Expression of aberrantly spliced oncogenic ikaros isoforms in childhood acute lymphoblastic leukemia.
OBJECTIVE
We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children.


METHODS
We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver-derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations.


RESULTS
In each of the ALL cases, we found high-level expression of a non-DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver-derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non-DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition.


CONCLUSIONS
Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non-DNA-binding Ikaros isoforms that are reminiscent of the non-DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.
Sun
L
L
Parker Hughes Cancer Center and Children's Cancer Group ALL Biology Reference Laboratory, Hughes Institute, St Paul, MN 55113, USA.
Goodman
P A
PA
Wood
C M
CM
Crotty
M L
ML
Sensel
M
M
Sather
H
H
Navara
C
C
Nachman
J
J
Steinherz
P G
PG
Gaynon
P S
PS
Seibel
N
N
Vassilev
A
A
Juran
B D
BD
Reaman
G H
GH
Uckun
F M
FM
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051740:Ikaros Transcription Factor; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013347:Subcellular Fractions; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497239:IKZF1 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; C497238:Zfpn1a1 protein, mouse; D051740:Ikaros Transcription Factor; D004247:DNA