Shuki Mizutani

Outcome of risk-based therapy for infant acute lymphoblastic leukemia with or without an MLL gene rearrangement, with emphasis on late effects: a final report of two consecutive studies, MLL96 and MLL98, of the Japan Infant Leukemia Study Group

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0–60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs) – severe combined immunodeficiency (SCID, n=11), Wiskott–Aldrich syndrome (WAS, n=11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n=8) – who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n=8). Since 1996, the donors have been HLA-matched related donors (MRD) (n=8), unrelated BM (UR-BM) (n=7) and unrelated cord blood (UR-CB) (n=7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Ataxia-telangiectasia-mutated dependent phosphorylation of Artemis in response to DNA damage.

Artemis plays a crucial role in the hairpin‐opening step of antigen receptor VDJ gene recombination in the presence of catalytic subunit of deoxyribonucleic acid (DNA)‐dependent protein kinase (DNA‐PKcs). A defect in Artemis causes human radiosensitive‐severe combined immunodeficiency. Cells from Artemis‐deficient patients and mice display increased chromosomal instability, but the precise function of this factor in the response to DNA damage remains to be elucidate. In this study, we show that Artemis is hyperphosphorylated in an Ataxia‐telangiectasia‐mutated (ATM)‐ and Nijmegen breakage syndrome 1 (Nbs1)‐dependent manner in response to ionizing radiation (IR), and that S645 is an SQ/TQ site that contributes to retarded mobility of Artemis upon IR. The hyperphosphorylation of Artemis is markedly reduced in ATM‐ and Nbs1‐null cells. Reintroduction of wild‐type ATM or Nbs1 reconstituted Artemis hyperphosphorylation in ATM‐ or Nbs1‐deficient cells, respectively. In support of this functional link, hyperphosphorylated Artemis was found to physically associate with the Mre11/Rad50/Nbs1 complex in an ATM‐dependent manner in response to IR‐induced DNA double strand breaks (DSB). Since deficiency of either DNA‐Pkcs or ATM leads to defective repair of IR‐induced DSB, our finding places Artemis at the signaling crossroads downstream of DNA‐PKcs and ATM in IR‐induced DSB repair. (Cancer Sci 2005; 96: 134–141)

Brap2 Functions as a Cytoplasmic Retention Protein for p21 during Monocyte Differentiation

ABSTRACT The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.

Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations

Topoisomerase II (Topo II) inhibitors are cell cycle-specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated-deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor-induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control.

Novel adopted immunotherapy for mixed chimerism after unrelated cord blood transplantation in Omenn syndrome.

Abstract:  Omenn syndrome is a variant form of severe combined immunodeficiency. It is fatal unless treated by allogeneic stem cell transplantation (SCT), which is the only curative approach. However, both treatment‐related complications and graft rejection are major obstacles to treatment success. This report describes a case with Omenn syndrome who developed mixed chimerism after unrelated cord blood transplantation (UCBT). This case was successfully treated by altering the patients immunosuppression and donor lymphocyte infusion (DLI) with donor cord blood‐derived activated CD4+ T cells ex vivo expanded from the cord blood cell residues in an infused bag. This novel development to expand CD4+ T‐lymphocytes from the donor cord blood unit for the use of DLI would serve as a useful method to overcome the risk of graft rejection in SCT for primary immunodeficiency disorders with residual cell‐mediated immunity without compounding graft‐vs.‐host disease, especially in the UCBT setting.

DNA damage-induced cell-cycle phase regulation of p53 and p21waf1 in normal and ATM-defective cells.

The ATM-dependent accumulation of p53 and induction of p21waf1 are key events for G1 cell-cycle checkpoint arrest following DNA damage. In ATM-null AT cells, even though the p53 and p21waf1 responses are kinetically delayed and quantitatively reduced, the G1 checkpoint is virtually disrupted, suggesting that these proteins arrive too late in G1 to enforce the arrest. As the precise mechanism remains unclear, we examined the response to DNA double-strand breaks generated by γ-radiation (IR), to determine if ATM deficiency affects the cell-cycle phase regulation of these molecules. We find that, after irradiation, whereas normal LCL-N cells markedly increase their levels of p53 in all phases of the cell cycle, AT cells fail to show any p53 increase in the G1 phase. In addition, whereas in LCL-N p21waf1 is induced in G1 and G2-M, in AT cells this induction is partly seen in G2-M, but not in G1, indicating a different cell-cycle phase regulation of p53 and p21waf1 as a result of ATM deficiency. The levels and catalytic activity of the p53-targeting kinases ATR and DNA-PK in LCL-N and AT cells are very similar throughout the cell cycle, both before and after IR, thus excluding a phase-specific activity for these kinases. Collectively, our findings demonstrate that, in ATM-deficient cells, the p53-dependent p21waf1 response to DNA damage is not only quantitatively reduced, but also specifically suppressed in the G1 phase, thus providing a mechanistic explanation for the severe disruption of the G1 checkpoint in AT cells.

Common gene expression signatures in t(8;21)- and inv(16)-acute myeloid leukaemia

Human acute myeloid leukaemia (AML) involving a core‐binding factor (CBF) transcription factor is called CBF leukaemia. In these leukaemias, AML1 (RUNX1, PEBP2αB, CBFα2)‐MTG8 (ETO) and CBFβ (PEBP2β)‐MYH11 chimaeric proteins are generated by t(8;21) and inv(16) respectively. We analysed gene expression profiles of leukaemic cells by microarray, and selected genes whose expression appeared to be modulated in association with t(8;21) and inv(16). In a pair‐wise comparison, 15% of t(8;21)‐associated transcripts exhibited high or low expression in inv(16)‐AML, and 26% of inv(16)‐associated transcripts did so equivalently in t(8;21)‐AML. These common elements in gene expression profiles between t(8;21)‐ and inv(16)‐AML probably reflect the situation that AML1‐MTG8 and CBFβ‐MYH11 chimaeric proteins affect a common set of target genes in CBF leukaemic cells. On the other hand, 38% of t(8;21)‐associated and 24% of inv(16)‐associated transcripts were regulated in t(8;21)‐ and inv(16)‐specific manners. These distinct features of t(8;21)‐ and inv(16)‐associated genes correlate with the bimodular structures of the chimaeric proteins (CBF‐related AML1 and CBFβ portions, and CBF‐unrelated MTG8 and MYH11 portions).

Analysis of serum soluble CD40 ligand (sCD40L) in the patients undergoing allogeneic stem cell transplantation: platelet is a major source of serum sCD40L.

Abstract:  CD40 ligand (CD40L) is expressed not only on activated T cells but also on activated platelets. A soluble CD40 ligand (sCD40L) is released from the activated T cells and platelets by ill‐defined proteolytic process in vitro. It has been reported that sCD40L is elevated in the serum of patients with systemic lupus erythematosus, unstable angina, essential thrombocythemia, and autoimmune thrombocytopenic purupura. However, source of sCD40L in vivo remains to be elucidated. We investigated the serial sCD40L in the serum in patients undergoing allogeneic stem cell transplantation and compared with the platelets number and soluble IL2R, which is a marker of activated T cells. The value of sCD40L was well correlated with platelet number or thrombopoiesis. In cases of severe graft vs. host disease with markedly increased sIL2R, sCD40L was not increased in vivo. These results indicate that sCD40L in vivo is released mainly from the platelets or in the process of platelet production but not from the activated T cells.

Knockdown of XAB2 Enhances All-Trans Retinoic Acid-Induced Cellular Differentiation in All-Trans Retinoic Acid-Sensitive and -Resistant Cancer Cells

Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.