Evi-1 is a transcriptional target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells.

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells.

Evi1 represses PTEN expression and activates PI3K/AKT/mTOR via interactions with polycomb proteins.

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly, high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However, mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here, we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow, which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model, Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore, we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation, which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1.

AML1/RUNX1 functions as a cytoplasmic attenuator of NF-κB signaling in the repression of myeloid tumors.

Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

A Phase 1 Study of KHK4083: A Single-Blind, Randomized, Placebo-Controlled Single-Ascending-Dose Study in Healthy Adults and an Open-Label Multiple-Dose Study in Patients With Ulcerative Colitis.

OX40 plays an essential role in maintaining late T-cell proliferation and survival by suppressing apoptosis and by inducing T-cell memory formation. Here, we report the results of the phase 1 study of KHK4083, a fully human antimonoclonal antibody specific for OX40. In this study, we aimed to assess the safety and tolerability of a single intravenous or subcutaneous administration of KHK4083 compared with placebo in healthy Japanese and Caucasian subjects and determined the pharmacokinetics (PK) and immunogenicity. Also, we assessed the preliminary efficacy and pharmacodynamics of multiple intravenous doses in Japanese patients with moderate to severe ulcerative colitis (UC). Drug-related treatment emergent adverse events occurred in 21 healthy subjects (58.3%) and 5 patients with UC (62.5%) after administration of KHK4083. There were no serious adverse events. The PK profile of a single intravenous dose of 10 mg/kg KHK4083 was similar in healthy Japanese and Caucasian subjects. Of 8 UC patients, a clinical response was observed in 3 patients (37.5%) and clinical remission in 2 patients (25.0%) in week 6. Our study demonstrated the safety and tolerability of single and multiple administrations of KHK4083 in healthy Japanese and Caucasian subjects and Japanese patients with moderate to severe UC. It also indicated favorable pharmacological properties of the drug.

Safety, tolerability and efficacy of repeated intravenous infusions of KHK4083, a fully human anti-OX40 monoclonal antibody, in Japanese patients with moderate to severe atopic dermatitis.

BACKGROUND: KHK4083, a fully human anti-OX40 monoclonal antibody, is a potential novel therapeutic option for moderate to severe atopic dermatitis (AD), targeting the immunopathogenic pathways. OBJECTIVE: Assess the safety and tolerability of repeated doses of KHK4083 in patients with moderate to severe AD, and investigate the pharmacokinetics and immunogenicity of KHK4083. Additionally, assess the clinical efficacy and pharmacodynamics as exploratory objectives. METHODS: In this phase 1, single-center, open-label, repeated-dose study, a total of 22 patients received KHK4083 10 mg/kg IV on Day 1, Day 15 and Day 29, and were followed until Day 155. RESULTS: There were no deaths, serious adverse events (SAEs), or discontinuations due to adverse events (AEs). Common treatment-emergent AEs were mild or moderate pyrexia (11 patients, 50.0 %), and chills (8 patients, 36.4 %). No clinically meaningful changes in the laboratory values, vital signs, and electrocardiogram recordings were observed. The Cmax was 267 ± 53 µg/mL and the t1/2 was 303 ± 88 h at Day 29. The overall assessment of antibodies against KHK4083 (immunogenicity) showed low positive responses. Continued improvement in the Eczema Area and Severity Index (EASI) and Investigator's Global Assessment (IGA) scores were observed throughout the study. The mean and median percent changes in thymus and activation-regulated chemokine (TARC) continued to decrease over time to -70.4 and -78.8 % until Day 155. CONCLUSION: Repeated intravenous infusion of KHK4083 had an acceptable safety profile in patients with moderate to severe AD. Sustained improvement in the symptoms of AD was observed after completion of KHK4083 treatment.

The novel CA IX inhibition antibody chKM4927 shows anti-tumor efficacy in vivo.

Carbonic anhydrase IX (CA IX) is an attractive target for cancer therapy. Many anti-CA IX antibodies have been reported but few have been shown to possess inhibition activity. Furthermore, effective use of CA IX-inhibition antibodies for cancer immunotherapy has not been well-validated since data are mainly limited to in vitro assays. In this study, we established that chKM4927, an anti-CA IX chimeric antibody, recognizes CA IX and has CA IX-specific inhibition activity. ChKM4927 also retains antibody-dependent cellular cytotoxicity (ADCC) activity against CA IX-expressing cancer cells. Compared to controls, chKM4927 treatment (10 mg/kg) showed anti-tumor activity in the VMRC-RCW xenograft model in vivo. ChKM4927-attenuated ADCC activity showed equally effective anti-tumor activity. These results suggest that the CA IX-inhibition antibody chKM4927 has an anti-tumor effect in the VMRC-RCW xenograft model via an ADCC-independent mechanism.

Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.

Ecotropic viral integration site 1 (Evi1), a transcription factor of the SET/PR domain protein family, is essential for the maintenance of hematopoietic stem cells (HSCs) in mice and is overexpressed in several myeloid malignancies. Here, we generate reporter mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus. Using these mice, we find that Evi1 is predominantly expressed in long-term HSCs (LT-HSCs) in adult bone marrow, and in the hematopoietic stem/progenitor fraction in the aorta-gonad-mesonephros, placenta, and fetal liver of embryos. In both fetal and adult hematopoietic systems, Evi1 expression marks cells with long-term multilineage repopulating activity. When combined with conventional HSC surface markers, sorting according to Evi1 expression markedly enhances purification of cells with HSC activity. Evi1 heterozygosity leads to marked impairment of the self-renewal capacity of LT-HSCs, whereas overexpression of Evi1 suppresses differentiation and boosts self-renewal activity. Reintroduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity. Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells.

