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In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug’s safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.

Summary Patients with acute myeloid leukemia (AML) carrying high-risk genetic lesions or high residual disease levels after therapy are particularly exposed to the risk of relapse. Here, we identified the long non-coding RNA CDK6-AS1 able to cluster an AML subgroup with peculiar gene signatures linked to hematopoietic cell differentiation and mitochondrial dynamics. CDK6-AS1 silencing triggered hematopoietic commitment in healthy CD34+ cells, whereas in AML cells the pathological undifferentiated state was rescued. This latter phenomenon derived from RUNX1 transcriptional control, responsible for the stemness of hematopoietic precursors and for the block of differentiation in AML. By CDK6-AS1 silencing in vitro, AML mitochondrial mass decreased with augmented pharmacological sensitivity to mitochondria-targeting drugs. In vivo, the combination of tigecycline and cytarabine reduced leukemia progression in the AML-PDX model with high CDK6-AS1 levels, supporting the concept of a mitochondrial vulnerability. Together, these findings uncover CDK6-AS1 as crucial in myeloid differentiation and mitochondrial mass regulation., Graphical abstract, Highlights • CDK6-AS1 acts in concert with CDK6 • High CDK6-AS1 levels trigger RUNX1 early differentiation arrest in myeloid cells • CDK6-AS1 controls mitochondrial mass of AML blasts • CDK6-AS1 levels impact on mitochondrial-targeted agents sensitivity, Molecular biology; Cell biology; Cancer

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but still >30% of patients relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the mesenchymal stromal cells (MSCs) role in the leukemic niche to define its contribution to the mechanisms of leukemia escape. We generated humanized three-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation, and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when co-cultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features, at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSCs selective CaV1.2 channel blocker drug, Lercanidipine, is able to impair leukemia progression in 3D niche both in vitro and when implanted in vivo, if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

The presence of CBFA2T3-GLIS2 fusion gene has been identified in childhood Acute Myeloid Leukemia (AML). In view of the genomic studies indicating a distinct gene expression profile, we evaluated the role of immunophenotyping in characterizing a rare subtype of AML-CBFA2T3-GLIS2 rearranged. Immunophenotypic data were obtained by studying a cohort of 20 pediatric CBFA2T3-GLIS2-AML and 77 AML patients not carrying the fusion transcript. Enrolled cases were included in the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP) AML trials and immunophenotypes were compared using different statistical approaches. By multiple computational procedures, we identified two main core antigens responsible for the identification of the CBFA2T3-GLIS2-AML. CD56 showed the highest performance in single marker evaluation (AUC = 0.89) and granted the most accurate prediction when used in combination with HLA-DR (AUC = 0.97) displaying a 93% sensitivity and 99% specificity. We also observed a weak-to-negative CD45 expression, being exceptional in AML. We here provide evidence that the combination of HLA-DR negativity and intense bright CD56 expression detects a rare and aggressive pediatric AML genetic lesion improving the diagnosis performance.

