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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.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function.systems immunobiology | bioinformatics | site-directed mutagenesis | signaling | molecular modeling

Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia.

The cytoplasmic spleen tyrosine kinase (SYK) is a key regulator of signal transduction events, apoptosis and orderly cell cycle progression in B-lineage lymphoid cells. Although SYK has not been linked to a human disease, defective expression of the closely related T-cell tyrosine kinase ZAP-70 has been associated with severe combined immunode®ciency. Childhood CD19 + CD10 7 pro-B cell acute lymphoblastic leukemia (ALL) is thought to originate from B-cell precursors with a maturational arrest at the pro-B cell stage and it is associated with poor prognosis. Since lethally irradiated mice reconstituted with SYK-de®cient fetal liver-derived lymphohematopoietic progenitor cells show a block in B-cell ontogeny at the pro-B to pre-B cell transition, we examined the SYK expression pro®les of primary leukemic cells from children with pro-B cell ALL. Here we report that leukemic cells from pediatric CD19 + CD10 7 pro-B cell ALL patients (but not leukemic cells from patients with CD19 + CD10 + common pre-pre-B cell ALL) have markedly reduced SYK activity. Sequencing of the reverse transcriptase-polymerase chain reaction (RT ± PCR) products of the Syk mRNA in these pro-B leukemia cells revealed profoundly aberrant coding sequences with deletions or insertions. These mRNA species encode abnormal SYK proteins with a missing or truncated catalytic kinase domain. In contrast to pro-B leukemia cells, pre-pre-B leukemia cells from children with CD19 + CD10 + common Blineage ALL and EBV-transformed B-cell lines from healthy volunteers expressed wild-type Syk coding sequences. Examination of the genomic structure of the Syk gene by inter-exonic PCR and genomic cloning demonstrated that the deletions and insertions in the abnormal mRNA species of pro-B leukemia cells are caused by aberrant splicing resulting in either missplicing, exon skipping or inclusion of alternative exons, consistent with an abnormal posttranscriptional regulation of alternative splicing of Syk pre-mRNA. Our ®ndings link for the ®rst time speci®c molecular defects involving the Syk gene to an immunophenotypically distinct category of childhood ALL. To our knowledge, this is the ®rst discovery of a speci®c tyrosine kinase de®ciency in a human hematologic malignancy. Oncogene (2001) 20, 3969 ± 3978.

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