Induction of CD33 Expression in B-Precursor ALL by E2A-HLF Fusion Transcription Factor Derived from t(17;19).

CD33 (Siglec-3), one of myeloid-specific antigens, belongs to a family of sialic acid-binding, immunoglobulin-like lectins (Siglecs), which is adhesion receptor for sialic-acid bearing ligands and regulates a signal transduction through its cytoplasmic tyrosine residues designated immunoreceptor tyrosine-based inhibitory motif (ITIM) as representatively well-characterized in CD22 (Siglec-2). CD33 is ectopically expressed in 5–20% of B-precursor ALL, and has been reported to be potentially associated with poor prognosis. This myeloid-specific antigen expression in B-precursor ALL is considered as lineage promiscuity or lineage infidelity, but underlying mechanism remains totally unknown. We have recently found that B-precursor childhood ALL with t(17;19), which has extremely poor prognosis, is principally CD33 positive, suggesting that CD33 expression might be regulated by E2A-HLF fusion transcription factor generated by t(17;19). To test this hypothesis, using zinc inducible expression vector, we have introduced E2A-HLF into Reh cells, which is a CD33-negative B-precursor ALL cell line. In flow cytometric analysis, by the addition of zinc, CD33 expression was gradually induced in Reh cells transfected with E2A-HLF but not in control cells, and returned to baseline after deprivation of zinc. Of note, mutant of E2A-HLF defective in its DNA-binding domain did not induce CD33 expression. Induction of CD33 expression was not due to “myeloid differentiation”, since upregulation of other myeloid markers including CD11b, CD13, CD14, CD15 and cytoplasmic myeloperoxidase was not observed. In real-time PCR analysis, despite the immediate expression of E2A-HLF protein after addition of zinc, the CD33 mRNA was slowly induced and progressively upregulated to the level approximately 20 times higher than baseline at 5 days after addition of zinc. In promoter region of the CD33 gene, there was no authentic HLF-binding consensus sequence. These observations suggest an indirect induction of CD33 transcription by E2A-HLF. As reported in myeloid cells, when CD33-positive t(17;19)-ALL cell lines were treated with a phosphatase inhibitor pervanadate and analyzed with immunoprecipitaions and Western blotting assay using anti-CD33 antibody, CD33 was confirmed to be tyrosine-phospholyrated and recruit both the protein-tyrosine phosphatases, SHP-1 and SHP-2. This is a first report indicating that an oncogenic fusion transcription factor is evidenced to be associated with ectopic expression of CD33 in B-precursor ALL and that CD33 might play some roles in signal transduction in B-precursor ALL cells.