Simple Summary Nucleophosmin (NPM1) protein regulates several cellular processes and is predominantly located in the nucleolus, owing to the localization signal provided by two tryptophan residues. In acute myeloid leukemia (AML), NPM1 gene is frequently mutated, leading to the aberrant translocation of the protein into cytoplasm. In the present work, we classified NPM1 mutations according to the loss of either one or both tryptophan residues as non-A-like and A-like mutations, respectively, and evaluated their biological features. We found that non-A-like mutations partially delocalize NPM1 protein into the cytoplasm, with a proportion of remaining nucleolar protein preserving p53 protein expression and downstream activity. Different HOXA and HOXB gene expression and cell death pathway activation between A-like and non-A-like NPM1-mutated cells were shown, with an enhanced sensitivity to chemotherapy for AML cells with non-A-like mutations. This study suggests the need for a sub-classification of NPM1-mutated AML, with subsequent implications in the therapeutic management. Abstract Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, predominantly located in the nucleolus, that regulates a multiplicity of different biological processes. NPM1 localization in the cell is finely tuned by specific signal motifs, with two tryptophan residues (Trp) being essential for the nucleolar localization. In acute myeloid leukemia (AML), several NPM1 mutations have been reported, all resulting in cytoplasmic delocalization, but the putative biological and clinical significance of different variants are still debated. We explored HOXA and HOXB gene expression profile in AML patients and found a differential expression between NPM1 mutations inducing the loss of two (A-like) Trp residues and those determining the loss of one Trp residue (non-A-like). We thus expressed NPM1 A-like- or non-A-like-mutated vectors in AML cell lines finding that NPM1 partially remained in the nucleolus in the non-A-like NPM1-mutated cells. As a result, only in A-like-mutated cells we detected HOXA5, HOXA10, and HOXB5 hyper-expression and p14ARF/p21/p53 pathway deregulation, leading to reduced sensitivity to the treatment with either chemotherapy or Venetoclax, as compared to non-A-like cells. Overall, we identified that the NPM1 mutational status mediates crucial biological characteristics of AML cells, providing the basis for further sub-classification and, potentially, management of this subgroup of patients.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Acute myeloid leukemia (AML) is a heterogeneous disease where selected subgroups of patients, linked by the presence of biological and clinical high-risk features, are candidates to receive allogenic hematopoietic stem cell transplantation HSCT) as post-remission consolidation treatment. The achievement of morphological complete remission (CR) before HSCT is an important pre-requisite to optimize the chance of successful post-transplant outcome. Minimal residual disease (MRD) assessment by quantitative polymerase chain reaction (q-PCR) has been shown to increase the ability to monitor therapy response in AML, improving prognostic accuracy and allowing to refine transplant strategies. Although MRD assessment was shown to have potential benefit when measured after induction and consolidation therapy courses, its role before HSCT remains to be fully elucidated. In order to contribute to better clarify this issue, we conducted a q-PCR I-BFM-AML collaborative study to measure MRD in bone marrow samples collected within 5 weeks prior to HSCT of 108 pediatric AML patients harboring one of the main recurrent AML aberrancies t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1q22)/t(16;16)(p13.1;q22); CBFB-MYH11, t(9;11)(p22;q23); KMT2A-MLLT3 or FLT3-ITD. Sixty patients underwent HSCT in first complete remission (CR1) with an overall survival (OS) of 84% versus 54% for the 48 transplanted in CR2 achieved after an initial relapse. Sixty patients showed q-MRD negativity (defined as a value lower than 2.1x10-4 calculated by ROC curve analysis with respect to diagnosis or relapse), whereas in 48 patients we detected q-MRD levels >2.1x10-4. Five-year OS after HSCT was 83% for patients with q-MRD negativity, while that of patients with q-MRD above the cutoff was 57% (p=0.012). As regards, cumulative incidence of relapse (CIR), q-MRD above the cutoff was associated with a high risk of recurrence (26% versus 10% for patients with q-MRD 2-log versus 73% for q-MRD2.1x10-4 and 2-log, HR). This combined stratification by q-MRD resulted into a better subdivision of the OS probability, which was 83%, 69% and 39% for LR, IR and HR respectively (p=0.004). Finally, a multivariate Cox regression model revealed that, together with CR status at time of the allograft (CR2, hazard ratio 4.4, p=0.001), q-MRD was an independent factor (hazard ratio 0.5, p=0.001) predicting HSCT outcome. In conclusion, this study supports the role of q-MRD pre-HSCT as a useful prognostic tool in childhood AML, able to provide information to tailor transplant strategies involving conditioning regimen intensity and graft-versus-host disease prophylaxis. Disclosures Reinhardt: AbbVie: Consultancy; Novartis: Consultancy, Other: Institutional Research Funding; Jazz: Consultancy, Other: Institutional Research Funding; Celgene: Consultancy, Other: Institutional Research Funding; bluebird bio: Consultancy; Roche: Consultancy, Other: Institutional Research Funding; Biotest: Other: Institutional Research Funding; Novo Nordisk: Other: Institutional Research Funding; Behring: Other: Institutional Research Funding. Merli:Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria; Sanofi-Genzyme: Honoraria; Atara Therapeutics: Honoraria.

