Kinuko Hirose

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.

Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

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.

Successful tandem (autologous-cord blood) SCT in advanced neuroblastomas with highly amplified MYCN

Although autologous tandem hematopoietic SCT has improved the prognosis of patients with advanced high-risk neuroblastoma, the results remain unsatisfactory. In an attempt to induce the graft-versus-tumor effect, we performed autologous PBSCT followed by allogeneic cord blood transplantation in three consecutive advanced neuroblastoma cases with marked BM infiltration and high MYCN amplification. Severe acute complications did not occur in any patient and they have maintained disease-free survival for 37–60 months. This strategy appears to be feasible and effective for the treatment of extremely high-risk neuroblastoma cases.

Natural pregnancy and delivery after allogeneic bone marrow transplantation in a Fanconi anaemia patient

Mohamad Mohty Jean El-Cheikh Philippe A. Cassier Catherine Faucher Sabine Fürst Norbert Vey Anne-Marie Stoppa Daniele Sainty Christine Arnoulet Luc Xerri Jean-Albert Gastaut Didier Blaise Reda Bouabdallah Unité de Transplantation et de Thérapie Cellulaire (UTTC), Institut Paoli-Calmettes, Département d’Hématologie, Institut Paoli-Calmettes, Département de Biopathologie, Institut Paoli-Calmettes, Université de la Méditerranée, Faculté de Médecine, and INSERM, UMR 599, Marseille, France. E-mail: mohtym@marseille.fnclcc.fr

BCR-ABL regulates death receptor expression for TNF-related apoptosis-inducing ligand (TRAIL) in Philadelphia chromosome-positive leukemia

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(−) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL.

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.

Specific induction of CD33 expression by E2A–HLF: the first evidence for aberrant myeloid antigen expression in ALL by a fusion transcription factor

Specific induction of CD33 expression by E2A–HLF: the first evidence for aberrant myeloid antigen expression in ALL by a fusion transcription factor

Induction of Impaired Membrane Phospholipid Asymmetry in Mature Erythrocytes after Chemotherapy

Senescent erythrocytes undergo a loss of phospholipid asymmetry in the plasma membrane and are removed from the circulation by phagocytosis. To examine the loss of phospholipid asymmetry in mature erythrocytes after chemotherapy, we monitored phosphatidylserine (PS)-exposing erythrocytes by using flow cytometry to detect annexin V-bound erythrocytes in the circulation of acute lymphoblastic leukemia patients after consolidation chemotherapy. Both the percentage and the absolute number of annexin V-positive erythrocytes gradually increased immediately after chemotherapy. This result paralleled the change in the serum levels of bilirubin, suggesting a direct induction of PS-externalization in mature erythrocytes and a subsequent increase in erythrocyte clearance by splenic macrophages. The PS-exposing erythrocyte counts showed negative correlations to platelet and reticulocyte counts, which reflect the hematopoietic potential in the bone marrow. This result suggests that bone marrow suppression could be responsible for the enlarged senescent population in the circulation. Moreover, PS-exposing erythrocytes in the circulation remained at relatively high levels even after the serum level of bilirubin normalized, indicating that the decreased clearance of senescent erythrocytes as a result of impaired phagocytosis following bone marrow suppression might also be involved in the increase in PS-exposing erythrocytes in the circulation late after chemotherapy.

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 × 109/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.