Impact of PAX5 alterations on gene expression and signaling pathways in ALL

Recently, PAX5 has been identified as a frequent target of mutation in BCP-ALL (around 30%). Among them 25% of the cases report deletions, 7% point mutations and 2-3% translocations that juxtapose PAX5 to several partner genes. To dissect the real frequency of PAX5 alterations in the Italian Cohort of BCP-ALL pediatric cases, we screened patients enrolled into AIEOP-IBFM-ALL2000 protocol, identifying alterations in the PAX5 locus, including deletions and translocations. To this purpose, we used different techniques in order to: a) select the patients carrying alterations on the chromosome 9p by cytogenetic analysis (e.g. karyotype analysis); b) describe the exact locus of the alterations and, in case of translocations, identify the partner genes; c) detect additional events, providing further elements to characterize PAX5 alterations as driver or passenger lesions. To study the functional properties of PAX5 fusion genes, we established an in vitro murine model of PAX5/ETV6 protein, as the most frequently reported example of PAX5 translocations. In particular, we aimed to widely characterize PAX5/ETV6 as an aberrant transcription factor, identifying the alterations induced in the gene expression profile and in the signaling pathway of primary populations of wt pre-BI cells co-cultured on OP9 BM stroma. Finally, to investigate the potential role of PAX5 as a tumor suppressor gene in ALL, we approached to study PAX5 deletions, in a xenograft model of primary BCP-ALL cells transplanted in NSG mice. We focused the attention on the Ph+ subgroup, because of the high frequency of PAX5 deletions in BCR/ABL1 positive patients. To this purpose we used different approaches: a) establishment of xenografts of 14 childhood BCP-ALL patients (Ph+ and Ph-, as control group); b) ChIP sequencing in order to identify PAX5 target genes in leukemic settings; c) analysis of the phosphorylation status; d) generation of an in vitro inducible vector model, in order to better study the effect of PAX5 and PAX5/ETV6 in human leukemic cells. Taken together, this study aimed to contribute to a better understanding of the role of PAX5 lesions in BCP-ALL leukemia with a comprehensive approach, from patients samples to the establishment of murine in vitro and human ex vivo models.