// Giuseppe Germano 1 , Giulia Morello 1 , Sanja Aveic 1 , Marica Pinazza 2 , Sonia Minuzzo 2 , Chiara Frasson 3 , Luca Persano 1 , Paolo Bonvini 1 , Giampietro Viola 3 , Silvia Bresolin 3 , Claudia Tregnago 3 , Maddalena Paganin 3 , Martina Pigazzi 3 , Stefano Indraccolo 4 , Giuseppe Basso 3 1 Foundation Institute of Pediatric Research Citta della Speranza, Padova, Italy 2 Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy 3 Department of Woman and Child Health, University of Padova, Italy 4 Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IRCCS, Padova, Italy Correspondence to: Giuseppe Germano, email: giuseppe.germano@unipd.it Keywords: ZNF521, acute myeloid leukemia, myeloid differentiation, transcription Received: August 12, 2016 Accepted: January 31, 2017 Published: February 16, 2017 ABSTRACT Zinc finger protein 521 (ZNF521) is a multiple zinc finger transcription factor and a strong candidate as regulator of hematopoietic stem cell homeostasis. Recently, independent gene expression profile studies have evidenced a positive correlation between ZNF521 mRNA overexpression and MLL -rearranged acute myeloid leukemia (AML), leaving open the question on the role of ZNF521 in this subtype of leukemia. In this study, we sought to analyze the effect of ZNF521 depletion on MLL -rearranged AML cell lines and MLL-AF9 xenograft primary cells. Knockdown of ZNF521 with short-hairpin RNA (shRNA) led to decreased leukemia proliferation, reduced colony formation and caused cell cycle arrest in MLL -rearranged AML cell lines. Importantly, we showed that loss of ZNF521 substantially caused differentiation of both MLL-rearranged cell lines and primary cells. Moreover, gene profile analysis in ZNF521 -silenced THP-1 cells revealed a loss of MLL-AF9 -directed leukemic signature and an increase of the differentiation program. Finally, we determined that both MLL-AF9 and MLL-ENL fusion proteins directly interacted with ZNF521 promoter activating its transcription. In conclusion, our findings identify ZNF521 as a critical effector of MLL fusion proteins in blocking myeloid differentiation and highlight ZNF521 as a potential therapeutic target for this subtype of leukemia.

The application of nanotechnologies to address biomedical questions is a key strategy for innovation in biomedical research. Among others, a key point consists in the availability of nanotechnologies for monitoring cellular processes in a real-time and label-free approach. Here, we focused on a grating-coupled Surface Plasmon Resonance (GC-SPR) sensor exploiting phase interrogation. This sensor can be integrated in a microfluidic chamber that ensures cell viability and avoids cell stress. We report the calibration of the sensor response as a function of cell number and its application to monitor cell adhesion kinetics as well as cell response to an external stimulus. Our results show that GC-SPR sensors can offer a valuable alternative to prism-coupled or imaging SPR devices, amenable for microfluidic implementation.

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. We report that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. Our data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects.

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. We report that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. Our data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement.

Chemotherapy still remains the pillar of treatment of children with AML, a disease in which refinements in diagnostic approaches, minimal residual disease monitoring, and patient stratification have resulted into remarkable progresses during the past decade. However, most of the recently tested, novel anti-leukemia agents failed during pre-clinical and clinical validation phases, and one main limit in AML field is the inappropriateness of current preclinical models used to study drug efficacy, this jeopardizing the advance of phase II and III clinical trials, especially for children. In light of this consideration, we aimed at creating novel robust in vitro and in vivo approaches to discover or to re-assess alternative treatments to improve the portfolio of agents active in childhood AML. For this purpose, we developed new protocols for long-term 3D-AML cultures to perform more predictable high throughput drug screening in vitro, and, once identified the best compounds, to create new pre-clinical in vivo models. We set up the bone marrow (BM) endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, where we seeded mesenchymal stromal cells derived either from AML patients (AML-MSCs) or from healthy BM donors (h-MSCs), together with osteoblasts, endothelial cells and finally AML blasts. We studied AML cell proliferation and clonogenicity cultured in 3D. We obtained results from twenty 3D long-term cultures of different primary AML, confirming blast proliferation up to 21 days. Clonogenic potential and immunophenotype preservation of the original AML blasts was also documented. At the same time, we compared AML-MSCs with h-MSCs, finding that AML-MSCs exhibited a higher proliferation rate (40% increase proliferation at 72 and 96 hours, p Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy.

