Coxsackievirus A11 is an immunostimulatory oncolytic virus that induces complete tumor regression in a human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Innovative treatment is required to improve overall survival rates for advanced NSCLC. Oncolytic virotherapy using enteroviruses has emerged as a promising anticancer strategy. To identify a novel, potent virotherapy with an improved safety profile, we assessed the oncolytic activity of 28 enteroviral strains and focused on coxsackievirus A11 (CVA11). CVA11 infection caused extensive oncolytic activity in all three of the examined human NSCLC cell lines, with high intercellular adhesion molecule-1 (ICAM-1) expression associated with greater CVA11-induced cytotoxicity. In vitro inhibition analysis using a pan-caspase inhibitor and western blot detection of cleaved poly (ADP-ribose) polymerase (PARP) indicated that apoptosis partly contributed to CVA11-driven cytotoxicity. CVA11 infection-induced immunogenic cell death in vitro was strongly suggested by substantial calreticulin expression and release of high mobility group box-1 protein (HMGB1). Moreover, in vivo treatment of human NSCLC xenografts with intratumoral CVA11 injection caused complete tumor regression in all treated mice, without significant weight loss. Our findings indicate that novel oncolytic virotherapy utilizing CVA11 may be less toxic and more effective than current treatments for human NSCLC, thus warranting further investigation in clinical trial settings, especially in combination with immunotherapy.

Development of New Oncolytic Virotherapy Targeting Breast Cancer Using Coxsackievirus B3.

BACKGROUND/AIM: Breast cancer is the most common cancer in women worldwide, and triple-negative breast cancer (TNBC) is highly refractory to current standard therapies. Oncolytic virotherapy has recently gathered attention as a new treatment candidate for refractory cancers. MATERIALS AND METHODS: We previously developed a new Coxsackievirus B3 (CVB3) virotherapy targeting lung cancers, and demonstrated that miRNA target sequence insertion into CVB3 reduced its pathogenicity, retaining its original oncolytic activity. In this study, we examined the oncolytic effects of CVB3 against breast cancer cells including TNBC cells. RESULTS: CVB3 infection killed breast cancer cells in a time- and titer-dependent manner, and induced apoptosis. Nude mice transplanted with human TNBC cells were successfully treated with both CVB3-WT and CVB3-HP. Importantly, mice treated with CVB3-HP showed very few adverse events. CONCLUSION: CVB3-HP is a strong oncolytic virus candidate for breast cancer, including TNBC, due to its remarkable oncolytic efficacy and improved safety profile.

A pilot study to establish human T-cell leukemia virus type 1 (HTLV-1) carrier model using common marmoset (Callithrix jacchus).

BACKGROUND: For the diagnosis and treatment of adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) are required therapeutic modalities urgently. Non-human primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non-human primate model using common marmosets (Callithrix jacchus). METHODS: We inoculated HTLV-1-producing MT-2 cells into 9-month-old marmosets, either intraperitoneally or intravenously. We next administrated MT-2 cells into 13-month-old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV-1 infection was determined by measuring HTLV-1 antibody titer in the common marmosets. RESULTS: The HTLV-1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study. CONCLUSION: We obtained HTLV-1 asymptomatic carriers of common marmosets by inoculating MT-2 cells.

Non-transmissible MV Vector with Segmented RNA Genome Establishes Different Types of iPSCs from Hematopoietic Cells.

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.

Extremely Low Organ Toxicity and Strong Antitumor Activity of miR-34-Regulated Oncolytic Coxsackievirus B3.

Oncolytic virotherapies have emerged as new modalities for cancer treatment. We previously reported that coxsackievirus B3 (CVB3) is a novel oncolytic virus (OV) with a strong ability to lyse human non-small cell lung cancer cells; however, its non-specific toxicity against normal cells remains to be resolved. To improve its safety profile, microRNA target sequences complementary to miR-34a/c, which is expressed preferentially in normal cells, were inserted into the 5' UTR or 3' UTR of the CVB3 genome. In the presence of miR-34a/c, the gene-modified CVB3 could not replicate in normal cells. We also found that the pathogenicity of CVB3 was reduced to a greater extent by targeting miR-34a than miR-34c; in addition, it was more effective to insert the target sequences into the 3' UTR rather than the 5' UTR of the viral genome. Ultimately, we developed a double-miR-34a targeting virus (53a-CVB) by inserting miR-34a targets in both the 5' UTR and 3' UTR of the virus. 53a-CVB was minimally toxic to cells in normal tissue, but maintained nearly its full oncolytic activity in mice xenografted with human lung cancer. 53a-CVB is the first miR-34-regulated OV and represents a promising platform for the development of safe and effective anti-cancer therapies.

