Yasushi Soda

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.

Dedifferentiation of Neurons and Astrocytes by Oncogenes Can Induce Gliomas in Mice

Cancer Stem Cells: A Moving Target? Glioblastoma multiforme (GBM) is a highly aggressive human brain tumor. The prevailing “cancer stem cell hypothesis” posits that GBMs arise primarily from neuronal stem cells. Now, Friedmann-Morvinski et al. (p. 1080, published online 18 October; see the Perspective by Krivtsov and Armstrong) suggest that this hypothesis may be too restrictive. In mouse models, brain tumors resembling human GBMs could form from fully differentiated cells such as astrocytes and neurons. Upon acquiring certain genetic alterations, mature brain cells may thus be capable of dedifferentiating into a progenitor-like cell, which could then initiate and maintain tumor growth. Murine brain tumors do not necessarily originate from neural stem cells but can arise from mature neurons and astrocytes. 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.

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.

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.

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34+ hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.

Targeting NF-κB in glioblastoma: A therapeutic approach

Inhibition of the transcription factor NF-κB or its target genes should be considered for the treatment of patients with glioblastoma multiforme. 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.

RNAi-mediated silencing of p190Bcr-Abl inactivates Stat5 and cooperates with imatinib mesylate and 17-allylamino-17-demetoxygeldanamycin in selective killing of p190Bcr-Abl-expressing leukemia cells.

The 190u2009kD (p190) and 210u2009kD (p210) Bcr-Abl proteins are responsible for the pathophysiology of Philadelphia chromosome (Ph)+ leukemia. We applied RNA interference (RNAi) to specific killing of p190+ cells, and determined the optimal sequences for gene silencing in the BCR, junctional and ABL regions of p190, respectively. Then, p190+ and p210+ cells were infected with lentiviral vectors encoding these shRNAs, resulting in efficient killing of p190+ cells, while p210+ cells were only sensitive to shBCR and shABL. In p190-transformed Ba/F3 cells, silencing of p190 specifically inhibited tyrosine phospohorylation of Stat5 prior to their death, but did not affect phosphorylation of Jak2, Akt or MEK1/2. In contrast, downregulation of p190 by their treatment with 17-allylamino-17-demetoxygeldanamycin (17-AAG) was associated with reduced protein levels of Jak2, Akt and MEK1/2. shRNA targeting p190 collaborated additively with imatinib and 17-AAG in growth inhibition of Ba/F3-p190wt and imatinib-resistant Ba/F3-p190Y253u2009H cells. Collectively, RNAi-mediated silencing of p190 is a promising option both for delineating signal transduction and for therapeutic application in 190+ leukemia.

Abstract A19: Role of NF-κB and gene targets of NF-κB in glioma cell plasticity

Glioblastoma (GBM) is the most common and lethal form of intracranial tumor. In the last century we have accumulated tremendous amounts of data on this type of cancer, but despite extensive study, few therapeutic targets have been identified for GBM. We have established a lentiviral-induced mouse model of malignant gliomas, which faithfully captures the pathophysiology and molecular signatures of human GBM. RNAseq analysis of these tumors revealed high NFκB activation showing enrichment of known NFκB target genes. Depletion of IKK2 in tumor derived cell lines attenuated tumor proliferation and prolonged mouse survival. We have previously shown that gliomas can originate by reprograming/dedifferentiation of terminally differentiated astrocytes and neurons following oncogenic insult. This tumor cell plasticity seems to be impaired when either cortical astrocytes or neurons derived from floxed-IKK2 mice are infected with a lentivirus expressing HRAS-iresCREerT2-shp53. The addition of tamoxifen to the infected cells induces IKK2 depletion and blocks the reprogramming process, manifested by inhibition of tumorspheres formation and retention of differentiation makers. When these cells are transplanted into mice, the control group (vehicle treated) succumbs to the disease, while induction of IKK2 depletion in the tamoxifen treated group (20 days post-transplantation) significantly prolonged the mice survival. Activation of NFκB requires the activity of IκB kinase (IKK) complex containing IKKα and IKKβ, and the regulatory protein NFκB essential modifier (NEMO). We tested a peptide corresponding to the NEMO-binding domain (NBD) of IKKα(IKK1) or IKKβ(IKK2), to specifically inhibit the induction of NFκB activation, and the mice treated with NBDwt peptide showed long-term survival compared to NBDmut control. We propose targeting the NFκB pathway as an attractive therapeutic strategy to treat GBM. Citation Format: Dinorah Friedmann-Morvinski, Rajesh Narasimamurthy, Yifeng Xia, Chad Myskiw, Yasushi Soda, Inder M. Verma. Role of NF-κB and gene targets of NF-κB in glioma cell plasticity. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A19.

Abstract 524: Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma

Glioblastoma (GBM) is the most common adult brain tumor and represents one of the most treatment refractory cancers. Although significant progress has been made in understanding the coding genomic alterations associated with GBM, patient survival rarely exceeds 12-14 months following diagnosis. Long non-coding RNAs (lncRNAs) are RNA species generally classified as > 200 base-pairs in length that lack protein coding potential and therefore exert their function as RNA. Several lncRNAs have been shown to play important roles in cancer biology. Despite this, the role of lncRNAs in cancer, and GBM in particular, remains relatively uncharacterized. We hypothesize that lncRNAs are differentially expressed in GBM and contribute to the pathogenesis of this disease. Using next generation sequencing, we have identified lncRNAs expressed in our mouse model of GBM, initiated by lentivirus-mediated expression of oncogenic Ras and a shRNA targeting p53. This resulted in the identification of 818 putative lncRNAs, the majority of which represent novel, uncharacterized transcripts. The majority of the lncRNAs identified were intergenic and not associated with any known protein coding gene. Of the total lncRNAs, 44% were differentially expressed in tumor tissue compared to normal mouse brain. Expression of a subset of these differentially expressed lncRNAs was validated by real-time PCR. Expression of several of these validated lncRNAs could be altered by direct activation of oncogenic signaling in normal mouse neuroprogenitor cells and astrocytes. Using a combination of computational and molecular biology approaches, we will identify candidate lncRNAs to test in functional assays for their role in glioblastoma biology. Citation Format: Chad Myskiw, Sabah Kadri, Eugene Ke, Alex Shishkin, Dinorah Friedmann-Morvinski, Yasushi Soda, Mitchell Guttman, Inder Verma. Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2014-524