In acute myeloid leukemia (AML), the assessment of post-induction minimal residual disease (MRD) is largely utilized for choosing post-remission therapies aimed at maintaining complete remission (CR) and preventing relapse. This latter is still the major cause of treatment failure in pediatric AML, and even if several efforts have been spent to validate MRD as a prognostic marker, numerous studies demonstrated that MRD negativity cannot be considered a completely reliable surrogate biomarker predicting outcome, since it does not exclude a relapse. The current interpretation is that disease relapse is due to mechanisms leading to therapy resistance mainly depending on driver chimeric or oncogenic protein-coding genes, which are monitored during treatment, and does not consider that chemotherapy resistance may arise from other genetic markers, phenomenon linked to methylation and non-coding RNAs genomic pressure. We, thus, hypothesized that other markers need to be explored to re-interpret leukemia progression. We showed an overall hyper-expression of the lncRNA BALR2 in 132 de novo AML bone marrow samples collected at diagnosis and analyzed the gene expression profile (GEP) of 58 cases. By unsupervised clustering analysis, we produced important advances in identifying BALR2 as a robust novel molecular marker of a new subgroup of AML characterized by a high rate of resistance to induction therapy, independently from the genetic lesions detected at diagnosis and any other prognostic clinical and genetic features. We demonstrated in vitro that BALR2 has a direct role in controlling bi-directionally its own and of its neighbor gene CDK6 promoter activity. This latter finding of high CDK6 expression was shown to sustain its complex with RUNX1 in order to inhibit RUNX1 binding to its target promoters, thus preventing the process of hematopoietic differentiation progression. To support BALR2 as a new proto-oncogene involved in the control of the myeloid differentiation program, we ranked the genes across the expression profile obtaining a signature of 337 transcripts able to cluster CD34+ human stem cell precursors (HSCPs) separately from more mature CD14+ cells. These in silico findings were validated in vitro by showing that, after BALR2 depletion, CD34+ cells had a skewed myeloid differentiation. Furthermore, we found that AML differentiation toward mature myeloid cells with increased phagocytic capacity was obtained through BALR2 level reduction, and enhanced by combinatorial differentiation stimuli. Our findings attribute a distinct role to BALR2 in the block of myeloid stem cell differentiation occurring during leukemogenesis. At the same time, we interrogated GEP ontology, finding that enrichments of genes involved in mitochondrial synthesis pathways were significantly correlated to patients with highest BALR2 levels, and confirmed the same mitochondriogenesis profile in the immature CD34+ HSCPs. We moved to deconvolute this feature and demonstrated that BALR2, by controlling mitochondria gene balance, was directly controlling the mitochondrial mass, which dramatically decreased after BALR2 silencing, this supporting the hypothesis that BALR2 would maintain mitochondrial functions to confer AML resistance to cytotoxicity. Consistently with this line of reasoning, we inhibited mitochondria by tigecycline, demonstrating that its activity was dramatically strengthened in BALR2 depleted cells, when used either alone or in combination with cytosine-arabinoside (Ara-C). Concomitantly, tigecycline treatment in BALR2 silenced AML cells reduced mitochondria depolarization, and increased the number of differentiated M-CFU colonies formation, confirming that BALR2, together with CDK6, forms novel transcriptional networks to create a circuit able to impair myeloid differentiation and to lower chemo-sensitivity in AML. We speculate that a novel therapeutic window of mitochondrial targeting in defined AML subgroups, identified through assessment of BALR2 levels at diagnosis or persistent MRD levels, could be envisaged to optimize the outcome of childhood AML. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development., Author summary In recent years, the role of microRNAs in regulating the endogenous circadian clock and its rhythmic outputs for behaviour/physiology has been recognized. We have observed that depletion or over-expression of miR-210 in Drosophila melanogaster modulates the phase of locomotor activity, without affecting the molecular oscillation of the pacemaker neurons. Moreover, miR-210 over-expression dramatically alters the pattern of projections from the PDF-positive Lateral Neurons (LNvs). Differentially expressed genes detected in miR-210 over-expressing flies implicated circadian processes, neuronal development, and photoreception. Taken together, our findings indicate the involvement of miR-210 in modulating circadian output and remodelling the projections of PDF clock neurons, and suggest that miR-210 may have pleiotropic effects on clock, light perception and neuronal development.