BRCA1/BARD1-dependent ubiquitination of NF2 regulates Hippo-YAP1 signaling.

Coordination of growth and genomic stability is critical for normal cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player in maintenance of genomic stability, its role in growth signaling remains elusive. Here, we show that BRCA1 facilitates stabilization of YAP1 protein and turning "off" the Hippo pathway through ubiquitination of NF2. In BRCA1-deficient cells Hippo pathway is "turned On." Phosphorylation of YAP1 is crucial for this signaling process because a YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 kinase recognition sites not only resists degradation but also rescues YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, an ectopic expression of the active Mt-YAP5SA, but not inactive Mt-YAP6SA, promotes EGF-independent proliferation and tumorigenesis in BRCA1-/- mammary epithelial cells. These findings establish an important role of BRCA1 in regulating stability of YAP1 protein that correlates positively with cell proliferation.

Targeting NF-κB in glioblastoma: A therapeutic approach.

Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas, which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2), expression of a IκBαM super repressor, or using a NEMO (NF-κB essential modifier)-binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1, one of the NF-κB target genes significantly up-regulated in GBM, was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM.

A versatile drug delivery system using streptavidin-tagged pegylated liposomes and biotinylated biomaterials.

Here we have developed a versatile liposome-mediated drug delivery system (DDS) allowing a strong bridge between the streptavidin-tagged liposome (SAL) and biotin (Bi)-tagged biomaterials which has strong affinity to surface proteins expressed in restricted cell lineages. This DDS was effective and specific for many leukemia cells in vitro and in vivo. When examining 6 human leukemia cell lines using calcein-encapsulated SALs in combination with Bi-granulocyte colony-stimulating factor (G-CSF), Bi-anti-CD33 monoclonal antibody (MAb) or Bi-anti-CD7 MAb, the fluorescent positive rate of each cell line was in almost proportion to degree of G-CSF receptor, CD33 or CD7 expression, respectively. More importantly, the binding ability was shown to be well maintained in a mouse xenograft model. Furthermore the cytosine arabinoside (AraC)-encapsulated SALs could kill the corresponding cells much more effectively in combination with Bi-biomaterials than free AraC, as expected. These findings strongly indicate that our SAL/Bi-biomaterial system could allow various types of medical agents to be delivered reliably and stably to the cells targeted.

Mechanisms of neovascularization and resistance to anti-angiogenic therapies in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and highly resistant to intensive combination therapies. GBM is one of the most vascularized tumors and vascular endothelial growth factor (VEGF) produced by tumor cells is a major factor regulating angiogenesis. Successful results of preclinical studies of anti-angiogenic therapies using xenograft mouse models of human GBM cell lines encouraged clinical studies of anti-angiogenic drugs, such as bevacizumab (Avastin), an anti-VEGF antibody. However, these clinical studies have shown that most patients become resistant to anti-VEGF therapy after an initial response. Recent studies have revealed some resistance mechanisms against anti-VEGF therapies involved in several types of cancer. In this review, we address mechanisms of angiogenesis, including unique features in GBMs, and resistance to anti-VEGF therapies frequently observed in GBM. Enhanced invasiveness is one such resistance mechanism and recent works report the contribution of activated MET signaling induced by inhibition of VEGF signaling. On the other hand, tumor cell-originated neovascularization including tumor-derived endothelial cell-induced angiogenesis and vasculogenic mimicry has been suggested to be involved in the resistance to anti-VEGF therapy. Therefore, these mechanisms should be targeted in addition to anti-angiogenic therapies to achieve better results for patients with GBM.

Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Transdifferentiation of glioblastoma cells into vascular endothelial cells.

