Expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia

Purpose: We sought to determine if molecular abnormalities involving the Ik aros gene could contribute to the development of acute lymphoblastic leukem ia (ALL) in children. Patients and Methods: We studied Ikaros gene expression in normal human bon e marrow, normal thymocytes, normal fetal liver-derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells f rom 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Ex pression of Ikaros protein and its subcellular localization were examined b y immunoblotting and confocal laser-scanning microscopy, respectively Polym erase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing o f splice junction regions of the Ikaros gene was performed in search for mu tations. Results: In each of the ALL cases, we found high-level expression of a nan- DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcell ular compartmentalization patterns. In contrast, only wild-type Ik-1 and lk -2 isoforms with normal subcellular localisation were found in normal bone marrow cells and thymus-derived or fetal liver-derived normal lymphocyte pr ecursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-ex on junctions between exons 6 and 7 yielded the wild-type sequence. We ident ified a single nucleotide polymorphism (SNP) affecting the third base of th e triplet codon for a proline (CCC or CCA) in the highly conserved bipartit e activation region (viz, A or C at position 1002 numbering from the transl ation start site of Ik-l) within our Ikaros clones. Bi-allelic expression o f truncated and/or non-DNA-binding isoforms along with wild-type isoforms w as observed in leukemic cells, which implicates trans-acting factor(s) affe cting splice site recognition. Conclusion: Our findings link specific molecular defects involving the Ikar os gene to childhood ALL. Posttranscriptional regulation of alternative spl icing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patient s, in contrast to normal lymphocyte precursors, express high levels of non- DNA-binding Ikaros isoforms that are reminiscent of the nan-DNA-binding Ika ros isoforms that lead to lymphoblastic leukemia in mice. J Clin Oncol 17:3 753-3766. (C) 1999 by American Society of Clinical Oncology.