Summary Mitochondria are key players in the regulation of T cell biology by dynamically responding to cell needs, but how these dynamics integrate in T cells is still poorly understood. We show here that the mitochondrial pro-fission protein Drp1 fosters migration and expansion of developing thymocytes both in vitro and in vivo. In addition, we find that Drp1 sustains in vitro clonal expansion and cMyc-dependent metabolic reprogramming upon activation, also regulating effector T cell numbers in vivo. Migration and extravasation defects are also exhibited in Drp1-deficient mature T cells, unveiling its crucial role in controlling both T cell recirculation in secondary lymphoid organs and accumulation at tumor sites. Moreover, the observed Drp1-dependent imbalance toward a memory-like phenotype favors T cell exhaustion in the tumor microenvironment. All of these findings support a crucial role for Drp1 in several processes during T cell development and in anti-tumor immune-surveillance., Graphical Abstract, Highlights • The pro-fission protein Drp1 sustains correct thymocyte maturation • Drp1 promotes T cell metabolic reprogramming and expansion upon activation • Drp1 allows efficient T cell extravasation from blood and infiltration into tumors • An optimal T cell anti-tumor response requires Drp1, Mitochondria are emerging as key players for optimal T cell functionality. Simula et al. demonstrate that the mitochondrial pro-fission factor Drp1 controls thymocyte maturation and plays multiple roles in mature T cells by promoting their proliferation, migration, and cMyc-dependent metabolic reprogramming upon activation; this activity sustains efficient anti-tumor immune-surveillance.

The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.

NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: a report from the AIEOP-AML group

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR

Purpose: The inducible cyclic adenosine monophosphate (cAMP) early repressor (ICER) is found downregulated in acute myeloid leukemia (AML), failing to control cAMP response element binding protein (CREB) transcriptional activity, recently demonstrated to mediate AML progression. We aimed to characterize ICER's role in drug sensitivity by treating myeloid cell lines and primary AML with chemotherapics. Experimental design: The effects on CREB target genes induced by ICER restoration and drug treatment were studied by quantitative real-time PCR (qRT-PCR) and western blot. Cell cycle and apoptosis analysis were performed. Possible ICER-evoked pathways were investigated in vitro. The mechanism involved in enhanced drug sensitivity was described in primary AML cultures by silencing ICER main target genes. Results: AML cell lines reduced cell growth and enhanced apoptotic behavior after chemotherapy treatment if ICER was expressed. A significantly lowered expression of CREB target genes involved in cell cycle control (CyA1, B1, D1), and in the mitogen-activated protein kinase signaling pathway (ERK, AKT, DUSP1/4), was found after Etoposide treatment. The dual-specificity phosphatases DUSP1 and DUSP4, directly repressed by ICER, activated the p38 pathway, which triggered enhanced caspase-dependent apoptosis. The silencing of DUSP1/4 in HL60 confirmed the same enhanced drug sensitivity induced by ICER. Primary AML cultures, silenced for DUSP1 as well as restored of ICER expression, showed DUSP1 downregulation and p38 activation. Conclusion: ICER mediates chemotherapy anticancer activity through DUSP1-p38 pathway activation and drives the cell program from survival to apoptosis. ICER restoration or DUSP1 inhibition might be possible strategies to sensitize AML cancer cells to conventional chemotherapy and to inhibit tumor growth. Clin Cancer Res; 17(4); 742–52. ©2011 AACR.

