IDENTIFICATION OF NOVEL NON-HODGKIN B-CELL LYMPHOMA DETERMINANTS THROUGH AN IN VIVO, CELL-SPECIFIC TRANSPOSON MUTAGENESIS SCREENING

Lymphomas represent the outgrowth of a clonal population of T- or B- lymphocytes. The majority of human lymphomas arise from mature B-cells recruited into germinal centers (GC). B-cell lymphomas fall within two major groups, Hodgkin and Non-Hodgkin (NH) lymphoma. Despite progress has been made toward the elucidation of the molecular basis of lymphomagenesis, few lymphoma determinants have been, to date, causatively linked to the pathogenesis of B-cell lymphomas. To identify novel genetic determinants of NHL, which include the most common and aggressive forms of B-cell lymphomas, we made use of a cell-type and stage-specific transposon-based, insertional mutagenesis system dependent on the Sleeping Beauty (SB) transposase. As transposon we used T2/Onc2, engineered to cause overexpression of proto-oncogenes or disrupt tumor suppressor genes, respectively. To restrict transposon mutagenesis to the mature B-cell compartment, we used conditional Rosa26-SBfl-stop mice, in which SB expression, controlled by the Rosa26 promoter, occurs upon Cre-mediated deletion of a loxP-flanked transcription termination sequence. SB expression was induced in mature and GC B-cells using CD21-Cre or Cγ1-Cre transgenes, respectively. The conditional SB system was also used to accelerate lymphomagenesis in mouse NHL models driven by deregulated Bcl6 and Bcl2 expression. Insertional mutagenesis in GC B-cells was sufficient to promote the occurrence of clonal B-cell tumors at high frequency. B-cell malignancies developing in compound mutants, represented a rather heterogeneous group of diseases originating from GC B-cells, as revealed by the accumulation of somatic mutations within clonal immunoglobulin gene rearrangements. Histological evaluation of tumors revealed close resemblance to human diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the most common forms of NHL. Also, we found that transposon mutagenesis efficiently cooperated with deregulated Bcl6 and Bcl2 to promote GC-derived DLBCL and FL respectively. High throughput sequencing of transposon integration sites from 29 GC-derived B-cell lymphomas identified 240 genes lying within Common Insertion Sites (CISs). Bioinformatic analysis revealed enrichment for genes belonging to the NF-κB signaling network, commonly deregulated in human NHL and in particular in DLBCL. The putative role of CISs as tumor suppressors or proto-oncogenes was assessed based on orientation and gene distribution of T2/Onc2 insertions. Moreover, analysis of gene-copy gain or loss in human lymphomas, occurrence of somatic mutations in human cancers of different type and expression in NHL contributed to select, among the 240 CISs, the strongest candidates to represent novel NHL determinants. Among them, we found genes controlling B-cell differentiation/identity (Ikaros, Aiolos, Bach2 and Swap-70), survival/apoptosis (NF-κB1, NIK, Traf3 Akt2, Rreb1, Zmat3), proliferation (Raf1), chromatin remodeling (Smarcc1) and protein glycosylation (B4galt1, Man2a).