ALTERNATIVE SPLICING OF IKAROS mRNA IS RESPONSIBLE FOR THE EXPRESSION OF DIFFERENT ABERRANT ISOFORMS IN PHILADELPHIA CHROMOSOME-POSITIVE (PH+) ADULT ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) PATIENTS

Background. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages at multiple stages, that acts both potentiating and repressing gene expression. By means of alternative splicing, Ikaros encodes several proteins that differ that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA non-binding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. An excess of short DNA nonbinding isoforms has been described in different human haematological malignancies. Aims. To detect and quantify different Ikaros transcript variants in Philadelphiapositive (Ph+) adult acute lymphoblastic leukaemia (ALL) patients by performing a high-throughput method based on capillary electrophoresis. Methods. We analyzed 46 adult de novo Ph+ ALL patients. Ikaros cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5’ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). The GeneMapper electrophoretograms display information about transcript length, peak height and peak area. The emitted fluorescence at each peak position reflects the number of amplified target sequence: peaks heights are correlated to the quantity of amplified PCR product and used as an indication of the Ikaros level expression in a sample. Results. In 19/46 (41%) of patients we identified a single peak of 255 bp corresponding to DNA non-binding isoform Ik6. In the remaining 59% patients we observed the co-expression of many fragments, corresponding to the different functional (Ik2) and non functional (Ik4, Ik4A, Ik5A, Ik6, Ik6D, Ik8) Ikaros transcript variants. Furthermore, a recurring 60-bp insertion immediately downstream of exon 3, was frequently detected in Ik2 and Ik4 isoforms either alone or together with an in-frame 10-amino acid deletion, due to a 30- bp deletion at the end of exon 7. Both the alterations are due to the selection of alternative splice sites which determine an impairment ability to form dimers and to bind the DNA target sequence. Summary and conclusions. Taken together, our findings demonstrated that alterations of the transcription factor Ikaros, involving both short spliced oncogenic isoforms and aberrant full-length isoforms are a common feature of Ph+ ALL. Because of the leukemic role of short and/or aberrant Ikaros splice variant, it is more important to use a sensitive method to detect them: in this study we develop a fast, sensitive, highthroughput method, to both detect and quantify splice variants. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.