ALBERT

Description: 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

Description: 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

Description: Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood. Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro. Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60(CREB+), where CREB was overexpressed. HL60(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60(CREB-) differentiated to a higher extent compared to HL60(CREB+).Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60(CREB-) behavior. Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: 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

Description: 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

Description: 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

Description: 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.

Description: Key Points MLL-AF6 leads to aberrant activation of RAS and its downstream targets. RAS targeting is a novel potential therapeutic strategy in AML patients carrying t(6;11).