Evi-1 has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Here, we show that hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are severely reduced in number with defective proliferative and repopulating capacity. Selective ablation of Evi-1 in Tie2(+) cells mimics Evi-1 deficiency, suggesting that Evi-1 function is required in Tie2(+) hematopoietic stem/progenitors. Conditional deletion of Evi-1 in the adult hematopoietic system revealed that Evi-1-deficient bone marrow HSCs cannot maintain hematopoiesis and lose their repopulating ability. In contrast, Evi-1 is dispensable for blood cell lineage commitment. Evi-1(+/-) mice exhibit the intermediate phenotype for HSC activity, suggesting a gene dosage requirement for Evi-1. We further demonstrate that disruption of Evi-1 in transformed leukemic cells leads to significant loss of their proliferative activity both in vitro and in vivo. Thus, Evi-1 is a common and critical regulator essential for proliferation of embryonic/adult HSCs and transformed leukemic cells.

Switching constant domains enhances agonist activities of antibodies to a thrombopoietin receptor.

We enhanced the activities of two agonist antibodies specific for the thrombopoietin receptor (c-MPL) by switching domains within their constant regions to those of different antibody isotypes. Our results suggest the importance of the hinge region in modulating agonist activity. The antibodies' thrombopoietin-like activity in vitro and in vivo, as well as the desirable pharmacokinetic profile conferred by retaining the whole-IgG structure, suggests that they provide a valuable option for treating thrombocytopenia.

Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries.

Immune cells, which constitute a significant cell mass in the corpora lutea (CLs), are considered to play critical roles in luteolysis, but the details are not fully understood. We histochemically investigated the changes in distribution and cell density of macrophages and T lymphocytes and in tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma, which can induce apoptosis in the luteal cells in murine CLs during luteal regression. No macrophages or T lymphocytes were observed in functionally healthy CLs. Abundant macrophages and increasing T lymphocytes were demonstrated in CLs at the functional regression stage (early stage of regression). At the structural regression stage (late stage of regression), abundant T lymphocytes but no macrophages were demonstrated in the CLs. A moderate amount of TNF-alpha was detected in all CLs at all stages. No IFN-gamma was detected in either healthy or early regressing CLs, but a large amount of IFN-gamma was detected at the late regression stage. Moreover, in cultured luteal cells, reactivity against Fas-ligand (FasL) was caused by pretreatment with TNF-alpha and IFN-gamma and apoptosis was induced by FasL treatment. These findings support the hypothesis that macrophages initiate T lymphocyte aggregation at the early stage of luteal regression, and then T lymphocytes induce apoptosis on luteal cells, which in turn develop sensitivity against FasL by TNF-alpha and IFN-gamma.

Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries.

During luteolysis, luteal cell apoptosis is induced by the Fas ligand (FasL)/Fas system. In murine luteal bodies, we demonstrated the expression of mRNA of soluble form of Fas (FasB), which binds to FasL and prevents apoptosis induction. By in situ hybridization, strong expression of FasB mRNA was observed in normal luteal bodies, in which no apoptotic cells were detected, but negative/trace expression in regressing luteal bodies, in which many apoptotic cells were observed. Immunohistochemical staining revealed that Fas and TNF-alpha were localized in both normal and regressing luteal bodies, but IFN-gamma was localized only in regressing luteal bodies. Apoptosis was induced in primary cultured luteal cells, when they were pretreated with TNF-alpha and IFN-gamma and then incubated with TNF-alpha, IFN-gamma, and mouse recombinant FasL (rFasL). However, no apoptosis was detected in the cells, when they were treated with rFasL alone, TNF-alpha alone, IFN-gamma alone, TNF-alpha and rFasL, IFN-gamma and rFasL, or TNF-alpha and IFN-gamma. Fas mRNA expression in cultured luteal cells was up-regulated by the treatment of TNF-alpha, IFN-gamma, or TNF-alpha and IFN-gamma. The expression of FasB mRNA was down-regulated, when the cells were treated with TNF-alpha and IFN-gamma, but its expression was not changed by the treatment of TNF-alpha alone or IFN-gamma alone. We conclude that FasB inhibits the apoptosis induction in luteal cells of normal luteal bodies, and that decreased FasB production induced by TNF-alpha and IFN-gamma made possible the apoptosis induction in the luteal cells of regressing luteal bodies.

Abnormal structural luteolysis in ovaries of the senescence accelerated mouse (SAM): expression of Fas ligand/Fas-mediated apoptosis signaling molecules in luteal cells.

Senescence accelerated mouse-prone (SAMP) mice with a shortened life span show accelerated changes in many of the signs of aging and a shorter reproductive life span than SAM-resistant (SAMR) controls. We previously showed that functional regression (progesterone dissimilation) occurs in abnormally accumulated luteal bodies (aaLBs) of SAMP mice, but structural regression of luteal cells in aaLB is inhibited. A deficiency of luteal cell apoptosis causes the abnormal accumulation of LBs in SAMP ovaries. In the present study, to show the abnormality of Fas ligand (FasL)/Fas-mediated apoptosis signal transducing factors in the aaLBs of the SAMP ovaries, we assessed the changes in the expression of FasL, Fas, caspase-8 and caspase-3 mRNAs by reverse transcription-polymerase chain reaction, and in the expression and localization of FasL, Fas and activated caspase-3 proteins by Western blotting and immunohistochemistry, respectively, during the estrus cycle/luteolysis. These mRNAs and proteins were expressed in normal LBs of both SAMP and SAMR ovaries, but not at all or only in trace amounts in aaLBs of SAMP, indicating that structural regression is inhibited by blockage of the expression of these transducing factors in luteal cells of aaLBs in SAMP mice.