Naomichi Arima

Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia

FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high ldh level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.

PD-1/PD-L1 expression in human T-cell leukemia virus type 1 carriers and adult T-cell leukemia/lymphoma patients

Adult T-cell leukemia/lymphoma (ATLL) develops after infection with human T-cell leukemia virus-1 (HTLV-1) after a long latency period. The negative regulatory programmed death-1/programmed death-1 ligand 1 (PD-1/PD-L1) pathway has been implicated in the induction of cytotoxic T-lymphocyte (CTL) exhaustion during chronic viral infection along with tumor escape from host immunity. To determine whether the PD-1/PD-L1 pathway could be involved in the establishment of persistent HTLV-1 infections and immune evasion of ATLL cells in patients, we examined PD-1/PD-L1 expression on cells from 27 asymptomatic HTLV-1 carriers (ACs) and 27 ATLL patients in comparison with cells from 18 healthy donors. PD-1 expression on HTLV-1-specific CTLs from ACs and ATLL patients was dramatically elevated. In addition, PD-1 expression was significantly higher on CD8+ T cells along with cytomegalovirus (CMV)- and Epstein–Barr virus (EBV)-specific CTLs in ATLL patients compared with ACs and control individuals. Primary ATLL cells in 21.7% of ATLL patients expressed PD-L1, whereas elevated expression was not observed in cells from ACs. Finally, in functional studies, we observed that an anti-PD-L1 antagonistic antibody upregulated HTLV-1-specific CD8+T-cell response. These observations suggest that the PD-1/PD-L1 pathway plays a role in fostering persistent HTLV-1 infections, which may further ATLL development and facilitate immune evasion by ATLL cells.

Oligomannose‐coated liposomes efficiently induce human T‐cell leukemia virus‐1‐specific cytotoxic T lymphocytes without adjuvant

Human T‐cell leukemia virus‐1 (HTLV‐1) causes adult T‐cell leukemia/lymphoma, which is an aggressive peripheral T‐cell neoplasm. Insufficient T‐cell response to HTLV‐1 is a potential risk factor in adult T‐cell leukemia/lymphoma. Efficient induction of antigen‐specific cytotoxic T lymphocytes is important for immunological suppression of virus‐infected cell proliferation and oncogenesis, but efficient induction of antigen‐specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV‐1‐specific CD8+ T‐cell response by oligomannose‐coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)‐A*0201‐restricted HTLV‐1 Tax‐epitope (OML/Tax). Immunization of HLA‐A*0201 transgenic mice with OML/Tax induced an HTLV‐1‐specific gamma‐interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA‐A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV‐1‐specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV‐1 carriers compared with epitope peptide alone, and these HTLV‐1‐specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen‐specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.

Azacitidine induces demethylation of p16INK4a and inhibits growth in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is one of the peripheral T-cell malignant neoplasms strongly associated with human T-cell leukemia virus type-I (HTLV-I). Although the viral transactivator protein Tax has been proposed to play a critical role in leukemogeneis, additional cellular events are required for the development of ATL. One of the genetic events of the disease is inactivation of tumor suppressor genes. The CDKN2A locus on chromosome 9p encodes 2 cell cycle regulatory proteins, p14ARF and p16INK4a, which share exon 2 using different reading frames. The p14ARF and p16INK4a genes have been implicated as tumor suppressor genes by their frequent mutation, deletion or promoter hypermethylation in a variety of human tumors. In this report, we describe the expression status of p14ARF and p16INK4a in 9 ATL cell lines (MT1, MT2, OKM3T, F6T, K3T, Oh13T, S1T, Su9T01 and HUT102). By reverse transcription polymerase chain reaction (RT-PCR), expression of p14ARF was not detected in one cell line (OKM3T), while expression of p16INK4a was not detected in 6 cell lines (OKM3T, MT1, MT2, Oh13T, S1T and Su9T01). In the OKM3T cell line, the shared exon 2 of the p14ARF/p16INK4a gene was deleted; however, the p16INK4a gene, was epigenetically inactivated in 5 other cells lines. In primary tumor cells obtained from ATL patients, p14ARF expression was absent in 6 of the 11 samples. We confirmed the methylation of the p16INK4a gene in MT1 and MT2 cells using the methylation-specific PCR (MSP) method. Treatment with 2.0 µM of Azacitidine (AZA), a demethylating agent, for 72 h restored p16INK4a transcript expression and induced growth inhibition in MT2 cells. Our results demonstrate that p16INK4a is epigenetically silenced in ATL. AZA offers a potential new therapeutic approach to improve the poor outcomes associated with ATL.

Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with the human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific cytotoxic T lymphocytes (CTLs) play an important role in suppressing proliferation of HTLV-1-infected or transformed T-cells in vitro. Efficient induction of antigen-specific CTLs is important for immunologic suppression of oncogenesis, but has evaded strategies utilizing poorly immunogenic free synthetic peptides. In the present study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by an HTLV-1/hepatitis B virus core (HBc) chimeric particle incorporating the HLA-A*0201-restricted HTLV-1 Tax-epitope. The immunization of HLA-A*0201-transgenic mice with the chimeric particle induced antigen-specific gamma-interferon reaction, whereas immunization with epitope peptide only induced no reaction as assessed by enzyme-linked immunospot assay. Immunization with the chimeric particle also induced HTLV-1-specific CD8+ T-cells in spleen and inguinal lymph nodes. Furthermore, upon exposure of dendritic cells from HLA-A*0201-transgenic mice to the chimeric particle, the expression of CD86, HLA-A02, TLR4 and MHC class II was increased. Additionally, our results show that HTLV-1-specific CD8+ T-cells can be induced by peptide with HTLV-1/HBc particle from ATL patient, but not by peptide only and these HTLV-1-specific CD8+ T-cells were able to lyse cells presenting the peptide. These results suggest that HTLV-1/HBc chimeric particle is capable of inducing strong cellular immune responses without adjuvants via effective maturation of dendritic cells and is potentially useful as an effective carrier for therapeutic vaccines in tumors, or in infectious diseases by substituting the epitope peptide.

HTLV-1 Specific CD8+ T Cell Function Augmented by Blockade of 2B4/CD48 Interaction in HTLV-1 Infection

CD8+ T cell response is important in the response to viral infections; this response though is regulated by inhibitory receptors. Expression of inhibitory receptors has been positively correlated with CD8+ T cell exhaustion; the consequent effect of simultaneous blockade of these inhibitory receptors on CD8+ T cell response in viral infections have been studied, however, the role of individual blockade of receptor-ligand pair is unclear. 2B4/CD48 interaction is involved in CD8+T cell regulation, its signal transducer SAP (signaling lymphocyte activation molecule (SLAM)-associated protein) is required for stimulatory function of 2B4/CD244 on lymphocytes hence, we analyzed 2B4/CD244 (natural killer cell receptor) and SAP (signaling lymphocyte activation molecule(SLAM)-associated protein) on total CD8+ and HTLV-1 specific CD8+T cells in HTLV-1 infection and the effect of blockade of interaction with ligand CD48 on HTLV-1 specific CD8+ T cell function. We observed a high expression of 2B4/CD244 on CD8+ T cells relative to uninfected and further upregulation on HTLV-1 specific CD8+ T cells. 2B4+ CD8+ T cells exhibited more of an effector and terminally differentiated memory phenotype. Blockade of 2B4/CD48 interaction resulted in improvement in function via perforin expression and degranulation as measured by CD107a surface mobilization on HTLV-1 specific CD8+ T cells. In the light of these findings, we thus propose an inhibitory role for 2B4/CD48 interaction on CD8+T cell function.

A second NOTCH1 chromosome rearrangement: t(9;14)(q34.3;q11.2) in T-cell neoplasia.

NOTCH1 encodes a transmembrane signaling protein that plays key roles in development and neoplasia. Its leukemogenic involvement was first revealed by analysis of t(7;9)(q34;q34.3) in a T-cell acute lymphocytic leukemia (T-ALL) cell line (SUP-T1), which juxtaposes truncated NOTCH1 with TCRB, directing overexpression of N-terminally truncated polypeptides (reviewed in Grabher et al). Wild-type NOTCH1 is initially cleaved (S1 cleavage) into twin polypeptides: an extracellular N-terminal subunit (NEC) and a transmembrane C-terminal

Successful Induction of Pluripotent Stem Cells From a Fabry Disease Mouse Model: Toward the Development of Safe Lentiviral Gene Therapy

Induced pluripotent stem (iPS) cells are now recognized as a valuable tool for cell repair through autologous transplantation. These cells are obtainable from somatic cells through the induction of the transcriptional factors Oct- 3/4, Klf4, and Sox2. In this study, we successfully established iPS cells from the tail-tip fibroblasts of a α-galactosidase A-knockout mouse, a well-known Fabry disease mouse model. These Fabry-iPS cells exhibited an embryonic stem (ES) cell phenotype, characterized by the expression of SSEA-1, increased alkaline phosphatase activity, silencing of the retroviral-transgene, and embryoid body (EB) formation. Subcutaneous inoculation of Fabry-iPS cells into nude mice resulted in teratoma formation. At day 6, EBs in differentiation media showed hematopoietic lineage-specific gene expression. In addition, we observed spontaneous contraction of EBs cultured on OP9 stroma cells for 5-7 days. RT-PCR demonstrated that various cardiac marker genes, such as Nkx2.5, Gata4, Tnnt2 (cardiac troponin T), and Mlc2a were more highly expressed in differentiation cultures of Fabry iPS cells than of control feeder cells. To assess their potential use for gene therapy, lentiviral transduction of Fabry-iPS cells with α-galactosidase-A cDNA, the therapeutic gene for Fabry disease, was performed. This transduction resulted in elevated intracellular and secreted α-galactosidase A activity. The ES cell-specific gene expression profile remained unaltered by lentiviral therapeutic gene transfer for more than 30 days post-transduction. These findings demonstrate that Fabry-iPS cells are readily obtainable and amenable for use in gene therapy.

Overexpression of SIRT1 and induction of apoptosis by its inhibition in adult T-cell leukemia cells

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide (NAD +)-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, due to its ability to deacetylate numerous substrates, such as histone and NFB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family, and degradation of SIRT1 in the nucleus. Interestingly, NAD+ augmented sirtinolinduced apoptosis in leukemic cell lines. Thus, the SIRT1 inhibitor acted as a tumor suppressor, where NAD+ accelerated the SIRT1 inhibitor-induced apoptosis. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.