Induction of Death Receptors for TRAIL, a Cytotoxic Factor for GVL Effect, by Oncogenic Fusion E2A-HLF Derived from t(17;19)-Positive Acute Lymphoblastic Leukemia.

Abstract

Graft versus leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT) is mediated by the cytotoxic factors on cytotoxic T-cells and NK cells. Recent analysis demonstrates that TNF-related apoptosis inducing ligand, TRAIL, plays an important role in the GVL effect. TRAIL induces apoptosis of leukemia cells by the binding to the death receptors, DR4 and DR5. Thus, although the precise mechanism remains unclarified, the regulation of DR4/DR5 expression could be one of critical factors for susceptibility of leukemia cells to the GVL effect. t(17;19)-positive acute lymphoblastic leukemia (ALL) has an extremely poor prognosis, but long-term disease-free survival was exceptionally observed in the case who underwent allo-SCT early in the first complete remission, suggesting that t(17;19)-ALL cells might be susceptible to TRAIL and eventually vulnerable to GVL effect. Accordingly, we examined the sensitivity of t(17;19)-ALL to recombinant human soluble (rhs) TRAIL using 4 cell lines. All 4 cell lines immediately underwent apoptosis by the treatment with rhsTRAIL, and the activation of Caspases 3 and 8, Bid, and PARP was confirmed with Western blotting. Of note, 4 t(17;19)-ALL cell lines showed a significantly higher TRAIL-sensitivity than did other 25 B-precursor ALL cell lines including Ph1, t(1;19), and MLL-rearrangement-positive ALLs. Cell surface expression of DR4/DR5, but not decoy receptors, was confirmed on all 4 cell lines by flow cytometry. Of importance, the levels of cell surface DR4/DR5 expression in 4 t(17;19)-ALL cell lines were significantly higher than those in other 25 B-precursor ALL cell lines. Real time RT-PCR analysis also demonstrated a significantly higher DR4/DR5 gene expression in t(17;19)-ALL cell lines. Since E2A-HLF chimeric transcription factor derived from t(17;19) plays an essential role in the leukemogenesis, we introduced E2A-HLF into the B-precursor ALL cell line 697, whose DR4/DR5 expression level is low, using Zn-inducible vector. When E2A-HLF expression was induced by Zn, the levels of DR4 and DR5 transcripts were immediately upregulated by 10-fold and 40-fold of baseline, respectively. The induction of DR4/DR5 expression was dependent on the DNA-binding and transactivation activity of E2A-HLF, since the mutants lacking either the DNA-binding or transactivation domains failed to induce DR4/DR5 expression. Further, binding of E2A-HLF to the consensus sites separated from the promoters of DR4/DR5 genes was demonstrated in EMSA. These observations indicate that E2A-HLF directly induces expression of DR4/DR5 and sensitizes leukemia cells to TRAIL and eventually GVL effect. This is the first presentation that oncogenic fusion product of translocation regulates the expression of death receptors.