The pre-leukemic state of the TEL-AML1 positive cell. TEL-AML1 fusion is the most frequent genetic abnormality in paediatric cancer, and it is usually an early or initiating and pre-natal event in childhood ALL. Transformation results in the generation of a persistent pre-leukemic clone, which converts to frank ALL post-natally (at 1-15 years) following the acquisition of secondary genetic alterations. The mechanism by which the transcriptional dysregulation imposed by TEL-AML1 impacts on the pre-leukemic phenotype and disease natural history is unknown. We showed that the TEL-AML1 protein inhibits the TGFb response pathway, facilitating the selective expansion of otherwise more slowly expanding TEL-AML1 expressing progenitors. We aim to further explore the pre-leukemic phase by analyzing the migration/adhesion properties of the TEL-AML1 positive cells, and their interactions with the bone marrow niche. 2) Functional characterization of PAX5 genomic lesions. PAX5 is a transcription factor essential for B-cell development. Recently, it has been found as frequent target of aberrancies in childhood ALL (30% of B-cell ALL cases), showing monoallelic loss, point mutations or chromosomal translocations. The role of these aberrancies is still poorly understood. We reported the first evidence of the PAX5/TEL fusion gene in an ALL patient with the t(9;12) translocation, and we further investigated the molecular and functional roles of PAX5/TEL protein in vitro, in murine wild type preBI cells. In vitro, PAX5/TEL protein has a dominant negative effect on target genes, it interferes with the process of B-cell differentiation and migration; and it induces resistance to apoptosis, key events in the process of B-cell transformation. We aim to further investigate the effect of the PAX5-TEL chimeric protein on the differentiation program of preBI cells, to evaluate the migration/adhesion properties of the PAX5 aberrant cells, and to study the in vivo role of the PAX5-TEL fusion in mice models. We are also characterizing additional PAX5 lesions in childhood ALL patients. 3) Identification and characterization of the �leukemia-initiating stem cell� in Infant ALL with t(4;11). The reciprocal t(4;11), giving rise to the expression of MLL/AF4 fusion protein, is the most frequent aberration involving the MLL gene, and it is mainly associated to infant ALL (less than 12 months old at diagnosis). These patients have an overall a very poor prognosis, and their cells have a peculiar gene signature and phenotype that confer distinct properties to the disease (different form other form of common leukemia), which may be driven and sustained by a unique clone of stem cells. Aim of this task is the identification of the subset of cells responsible for initiating, expanding and maintaining the tumor, the �cancer stem cell� of Infant ALL/t(4;11). Identifying the nature of this cell not only may be key to unravelling the origin and the dismal prognosis of the disease, but also it is crucial to develop new therapies able to target these cells in patients. We started a combined multiparameter immunophenotype and FISH analyses on different subpopulations of PB/BM-derived cells from infant ALL/t(4;11) patients at diagnosis. We will set up an in vitro assay to confirm the clonogenic potential and self-replating ability of these cell. The NOD/SCID repopulating assay, by injecting purified sub-populations, will assess the ability of the candidate leukemia stem cell to transfer the leukemia in vivo.

Leukemias are the most common cancers affecting children while malignant lymphomas, including non-Hodgkin lymphomas (NHL), comes in third position after brain tumors. A significant number of children with leukemia/lymphomas still fail current therapies. The aim of the CHILDHOPE project is to develop a safe and efficient adoptive immunotherapy for children with advanced or refractory malignancies. CHILDHOPE particularly focuses on three pediatric tumors: acute B-lineage lymphoblastic leukemia, non-hodgkin B-lineage lymphoma and acute myeloid leukemia. The CHILDHOPE project is a new approach in pediatric cancer treatment since it brings from bench to bedside (and back) an innovative technology as yet never applied in children with advanced or refractory hematopoietic malignancies. The CHILDHOPE translational research project will focus on: 1. Improving and testing the efficacy and the safety of anti-leukemia/lymphoma chimeric T cells in relevant preclinical models in vitro and in vivo in mice. 2. Scaling-up this technology to numbers suitable for a clinical application in children with hematopoietic malignancies. Based on biological material obtained from our preclinical models and from children treated with these genetically engineered T cells, dissecting the interface between the host and tumor and immune cells and use this knowledge to understand the mechanisms of anti-tumor action, validate novel targets and diagnostic tools specific to children affected with leukemia or lymphomas. The CHILDHOPE project is built on the excellence of a network of EU-based partners with a broad experience in the field of pediatric hematology and oncology, immunology and cell & gene therapies and integrates the international confederation of parents of children with cancer and an SME specialized in the project management.