Safety and Effectiveness of Natalizumab: First Report of Interim Results of Post-Marketing Surveillance in Japan.

INTRODUCTION: Natalizumab, a humanized anti-α4 integrin monoclonal antibody, received marketing approval in Japan in 2014 for the treatment of multiple sclerosis (MS). Because the previous large-scale clinical trials of natalizumab were mainly conducted in Europe and North American countries, and data in patients with MS from Japan were limited, we conducted an all-case post-marketing surveillance of natalizumab-treated MS patients from Japan to investigate the safety and effectiveness of natalizumab in a real-world clinical setting in Japan. Here, we report the results of an interim analysis. METHODS: During the observation period of 2 years, all patients who were treated with natalizumab subsequent to its approval in Japan were followed. The effectiveness of natalizumab was assessed by examining the changes in expanded disability status scale (EDSS) score and annualized relapse rate (ARR) from baseline. Safety was assessed by analyzing the incidence of adverse drug reactions (ADRs). RESULTS: The safety analysis included 106 patients (mean age 39.3 years; women 62.3%) whose data were collected until the data lock point (February 7, 2016). The effectiveness analysis included 75 patients. The majority of patients had relapsing-remitting MS (93/106 patients; 87.7%). The mean length of treatment exposure in the present study was 6.6 months. During the 2-year observation period, no significant change in the EDSS was observed, while the ARR decreased significantly from baseline (72.9% reduction, p = 0.001). ADRs and serious ADRs were observed in 11.3% and 3.8% of patients, respectively; however, no new safety concerns were detected. No patient had progressive multifocal leukoencephalopathy (PML) during the present study period. CONCLUSION: The safety and effectiveness of natalizumab were confirmed in Japanese patients with MS in clinical practice. Nevertheless, potential risks including PML require continuous, careful observation. FUNDING: Biogen Japan Ltd (Tokyo, Japan).

Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity.

Disrupted-in-schizophrenia 1 (DISC1) is a susceptibility gene for major psychiatric disorders, including schizophrenia. DISC1 has been implicated in neurodevelopment in relation to scaffolding signal complexes. Here we used proteomic analysis to screen for DISC1 interactors and identified several RNA-binding proteins, such as hematopoietic zinc finger (HZF), that act as components of RNA-transporting granules. HZF participates in the mRNA localization of inositol-1,4,5-trisphosphate receptor type 1 (ITPR1), which plays a key role in synaptic plasticity. DISC1 colocalizes with HZF and ITPR1 mRNA in hippocampal dendrites and directly associates with neuronal mRNAs, including ITPR1 mRNA. The binding potential of DISC1 for ITPR1 mRNA is facilitated by HZF. Studies of Disc1-knockout mice have revealed that DISC1 regulates the dendritic transport of Itpr1 mRNA by directly interacting with its mRNA. The DISC1-mediated mRNA regulation is involved in synaptic plasticity. We show that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity.

Functional analysis of a novel glioma antigen, EFTUD1.

BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas. METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed. RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone. CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.

Downregulation of KIF23 suppresses glioma proliferation.

To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimera-treated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse.

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

Epistatic and functional interactions of catechol-o-methyltransferase (COMT) and AKT1 on neuregulin1-ErbB signaling in cell models.

BACKGROUND: Neuregulin1 (NRG1)-ErbB signaling has been implicated in the pathogenesis of cancer and schizophrenia. We have previously reported that NRG1-stimulated migration of B lymphoblasts is PI3K-AKT1dependent and impaired in patients with schizophrenia and significantly linked to the catechol-o-methyltransferase (COMT) Val108/158Met functional polymorphism. METHODOLOGY/PRINCIPAL FINDINGS: We have now examined AKT1 activation in NRG1-stimulated B lymphoblasts and other cell models and explored a functional relationship between COMT and AKT1. NRG1-induced AKT1 phosphorylation was significantly diminished in Val carriers compared to Met carriers in both normal subjects and in patients. Further, there was a significant epistatic interaction between a putatively functional coding SNP in AKT1 (rs1130233) and COMT Val108/158Met genotype on AKT1 phosphorylation. NRG1 induced translocation of AKT1 to the plasma membrane also was impaired in Val carriers, while PIP(3) levels were not decreased. Interestingly, the level of COMT enzyme activity was inversely correlated with the cells' ability to synthesize phosphatidylserine (PS), a factor that attracts the pleckstrin homology domain (PHD) of AKT1 to the cell membrane. Transfection of SH-SY5Y cells with a COMT Val construct increased COMT activity and significantly decreased PS levels as well as NRG1-induced AKT1 phosphorylation and migration. Administration of S-adenosylmethionine (SAM) rescued all of these deficits. These data suggest that AKT1 function is influenced by COMT enzyme activity through competition with PS synthesis for SAM, which in turn dictates AKT1-dependent cellular responses to NRG1-mediated signaling. CONCLUSION/SIGNIFICANCE: Our findings implicate genetic and functional interactions between COMT and AKT1 and may provide novel insights into pathogenesis of schizophrenia and other ErbB-associated human diseases such as cancer.

