Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursor ALL with t(17;19).

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

[Chronic graft-versus-host disease with multiple serositis after bone marrow transplantation from non-inherited maternal antigen-complementary sibling donor].

We report a patient who developed multiple serositis during chronic graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation from a non-inherited maternal antigen (NIMA) -complementary sibling donor. The patient was a 9-year-old boy with myelodysplastic syndrome, who urgently underwent bone marrow transplantation from his NIMA-complementary HLA two-locus-mismatched sister following graft failure of cord blood transplantation. Engraftment was successfully confirmed and no acute GVHD developed. After withdrawal of tacrolimus to prevent recurrent viral infection, he developed pleural effusion, ascites and edema approximately 6 months after transplantation. His clinical symptoms were resolved by methylprednisolone pulse therapy, but he subsequently progressed to develop pericardial effusion, pneumothorax and truncal panniculitis. Pleural and pericardial effusion contained numerous lymphocytes, which gradually subsided with continuous drainage. His symptoms were thereafter controlled by the addition of mycophenolate mofetil (MMF) administration, and his current performance status is almost perfect by the administration of prednisolone (5 mg/day) and MMF at 6 years after transplantation. Although multiple serositis associated with GVHD is known to have a poor prognosis, the multiple symptoms of this patient improved gradually, probably owing to a lack of acute GVHD and the effect of MMF.

Fms-like tyrosine kinase 3 ligand stimulation induces MLL-rearranged leukemia cells into quiescence resistant to antileukemic agents.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in acute lymphoblastic leukemia with the mixed-lineage leukemia (MLL) gene rearrangement refractory to chemotherapy. We examined the biological effect of FLT3-ligand (FL) on 18 B-precursor leukemic cell lines with variable karyotypic abnormalities, and found that nine of nine MLL-rearranged cell lines with wild-type FLT3, in contrast to other leukemic cell lines, are significantly inhibited in their proliferation in a dose-dependent manner by FL. This inhibition was due to induction of the G0-G1 arrest. A marked up-regulation of p27 by suppression of its protein degradation and an abrogation of constitutive signal transducers and activators of transcription 5 phosphorylation were revealed in arrested leukemia cells after FL stimulation. Importantly, FL treatment rendered not only cell lines but also primary leukemia cells with MLL rearrangement resistant to chemotherapeutic agents. MLL-rearranged leukemia cells adhering to the bone marrow stromal cell line, which expresses FL as the membrane-bound form, were induced to quiescent state resistant to chemotherapeutic agents, but their chemosensitivity was significantly restored in the presence of neutralizing anti-FL antibody. The FL/FLT3 interaction between leukemia cells and bone marrow stromal cells expressing FL at high levels should contribute, at least in part, to persistent minimal-residual disease of MLL-rearranged leukemia in bone marrow.

Endoplasmic reticulum stress inducers, but not imatinib, sensitize Philadelphia chromosome-positive leukemia cells to TRAIL-mediated apoptosis.

Philadelphia-chromosome (Ph1)-positive leukemia cells frequently express death receptors DR4/DR5 for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and show high TRAIL-sensitivity. It has been reported that imatinib damaged cardiomyocytes by triggering endoplasmic reticulum (ER) stress and that ER stress inducers intensified TRAIL-sensitivity of some cancer cells by upregulating DR4/DR5 expression. In fact, ER stress inducers enhanced TRAIL-sensitivity of Ph1-positive leukemia cells by upregulating DR4/DR5 expression. In contrast, imatinib did not induce ER stress responses and unexpectedly downregulated DR4/DR5 expression, indicating that sensitization of Ph1-positive leukemia cells to TRAIL-mediated cellular immunity by imatinib through upregulation of DR4/DR5 expression is unlikely.

Resistance of T-cell acute lymphoblastic leukemia to tumor necrosis factor--related apoptosis-inducing ligand-mediated apoptosis.

OBJECTIVE: Cytotoxic ligands are involved in tumor immunity and graft-vs.-leukemia effect after allogeneic stem cell transplantation for leukemia. To clarify the susceptibility of T-cell acute lymphoblastic leukemia (T-ALL) to tumor immunity, sensitivity to recombinant human soluble Fas ligand (rhsFasL) and tumor necrosis factor-related apoptosis-inducing ligand (rhsTRAIL) was determined. MATERIALS AND METHODS: Sensitivity to rhsFasL and rhsTRAIL and cell surface expression of their receptors were tested in T-ALL cell lines (n = 7) and patients' samples (n = 17) and compared with those in B-precursor ALL cell lines (n = 30). Expression of components of the death-inducing signaling complex and the TRAIL receptor genes (DR4/DR5), and the methylation status and promoter activity of the DR4/DR5 gene were tested in T-ALL cell lines. RESULTS: T-ALL cell lines showed higher level of Fas expression and higher sensitivity to rhsFasL than did B-precursor ALL cell lines. Despite comparable expression of components of death-inducing signaling complex, cell lines and patients' samples of T-ALL showed TRAIL-resistance associated with low cell surface expression of DR4/DR5. Gene expression of DR4/DR5 in T-ALL cell lines was significantly lower than that in B-precursor ALL cell lines, and the methylation status of the gene promoter in T-ALL cell lines was associated with the gene expression level at least for DR4. The demethylating agent, 5-aza 2'deoxycytidine, upregulated the gene expression of DR4/DR5, but was insufficient for their surface expression due to low basal promoter activity. CONCLUSIONS: In contrast to higher sensitivity to FasL, T-ALL showed resistance to TRAIL, which might be responsible for resistance to TRAIL-mediated cellular immunity.

Monitoring neutrophil engraftment in allogeneic stem cell transplantation by flow cytometric analysis of neutrophil-specific antigens NA1 and NA2.

Neutrophil-specific antigen (NA) expression on neutrophils was analysed in 18 Japanese children before and after allogeneic stem cell transplantation (allo-SCT) with myeloablative regimen. Donor-recipient NA-incompatibility was present in one of eight NA1/NA2 heterozygous patients and eight of 10 NA1/NA1 or NA2/NA2 homozygous patients. After allo-SCTs from NA-incompatible donors, a neutrophil recipient-to-donor conversion was confirmed in all cases. Conversion to donor NA type was complete before the absolute neutrophil count reached 0.1 x 10(9)/l. These observations indicate that flow cytometric analysis of NA antigens is a simple and useful method for monitoring neutrophil engraftment in NA-incompatible allo-SCT.