Intronic CD22 Gene Mutations as A Pathogenic Mechanism of Human B-Precursor Leukemia

Abstract 149

CD22 is an inhibitory co-receptor of B-cells and B-cell precursors that acts as a negative regulator of multiple signal transduction pathways critical for B-cell homeostasis. The inhibitory and apoptosis-promoting signaling function of CD22 is dependent on recruitment of the Src homology 2 domain–containing tyrosine phosphatase (SHP)-1 to the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of its cytoplasmic domain. Collective genetic evidence from CD22-deficient or SHP-1-deficient mice shows that disruption of the CD22-SHP1 signaling network can result in development of a B-cell lymphoproliferative state associated with defective apoptosis and maturation. However, direct genetic evidence for the functional significance of CD22 in human B-cell ontogeny or its implied tumor suppressor role has been lacking. Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia (BPL) express a truncated and functionally defective CD22 co-receptor protein that is unable to transmit apoptotic signals. 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 co-receptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22DE12). CD22DE12 involves an exact splice with no other mutation at the splice junction. The deletion of exon 12 in CD22 mRNA results in a truncating frameshift mutation starting at K736 with an insertion of 15 amino acids not seen in wild-type CD22 sequence followed by a TGA termination codon. Mutant CD22DE12 protein lacks the conserved tyrosines and ITIMs that provide docking sites for the SH2 domains of the tyrosine phosphatase SHP1. It also lacks regions homologous to ITAMs, tyrosine-based activation motifs, which are docking sites for SH2 containing proteins. Thus, CD22DE12 mRNA encodes a truncated CD22 protein lacking most of the intracellular domain including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. 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 CD22DE12 protein in transgenic mice, under control of the immunoglobulin enhancer Eμ that is activated in early B-cell ontogeny prior to Ig gene rearrangements, perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia at 6 weeks of age and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. This CD22DE12-associated unique 12-gene signature transcriptome included (i) tumor suppressor genes TP53 (as well as TP53 regulator MDM2), neurofibromatosis 2 (NF2) (as well as NF2 regulator RAC1), and the adenomatous polyposis coli (APC) gene, a tumor suppressor known to regulate the Wnt/beta-catenin signaling, (ii) genes for chromatin remodeling/global gene expression regulators with a tumor suppressor function IKZF1/IKAROS and SATB1, as well as (iii) cell cycle regulatory genes CDKN1C, CCNG1, and NOTCH4. By comparing gene expression profiles of primary leukemia cells from 31 infants vs. 30 non-infant children with ALL we found that reduced expression levels of 6 of the 9 CD22DE12 signature genes that were represented on the human cDNA arrays, including TP53 and APC as well as MDM2, SATB1, CCNG1 and GNB2 discriminated infant BPL from non-infant BPL. The documented molecular and functional abnormalities involving CD22 in primary leukemic cells from patients with newly diagnosed infant BPL uniquely implicate deficiency of this B-lineage restricted co-receptor protein in the pathogenesis of infant leukemia. This collection of experimental data is unprecedented in that it links a genetic defect involving a B-lineage specific regulatory gene to the most aggressive human B-lineage lymphoid malignancy that arises from an uncoupling of proliferation and differentiation of B-precursors during the earliest stages of B-cell ontogeny.