A rare location, t(6;11)(q27;q23) (MLL-AF6), is associated with poor outcome in childhood acute myeloid leukemia (AML). The described mechanism by which MLL-AF6, through constitutive self-association and in cooperation with DOT-1L, activates aberrant gene expression does not explain the biological differences existing between t(6;11)-rearranged and other MLL-positive patients nor their different clinical outcome. Here, we show that AF6 is expressed in the cytoplasm of healthy bone marrow cells and controls rat sarcoma viral oncogene (RAS)-guanosine triphosphate (GTP) levels. By contrast, in MLL-AF6-rearranged cells, AF6 is found localized in the nucleus, leading to aberrant activation of RAS and of its downstream targets. Silencing MLL-AF6, we restored AF6 localization in the cytoplasm, thus mediating significant reduction of RAS-GTP levels and of cell clonogenic potential. The rescue of RAS-GTP levels after MLL-AF6 and AF6 co-silencing confirmed that MLL-AF6 oncoprotein potentiates the activity of the RAS pathway through retention of AF6 within the nucleus. Exposure of MLL-AF6-rearranged AML blasts to tipifarnib, a RAS inhibitor, leads to cell autophagy and apoptosis, thus supporting RAS targeting as a novel potential therapeutic strategy in patients carrying t(6;11). Altogether, these data point to a novel role of the MLL-AF6 chimera and show that its gene partner, AF6, is crucial in AML development.

Purpose. Among pediatric acute myeloid leukemia (AML), the t(6;11)(q27;q23) MLL-AF6 translocation accounts for 26% of MLL-rearranged AML, and is associated with a worse prognosis (event-free survival of 23.3% at 3-years) compared to other forms of MLL-rearranged AML1. Gene expression profile analysis revealed a specific transcriptional signature, and this peculiarity has been explained by the mislocalization of AF6 protein into the nucleus with a consequent hyperactivation of the RAS pathway in these patients. The uncovered involvement of the RAS pathway in this AML subgroup provides the rationale for searching new therapeutical strategies to selectively target MLL-AF6-rearranged cells. Methods. We established a cell-based drug screening assay, by testing a library of 1,280 pharmacologically active compounds (Lopac library, Sigma-Aldrich) on t(6;11)-rearranged ML2 and SHI-1 cell lines. Compounds (used at 10μM) which decreased cell viability by at least 50% (by ATP measurement) were further tested in different AML cell lines (HL60, as well as NOMO1 and THP1, both t(9;11)MLL-AF9 rearranged), to exclude those with broad anti-leukemic activity and to focus specifically over MLL-AF6 action. Finally, functional studies were performed for the compounds resulted selective for the MLL-AF6 rearrangement in cell lines and patient's primary blast cultures and the most promising drug was tested in vivo using NSG mice. Results. Of 1,280 compounds, 104 and 93 impaired cell proliferation of ML2 and SHI-1, respectively. 73 were found efficacious over HL60 and, thus, excluded. Then, the remaining 20 compounds were evaluated in other MLL-cell lines (NOMO-1 and THP-1), and finally 10/20 resulted active selectively on t(6;11)-rearranged cell lines. The selected compounds were Arvanil, CP-100356 monohydrochloride, Fluspirilene, CID2858522, Eupatorin, ANA-12, BAY 61-3606 hydrochloride hydrate, Ara-G hydrate, Tyrphostin 47, Thioridazine hydrochloride and were also confirmed to impair viability over t(6;11) primary blast cultures from patients. Among them, we were particularly interested in Fluspirilene and Thioridazine, these compounds being both antipsychotics working as dopamine receptor (DR) antagonists and FDA approved. By flow cytometry we showed the DRs (DR-1 to DR-5) expression in ML2, SHI-1, NOMO-1, THP1 and SKNO-1 whereas HL60 resulted devoid of DRs. Blasts from t(6;11)-rearranged patients (n=3) expressed DRs as well. Treatment of the cell lines with Fluspirilene and Thioridazine triggered apoptosis induction in SHI-1, and to a lesser extent in ML2, due to autophagy activation, whereas no effects were observed on HL60, NOMO-1, THP1 and SKNO-1. Clonogenic assay showed that after 24 hours of treatment self-renewal ability of SHI-1 and ML-2 significantly decreased, with no effects observed in other cell lines. Same results were obtained in primary cultures from patients t(6;11), without toxic effects on healthy bone marrow cells, confirming the drug specific activity over leukemia proliferation, and with Thioridazine being more active. NSG mice were then flank injected with t(6;11) cells and treated with Thioridazine 12 mg/kg; treatment significantly inhibited tumor growth in vivo (compared to mice treated with DMSO, p Conclusions. This study led to the identification of DRs expression in myeloid blasts, and revealed their role in leukemia maintenance exclusively of the t(6;11)-rearranged AML. We identified a series of new compounds to be prioritized for further analysis in MLL-AML; in particular Thioridazine deserves further investigation as a novel therapeutic strategy to improve outcome of t(6;11)-rearranged patient's. 1 Pigazzi M, et al Leukemia. 2011 Mar;25(3):560-3. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients1. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the context of the clonal architecture dynamic evolution. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. The importance of the identification of molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse could be instrumental to improve their cure rate. We performed high throughput DNA methylation profiling (RRBS-seq) and integrated results with gene expression profiling (Affymetrix HTA 2.0) of 16 isolated t(8;21) AML samples collected at diagnosis, and analyzed data by comparing patients who did or did not experience relapse. We applied a logistic regression algorithm to identify differentially methylated regions (DMRs) considering a minimum change in methylation level of 25%. We validated results in a proteome context by reverse phase protein array (RPPA) in an independent cohort of 35 t(8;21) AML patients. DNA methylation profiling analysis identified 337 DMRs able to correctly predict t(8;21) patients who did relapse from those who did not. In particular, 23 DMRs (7%) were located at promoters, while most of them were equally distributed between intronic (48%) and exonic (45%) regions. Globally, we found hypomethylated DMRs being significantly enriched in relapsed patients, in particular in repetitive elements regions of the genome (LINE, SINE, DNA transposon: 38.9% vs 52.4%; p We then considered the role of methylation over gene expression and found a weak correlation between DMRs (mostly at promoters) and their associated gene levels (14.5% of DMRs with an inverse correlation r These data show that the methylation signature may be considered a novel, emerging diagnostic tool making possible to better stratifying t(8,21)-rearranged patients through the identification, already at diagnosis, of those who are prone to relapse . Preliminary data of functional analysis suggest that epigenome of t(8;21) blasts may control cell adhesion properties at bone marrow niche and treatment response, contributing to patients relapse. 1 Pession A, Blood. 2013;122(2):170-8. Disclosures No relevant conflicts of interest to declare.

MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and contributes to myeloid transformation from both healthy bone marrow and myelodysplastic syndromes. We identified a subset of CREB target genes that represents a novel transcriptional network that may control myeloid transformation.

Purpose. While most of the recurrent molecular marker of AML have been already discovered and used for risk stratification in clinical protocols, post-treatment monitoring of these abnormalities can be useful in the clinical management of patients. In particular, monitoring of minimal residual disease (MRD), largely used in acute lymphoblastic leukemia, is gaining popularity also in AML, in the perspective of optimizing risk stratification of patients in terms of subsequent clinical relapse. While MRD monitoring in AML is mainly based on flow-cytometry approaches, molecular MRD measurements are not currently routinely used for taking clinical decision. Here we examine if the molecular MRD and Allelic Ratio (AR) levels monitoring may influence the survival of FLT3 internal tandem duplication (ITD)-mutated AML patients. Patients and methods. We retrospectively analyzed 507 children with de novo AML for FLT3ITD mutation by RT-PCR. Mutation was sequenced and the AR was calculated by Genescan. Bone marrow samples after induction treatment were analyzed for MRD levels by Real-Time PCR. We correlated these parameters with both patient event-free survival (EFS) and gene expression profile (GEP) findings. Results. 54/507 patients (10.6%) harboured FLT3ITD mutation. AR was calculated both at cDNA and DNA levels showing a reliable correlation (R=0.68), even if only AR measured on cDNA was found to be a significant poor prognostic feature. This latter observation supports the concept that expression of the mutation is more important than genetic bulk architecture at diagnosis. Patients with high AR showed a significant worse EFS as compared to those with low AR (19.2% for AR>0.51, vs 63.5% for AR Conclusion. Our results show that ITD-AR and MRD status are important independent prognostic factors for the management of of FLT3ITD patients, to be taken into consideration in planning the post-induction treatment. The high expression of the FLT3ITD mutation and the persistence of the mutated clone confer an aberrant hyperactivation of the downstream oncogenic FLT3 pathway influencing the outcome within these AML. Transcriptional profiles opens for further consideration of epigenetic targeting for FLT3ITD patients. Disclosures No relevant conflicts of interest to declare.