Evidence that the BLOC-1 protein dysbindin modulates dopamine D2 receptor internalization and signaling but not D1 internalization.

The schizophrenia susceptibility gene dystrobrevin-binding protein 1 (DTNBP1) encodes dysbindin, which along with its binding partner Muted is an essential component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Dysbindin expression is reduced in schizophrenic brain tissue, but the molecular mechanisms by which this contributes to pathogenesis and symptomatology are unknown. We studied the effects of transfection of DTNBP1 siRNA on cell surface levels of dopamine D2 receptor (DRD2) in human SH-SY5Y neuroblastoma cells and in rat primary cortical neurons. DTNBP1 siRNA decreased dysbindin protein, increased cell surface DRD2 and blocked dopamine-induced DRD2 internalization. MUTED siRNA produced similar effects. In contrast, decreased dysbindin did not change dopamine D1 receptor (DRD1) levels, or its basal or dopamine-induced internalization. The DRD2 agonist quinpirole reduced phosphorylation of CREB (cAMP response element-binding protein) in dysbindin downregulated cells, demonstrating enhanced intracellular signaling caused by the upregulation of DRD2. This is the first demonstration of a schizophrenia susceptibility gene exerting a functional effect on DRD2 signaling, a pathway that has long been implicated in the illness. We propose a molecular mechanism for pathogenesis in which risk alleles in DTNBP1, or other factors that also downregulate dysbindin, compromise the ability of BLOC-1 to traffic DRD2 toward degradation, but has little effect on DRD1 trafficking. Impaired trafficking of DRD2 decreases dopamine-induced internalization, and with more receptors retained on the cell surface, dopamine stimulation produces excess intracellular signaling. Such an increase in DRD2 signaling relative to DRD1 would contribute to the imbalances in dopaminergic neurotransmission characteristic of schizophrenia.

Identification of a glioma antigen, GARC-1, using cytotoxic T lymphocytes induced by HSV cancer vaccine.

Despite several ongoing clinical trials of immunotherapies against glioma, few glioma-specific antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified. We recently demonstrated that intratumoral inoculation with herpes simplex virus (HSV) as a cancer vaccine activates tumor-specific CTLs. To identify glioma antigens recognized by CTLs, we used the HSV cancer vaccine to vaccinate mice harboring a syngeneic mouse glioma cell line, GL261. From the splenocytes of the immunized mice, we generated an H-2Db-restricted CTL line, GCL-1, that was specific for GL261. Then, a cDNA expression library generated from GL261 was screened with GCL-1, and a new gene encoding glioma antigen, GARC-1, was isolated. Sequence analysis revealed that the GARC-1 gene isolated from GL261 had a point mutation causing an amino acid change (Asp to Asn at position 81). T-cell epitope analysis revealed that the mutated peptide GARC-1(77-85) (AALLNKLYA) but not the wild-type peptide (AALLDKLYA), was recognized by GCL-1. These results suggest that HSV cancer vaccination may be a useful method for inducing tumor-specific CTLs and identifying tumor antigens. Furthermore, this GL261/GARC-1 murine glioma model may be useful for the development of immunotherapy for brain tumors.

Augmentation of antitumor immune responses by multiple intratumoral inoculations of replication-conditional HSV and interleukin-12.

Intratumoral inoculation with a herpes simplex virus (HSV) mutant, G207, as an in situ cancer vaccine has been shown to inhibit tumor growth by inducing tumor-specific immune responses. Here, as a step toward the clinical application of this therapeutic approach, we evaluated different protocols for enhancing the antitumor effect. First, in a bilaterally established tumor model with CT26 colon carcinoma, we demonstrated that multiple intratumoral inoculations with G207 induced a greater antitumor effect on both the inoculated and distant tumors than did 1 or 2 inoculations. Second, to boost this antitumor effect, we developed 2 strategies: multiple in situ cancer vaccines with G207 in combination with systemic administration of recombinant interleukin-12 (rIL-12) (G207/systemic rIL-12) or local administration of rIL-12 (G207/local rIL-12). The antitumor effects in both the inoculated and distant tumors by the combined treatments were significantly greater than by either G207 or rIL-12 treatment alone. G207/systemic rIL-12 and G207/local rIL-12 mediated the complete regression of both the inoculated and distant tumors in 67% and 79% of the animals, respectively. These results indicate that multiple intratumoral inoculations of G207 and systemic or local rIL-12 administration work synergistically to facilitate tumor regression and that this combination of treatments may have potential for treating cancer metastasis.

