and were shown to contain mRNA transcripts. Unexpectedly in most cases MVs isolated from patients did not show leukaemia cell surface antigens but were found to contain mRNA. MVs' RNA was extracted from 27 patients and from 4 controls by Trizol, according to manufacturer instructions. Standard RT-PCR was performed to detect osteoclast and osteoblast markers as RANK, RANKL and TNFα. The presence at different extent of RANK/RANKL/TNFα within cMVs from patients and in none of control samples suggests that this pathway, mainly involved in bone remodelling, is contributing to signalling between the hematopoietic niche and the bone marrow microenvironment in leukaemia samples. Other reports have demonstrated that osteoclasts can induce the mobilization of haematopoietic progenitor cells under stress conditions. The role of the RANK/RANKL/TNFα messengers carried by PB derived MVs in ALL patients and how these might affect leukaemia progression and maintenance needs to be further explored. Moreover we found that some of the MVs isolated from patients known to carry specific translocations at blast cell levels e.g t(12:21) and t(1:19), presented the same genetic aberration at MVs mRNA transcripts. MVs from one of the above patients (t(12:21)positive at blasts) were also positive for blast cells surface antigen (CD19), as seen by flow cytometry. For the other patient (t(1:19)) we did not perform parallel flow cytometry analysis due to lack of material. These preliminary observations suggest that peripheral blood plasma contains MV populations with different features and functions contributing to transfer of messages to distal cells. In conclusion this is the first study on cMV populations in paediatric ALL patients and our date seem to underscore a role for the bone microenvironment including cMVs in this disease. Disclosures: No relevant conflicts of interest to declare.

EBV-seronegative solid organ transplant recipients, or recipients of T-cell depleted hematopoietic stem cell from a EBV-seronegative donor, are cohorts at high risk for the development of EBV-associated posttransplant lymphoproliferative disease (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. Reported experiences have shown that CTL obtained from EBV-seronegative individuals are almost exclusively HLA-class II-restricted CD4+ cytotoxic T cells, and efficiency of this CTL subset in the control of PTLD has not yet been demonstrated in vivo. Conversely, the central role of CD8+ CTL in the cell-mediated containment of EBV infection is well established. Therefore, we endeavoured to develop a protocol of EBV-CTL priming that would allow optimal expansion and high specific cytotoxic activity of CD8+ CTL. We employed naïve lymphocytes obtained from 5 EBV seronegative patients with end-stage renal failure receiving dialysis while listed for a kidney transplantation (4 children, 1 adult). In particular, we compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on LCL stimulation potentially enhanced by either LCL presentation through DC, or selection of IFNg+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, to the standard protocol for reactivation of EBV-specific CTL from EBV-seropositive individuals. We found that while all protocols employed, with the exception of IFN-g+ T cell selection, allowed for the amplification of CD8+ and CD4+ mediated EBV-specific cytotoxic T cell responses in the EBV-seronegative adult, only specific EBV-transformed lymphoblastoid cell lines (LCL) stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly generate and expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children (mean lysis at a E:T ratio of 10:1: 14±10% with the standard protocol vs. 66±30% with the addition of rhIL-7 and rhIL-12). The lines thus activated showed a higher percentage CD8+ T cells, with less than 10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7- T cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, stimulation with LCL in the presence of IL-12 and IL-7 activates EBV-specific, highly cytotoxic CD8+ CTL from EBV-seronegative subjects, this opening new perspectives for prevention/treatment of PTLD.

2512 Background: nasopharyngeal carcinoma (NPC) is an EBV-related neoplasm expressing a limited number of viral antigens, including latent membrane protein 2 (LMP-2). Currently, the outcome of pati...

SummaryCancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble—or induction of the epithelial-mesenchymal transition (EMT)—disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.