Abstract 4727 Introduction. Transcription factors has been widely confirmed to play a central role in acute leukemia, and among them the cyclic-adenosine monophosphate response element-binding protein (CREB) was identified to be involved in triggering acute myeloid leukemia. CREB protein overexpression has been found in the bone marrow of most pediatric patients with acute leukemia, and it has been shown to induce myeloid leukemia progression in vitro and in vivo by driving the upregulation of a series of target genes. CREB is evolutionarily conserved from invertebrates to human, and although the main focus of zebrafish research has traditionally been developmental biology, this model is currently used for cancer research. In fact, tumors induced in ZF have similar morphology and activated signaling pathways of human cancers. Aim. We aimed to develop a ZF harboring CREB overexpression in myeloid precursors, and by monitoring the induced myeloid malignancy, we will characterize CREB signaling and its involvement in the myeloid transformation process. Results. Using a Multisite Gateway System we constructed a vector containing human-CREB gene fused to EGFP, driven by an early myeloid promoter, zPu.1, to induce CREB overexpression specifically in the myeloid lineage of ZF. We injected the EGFP-CREB plasmid into one-cell stage zebrafish embryos, and monitored its expression during early development. Results showed that CREB was expressed in ZF zones typical of myelopoiesis, such as in the intermediate cell mass and anterior lateral mesoderm migrating through the yolk from 12 to 48 hours post-fertilization (hpf), and few cells circulating throughout the embryo from 24 hpf. CREB transcriptional activity on cAMP response elements (CREs) was measured and confirmed by luciferase assay. The co-localization of EGFP-CREB with the CRE-mCherry reporter was seen by fluorescence microscopy analysis. To test CREB target gene expression, embryos injected with CREB or Empty vector were subjected to RNA extraction and RQ-PCR. CREB over-expression was documented (up to 103 fold), and c-myb, stat3, rb, runx1, cyclins A, B, D1 and E2 were found upregulate at 24 and 48 hpf. By RNA whole mount in situ hybridization, we revealed pu.1, mpo, gata1 and fli.1 increased signals, suggesting a general disruption of the main hematopoietic factors. Ten CREB-injected zebrafish have been grown to adulthood and all of them displayed an abnormal/sick phenotype with abdominal enlargement and swelling from 10 to 12 months. Histochemical H&E staining performed on paraffin sections revealed an abdominal tumor and metastatic infiltration in kidney, skin, gills, muscles, adipose tissue. PAS staining identified the myeloid character of the tumor mass and kidney marrow. Wright-Giemsa and ANAE staining showed the predominance of clonal monocytes on appositions of tumor mass and kidney marrow of sick ZF. Tumor mass cell sorting displayed a clonal feature of the tumor with an enrichment of the myeloid-monocitic compartment. RNA extraction from the mass shows CREB overexpression as well as its target genes (c-myb, runx1, cyclin A1, cyclin B1, cyclin B2, cyclin E1), confirming CREB involvement in this tumor formation. Conclusion. We demonstrated that CREB overexpression recapitulates myeloproliferative disorder in ZF, supporting the ZF as a suitable model for studying CREB-induced leukemia. The discovery through which targets CREB would preferentially mediate the myeloid transformation will help to unravel leukemogenesis. ZF model might be useful to test CREB directed drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract 538 The cAMP response element binding protein (CREB) is a nuclear transcription factor downstream of various stimuli and is critical for the pathogenesis of leukemia. CREB overexpression promotes abnormal proliferation, cell cycle progression, and clonogenic potential in vitro and in vivo. We found that CREB deregulation in Acute Myeloid Leukemia (AML) is due to both genomic amplification and aberrant miRNA expression. CREB has been shown to be a direct target of the microRNA, miR-34b. The inverse correlation between CREB and miR-34b expression has been described in myeloid leukemic cell lines. Mir-34b restoration reduced CREB levels and leukemia proliferation in vitro. One reason for the lower expression of miR-34b in myeloid leukemia cell lines is the hypermethylation of its promoter. Our goal was to characterize the role of miR-34b in AML progression using primary cells and mouse models. We also studied the regulation of miR-34b expression in cells from patients with AML and myelodysplastic syndromes (MDS). Primary AML cells transiently overexpressing miR-34b had decreased clonogenicity, as well as increase in apoptosis (9.9 vs. 25.5%, p Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.