Identification of a human glioma antigen, SOX6, recognized by patients' sera.

To identify tumor antigens for glioma, a human testis cDNA library was screened by serological identification of antigens by recombinant expression cloning with sera from glioma patients. In this screening, the most frequently isolated antigen was SOX6, an Sry-related high-mobility group (HMG) box-containing gene. SOX6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. IgG antibodies against SOX6 were detected in sera from 12 of 36 glioma patients (33.3%), 0 of 14 patients with other brain disease (0%), and one of 54 other cancer patients (1.9%). In sera from 37 healthy individuals, no IgG responses against SOX6 were detected, except in an elderly female. Furthermore, Western blot and ELISA analyses with sera from glioma patients revealed that the DNA-binding domain, the HMG box of SOX6, might be a dominant epitope of IgGs against SOX6. RT-PCR and Northern blot analysis revealed that the SOX6 gene was more highly expressed in glioma tissues than in normal adult tissues, except testis. Western blot analysis with an anti-SOX6 antibody demonstrated that the SOX6 protein was expressed in glioma tissues, but not in normal adult brain tissue. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analysed expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in non-neoplastic tissues from the cerebral cortex. In summary, these results indicate that the developmentally regulated transcription factor SOX6 is aberrantly expressed in glioma and specifically recognized by IgGs from glioma patients' sera.

Immuno-viral therapy of brain tumors by combination of viral therapy with cancer vaccination using a replication-conditional HSV.

Here we developed an effective therapeutic approach using a replication-conditional mutant of herpes simplex virus (HSV), G207, for the treatment of metastatic tumors in the immunologically privileged central nervous system. An experimental model of brain metastasis was developed using BALB/c mice that harbored both intracranial (i.c.) and subcutaneous (s.c.) mouse CT26 colon adenocarcinoma tumors. Intratumoral injections of G207 into s.c. tumors elicited cytotoxic T-cell responses not only to HSV but also to a tumor antigen; however, only a limited antitumor effect was observed on metastatic brain tumors. To improve this antitumor effect, G207 was also injected into the brain tumor. After intratumoral injections of G207 into both i.c. and s.c. CT26 tumors, a significant antitumor effect was observed in the metastatic brain tumors. This therapeutic efficacy was absent in athymic mice, indicating that the antitumor effect could be mediated by T cells. Cytotoxic T-cell responses to HSV and the tumor antigen were induced by injections of G207 into i.c. and s.c. CT26 tumors. These results suggest that HSV-infected brain tumors may be efficiently eliminated by the induced anti-HSV T cells as well as by antitumor T cells. Therefore, this strategy of immuno-viral therapy, involving direct viral oncolytic activities and inducing antitumor and antiviral immune responses, may be useful for the treatment of tumors in the immunologically privileged central nervous system.

In situ cancer vaccination with a replication-conditional HSV for the treatment of liver metastasis of colon cancer.

In this study, we investigated the therapeutic efficacy of a replication-conditional mutant HSV, G207, for the treatment of liver metastasis of colon carcinoma. Three liver metastasis models in syngeneic BALB/c mice were developed: (i) splenic injection, (ii) splenic and subcutaneous (s.c.) injection, and (iii) orthotopic implantation of CT26 colon carcinoma. In the splenic injection model, G207 was injected into the established splenic tumor on day 7. In the splenic and s.c. injection model, G207 were injected into the established s.c. tumor on days 5 and 8. In the orthotopic implantation model, a piece of CT26 tumor tissue was transplanted onto the wall of the cecum and G207 was injected in the established cecum tumor on day 7. On day 21 or 28, animals were sacrificed and liver metastases were evaluated. In all three models in immunocompetent mice, liver metastases were significantly reduced by intratumoral inoculation with G207 compared to the control. In athymic mice, however, there was no significant therapeutic effect of intratumoral inoculation with G207 on liver metastases. Tumor-specific cytotoxic T-lymphocyte responses were induced in mice treated with G207 in the orthotopic implantation model. These results suggest that intratumoral inoculation of G207, as an in situ cancer vaccine, can be an effective approach against liver metastasis of colon cancer and the efficacy involves tumor-specific T-cell responses.