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Acute Myeloid Leukemia - Biology 2 with Pmscv-NUP98-NSD1-Neo Or Pmscv-FLT3-ITD-GFP Retroviral Vectors Or Both

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Acute Myeloid Leukemia - Biology 2 with Pmscv-NUP98-NSD1-Neo Or Pmscv-FLT3-ITD-GFP Retroviral Vectors Or Both Milan, Italy, June 12 – 15, 2014 Methods: Lineage marker-negative murine bone marrow cells were transduced Acute myeloid leukemia - Biology 2 with pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP retroviral vectors or both. Transduced cells were analyzed for their clonogenic potential by serial plating in growth-factor containing methylcellulose followed by expansion and P142 injection into sublethally irradiated syngeneic recipients. Results: Expression of NUP98-NSD1 provided aberrant self-renewal poten - Abstract withdrawn tial to bone marrow progenitor cells. Co-expression of FLT3-ITD increased proliferation but impaired self-renewal in vitro . Transplantation of cells expressing NUP98-NSD1 and FLT3-ITD into mice resulted in transplantable P143 AML after a short latency. The leukaemic blasts expressed myeloid markers + + + lo lo THE CAMP RESPONSE ELEMENT BINDING PROTEIN (CREB) OVEREX - (Mac-1 /Gr-1 /Fc γRII/III /c-kit /CD34 ). Splinkerette-PCR revealed similar PRESSION INDUCES MYELOID TRANSFORMATION IN ZEBRAFISH patterns of potential retroviral integration sites of in vitro immortalized bone C Tregnago 1,* V Bisio 1, E Manara 2, S Aveic 1, C Borga 1, S Bresolin 1, G Germano 2, marrow cells before and after expansion in vivo . Mice that received cells G Basso 1, M Pigazzi 2 expressing solely NUP98-NSD1 did not develop AML but showed signs of a 1Dipartimento di salute della donna e del bambino, Università degli Studi di myeloid hyperplasia after long latency, characterized by expansion of Mac- Padova, 2Dipartimento di salute della donna e del bambino, IRP, Padova, Italy 1+/Gr-1 + cells in the bone marrow and active extramedullary haematopoiesis in the spleen. Interestingly, similar to what has been observed in patients car - Background: Pediatric acute myeloid leukemia (AML), is a heterogeneous rying NUP98-NSD1, an increased Flt3-ITD to wildtype Flt3 mRNA expression disease characterized by multiplicity of genetic or epigenetic events that can ratio was found to be associated with a more aggressive disease induced by lead to transformation. Identifying specific leukemogenic aberrations may guide co-expression of NUP98-NSD1 and FLT3-ITD. Additionally, the shorter laten - the development of targeted therapy approaches for selected patient groups. cy of AML induction correlated with increased activation of the FLT3-STAT5 We previously identified and characterized one AML proto-oncogene: the cAMP signaling axe as shown by increased pFLT3 levels and increased basal and response element binding protein (CREB). Its overexpression has been found GM-CSF induced pSTAT5 levels, as assessed by flow cytometry. To further in 66% of pediatric AML patients, and represents a poor prognostic marker in underline the importance of FLT3 signaling for NUP98-NSD1 induced AML we human AML. In vitro and in vivo studies demonstrated that its overexpression treated ex vivo immortalized progenitors as well as in vivo established leads to abnormal cell proliferation, cell cycle progression and higher clonogenic leukemic blasts with a small molecule FLT3 inhibitor (PKC-412) and found that potential, through an aberrant transcriptional regulation of its target genes. proliferation of all cells co-expressing NUP98-NSD1 and FLT3-ITD was sig - However the molecular mechanism and the transcriptional networks by which nificantly impaired by PKC-412. CREB triggers leukemogenesis has never been underpinned. Summary and Conclusions: Our data revealed a potent cooperation between Aims: The aim of this study is to investigate the effects of CREB overexpression NUP98-NSD1 and FLT3-ITD for the induction of AML disease in vivo and under - on hematopoiesis using the zebrafish in vivo model. Moreover, the identification lined the significance of aberrant FLT3 signaling for maintenance of NUP98- of the main deregulated CREB targets will be pursued, in order to discover rele - NSD1-positive AML. In addition, we provided the rationale and a mouse mod - vant pathways which will be considered for further therapeutic targeting. el that allows to functionally explore the use of small molecule FLT3 inhibitors Methods: We developed a zebrafish model that overexpressed human CREB for NUP98-NSD1 positive AML. in myeloid precursor cells, by injecting a plasmid which expressed CREB under control of pu.1 promoter into 1-cell stage zebrafish embryos. The plasmid was constructed using the site-specific recombination-based cloning (multisite Gate - P145 way technology) Tol2kit system. Gene expression studies were conducted by RQ-PCR and whole mount in situ hybridization. Hematopoiesis after CREB AN INDUCIBLE MOUSE MODEL FOR MLL-ENL