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53(+/-) heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP(+) vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

Development of a novel mouse glioma model using lentiviral vectors.

We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP-controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein-positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme-like tumors, which contained CD133(+) cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP-controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.

Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells.

We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT+Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate.

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals.

PURPOSE: The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. METHODS: Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. RESULTS: The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. CONCLUSION: Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity.

Unrelated cord blood transplantation after myeloablative conditioning for adult patients with refractory anemia.

We report the results of unrelated cord blood transplantation (CBT) after myeloablative conditioning in 3 patients with myelodysplastic syndrome-refractory anemia (MDS-RA). All patients were treated with total body irradiation, cytosine arabinoside (Ara-C), and cyclophosphamide, followed by unrelated HLA-mismatched CBT. Granulocyte colony-stimulating factor was infused continuously, starting 12 hours before Ara-C therapy and continuing until the end of Ara-C therapy. All patients received standard cyclosporine and methotrexate therapy as graft-versus-host disease prophylaxis. All patients had myeloid reconstitution, and the times to reach an absolute neutrophil count >0.5 x 10(9)/L were 23, 20, and 26 days. All patients showed full donor chimerism at the time of the first bone marrow examination (on day +42, +43, and +62) after CBT. All patients are alive and free of disease at between 17 and 39 months after CBT. These results suggest that adult MDS-RA patients without suitable related or unrelated bone marrow donors should be considered as candidates for CBT.

The synthetic peptide derived from the NH2-terminal extracellular region of an orphan G protein-coupled receptor, GPR1, preferentially inhibits infection of X4 HIV-1.

Several G protein-coupled receptors (GPCRs) serve as co-receptors for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. Here we report that a synthetic peptide derived from the NH2-terminal extracellular region of an orphan GPCR, GPR1 (GPR1ntP-(1-27); MEDLEETLFEEFENYSYDLDYYSLESC), inhibited infection of not only an HIV-1 variant that uses GPR1 as a co-receptor, but also X4, R5, and R5X4 viruses. Among these HIV-1 strains tested, viruses that can utilize CXCR4 as their co-receptors were preferentially inhibited. Inhibition of early steps in X4 virus replication was also detected in the primary human peripheral blood lymphocytes. GPR1ntP-(1-27) directly interacted with recombinant X4 envelope glycoprotein (rgp120). This interaction was neither inhibited nor enhanced by the soluble CD4 (sCD4) but inhibited by the anti-third variable (V3) loop-specific monoclonal antibody and heparin known to bind to the V3 loop. Although the conformational changes in gp120, including the V3 loop, have been reported to be required for its interaction with a co-receptor after binding of gp120 to CD4, it has also been reported that the V3 loop is already exposed on the surface of virions before interaction with CD4. We found that GPR1ntP-(1-27) blocked binding of virus to the cells, and this peptide equally bound to rgp120 in the presence or absence of sCD4. Because we detected the binding of GPR1ntP-(1-27) to the highly purified virions even in the absence of sCD4, GPR1ntP-(1-27) probably recognized the V3 loop exposed on the virions, and this interaction was responsible for the anti-HIV-1 activity of GPR1ntP-(1-27).

Atypical hypersensitivity to mosquito bites without natural killer cell proliferative disease in an adult patient.

Hypersensitivity to mosquito bites (HMB) is a rare disorder that occurs in the first 2 decades of life and is considered to be associated with chronic Epstein-Barr virus (EBV) infection and natural killer (NK) cell leukemia/lymphoma. EBV-encoded small nuclear RNA (EBER)-positive NK cells infiltrate the skin lesion at the site of the mosquito bite. In this report, we present the case of an adult patient with mantle cell lymphoma complicated by atypical HMB. The anti-EBV antibody titer of the patient indicated reactivation of chronic infection with this virus, and EBV DNA in the peripheral blood mononuclear cells was detected after chemotherapy by quantitative polymerase chain reaction analysis. However, an in situ hybridization analysis did not detect EBER-positive cells in the skin lesion at the bite site or in the lymph node. Peripheral NK cell lymphocytosis and EBV-associated lymphoproliferative disease did not develop. These findings suggest that some patients with chronic EBV infection may develop HMB without NK cell proliferative disease.