MIXED-LINEAGE LEUKEMIA enforced expression was investigated by flow cytometry analysis of kidney mar - V Stavropoulou 1,* S Juge 1, JF Spetz 2, A Tzankov 3, L Brault 1, M Iacovino 4, row cells, by immunohistochemistry. RNA extracted from kidney of 5 CREB M Kyba 4, A H Peters 2, J Schwaller 1 and 5 control zebrafish were analyzed for gene expression (GeneChip Zebrafish 1Dept. of Biomedicine, University Children’s Hospital, 2Friedrich Miescher Insti - Genome Arrays, Affymetrix, Santa Clara, CA, USA). tute for Biomedical Research, 3Institute for Pathology, University Hospital Basel, Results: Results showed that CREB recognized the cyclic-AMP responsive ele - Basel, Switzerland, 4Department of Pediatrics, Lillehei Heart Institute, Univer - ments at genes promoter modifying gene expression. We documented the up- sity of Minnesota, Minneapolis, United States regulation of its targets, particularly of bcl-2 and jun . Then, we controlled gene expression of genes involved in myelopoiesis and found increased levels of Background: The t(11;19)(q23;p13.3) translocation leading to expression of the pu.1 , mpo and gata1 during the primitive hematopoiesis. Then, we monitored MLL-ENL fusion protein is one of the most prevalent MLL gene alterations, the kidney marrow of zebrafish during growth: 6-8 months old CREB zebrafish mostly associated with mixed lineage or B-cell acute lymphoblastic leukemia. showed an impairment of myelopoiesis with a loss of cell precursors and The existing retroviral and knock-in models do not closely recapitulate the increased myelo-monocytes by flow cytometry analysis. From 9 to 14 months, human disease, possibly because of a failure to activate MLL-ENL expression CREB overexpressing zebrafish developed a fatal myeloproliferative neoplasm in the appropriate hematopoietic compartment. (MPN) with 41% mortality rate. A clonal expansion of mature myelo-monocitic Aims: To establish an inducible mouse model for MLL-ENL that recapitulates cells were present in kidney marrow with an impairment of lymphocyte produc - the human disease and thereby facilitates the dissection of the mechanisms tion. Histological analysis revealed that myeloid cells infiltrated extramedullary underlying MLL-ENL leukemogenesis. organs, such as heart, liver, and abdominal organs, leading to zebrafish death. Results: We used the doxycycline (DOX)-induced gene expression system to Immunohistochemistry assays displayed that disseminated blasts expressed establish transgenic rtTA;MLL-ENL mice. DOX-induced ex vivo expression of human CREB. Microarray analysis identified 92 CREB target genes differen - MLL-ENL provided bone marrow and hematopoietic stem and progenitor cells tially expressed, belonging to proliferation/differentiation MAPK and cell with a strong long-term self-renewal capacity, causing the accumulation of cycle/DNA repair pathways. immature blast-like cells upon serial replating in methylcellulose cultures. In the Summary and Conclusions: This represents the first evidence that CREB presence of factors favoring myelopoiesis, like IL-3, DOX removal resulted in overexpression triggers myeloid transformation in zebrafish. This model will a complete differentiation towards the granulocytic-monocytic, lineages serve to identify CREB mediated genes/pathways that contribute to the myeloid expressing high levels of Mac1/Gr-1, whereas IL-7 favored differentiation transformation, with the final aim of identifying novel therapeutic targets. towards the B-cell lineage characterized by the expression of high levels of B220. Induction of MLL-ENL expression in newborn or adult mice resulted in 100% of the mice in a leukemic phenotype that phenocopied pre-B- and myeloid P144 mixed lineage leukemia. Diseased mice had excessive splenomegaly, mas - POTENT COOPERATION BETWEEN THE NUP98-NSD1 FUSION AND THE sive lymph node and multi-organ infiltration characterized by the coexistence FLT3-ITD MUTATION IN ACUTE MYELOID LEUKAEMIA INDUCTION of at least two types of blasts of various sizes and granularity that expressed A Thanasopoulou 1,* A Tzankov 2, J Schwaller 1 intermediate levels of myeloid markers or lymphoid markers. Transplantation high 1Department of Biomedicine, University Children’s Hospital, 2Institute for Pathol - of B220 , blasts into sub-lethally irradiated recipients rapidly induced the dis - ogy, University Hospital, Basel, Switzerland ease (15.6±0.5 days) whereas transplantation of B220 low blasts resulted in sig - nificantly longer latency (22.5±1.1 days) suggesting that the B220 high popula - Background: The NUP98-NSD1 fusion, resulting from an acute myeloid tion is enriched for leukemia initiating cells. Expression of the fusion gene and leukaemia (AML)-associated cytogenetically silent chromosomal translocation, disease induction was DOX dosage dependent and fully reversible upon DOX is frequently found in pediatric cytogenetically normal AML and associated with removal.
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