Dinorah Friedmann-Morvinski

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.

Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma

Antiangiogenic therapy can produce transient tumor regression in glioblastoma (GBM), but no prolongation in patient survival has been achieved. We have constructed a nanosystem targeted to tumor vasculature that incorporates three elements: (i) a tumor-homing peptide that specifically delivers its payload to the mitochondria of tumor endothelial cells and tumor cells, (ii) conjugation of this homing peptide with a proapoptotic peptide that acts on mitochondria, and (iii) multivalent presentation on iron oxide nanoparticles, which enhances the proapoptotic activity. The iron oxide component of the nanoparticles enabled imaging of GBM tumors in mice. Systemic treatment of GBM-bearing mice with the nanoparticles eradicated most tumors in one GBM mouse model and significantly delayed tumor development in another. Coinjecting the nanoparticles with a tumor-penetrating peptide further enhanced the therapeutic effect. Both models used have proven completely resistant to other therapies, suggesting clinical potential of our nanosystem.

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.

Dedifferentiation and reprogramming: origins of cancer stem cells

Regenerative medicine aims to replace the lost or damaged cells in the human body through a new source of healthy transplanted cells or by endogenous repair. Although human embryonic stem cells were first thought to be the ideal source for cell therapy and tissue repair in humans, the discovery by Yamanaka and colleagues revolutionized the field. Almost any differentiated cell can be sent back in time to a pluripotency state by expressing the appropriate transcription factors. The process of somatic reprogramming using Yamanaka factors, many of which are oncogenes, offers a glimpse into how cancer stem cells may originate. In this review we discuss the similarities between tumor dedifferentiation and somatic cell reprogramming and how this may pose a risk to the application of this new technology in regenerative medicine.

Mesenchymal high-grade glioma is maintained by the ID-RAP1 axis

High-grade gliomas (HGGs) are incurable brain tumors that are characterized by the presence of glioma-initiating cells (GICs). GICs are essential to tumor aggressiveness and retain the capacity for self-renewal and multilineage differentiation as long as they reside in the perivascular niche. ID proteins are master regulators of stemness and anchorage to the extracellular niche microenvironment, suggesting that they may play a role in maintaining GICs. Here, we modeled the probable therapeutic impact of ID inactivation in HGG by selective ablation of Id in tumor cells and after tumor initiation in a new mouse model of human mesenchymal HGG. Deletion of 3 Id genes induced rapid release of GICs from the perivascular niche, followed by tumor regression. GIC displacement was mediated by derepression of Rap1gap and subsequent inhibition of RAP1, a master regulator of cell adhesion. We identified a signature module of 5 genes in the ID pathway, including RAP1GAP, which segregated 2 subgroups of glioma patients with markedly different clinical outcomes. The model-informed survival analysis together with genetic and functional studies establish that ID activity is required for the maintenance of mesenchymal HGG and suggest that pharmacological inactivation of ID proteins could serve as a therapeutic strategy.

Proapoptotic Peptide-Mediated Cancer Therapy Targeted to Cell Surface p32

Antiangiogenic therapy is a promising new treatment modality for cancer, but it generally produces only transient tumor regression. We have previously devised a tumor-targeted nanosystem, in which a pentapeptide, CGKRK, delivers a proapoptotic peptide into the mitochondria of tumor blood vessel endothelial cells and tumor cells. The treatment was highly effective in glioblastoma mouse models completely refractory to other antiangiogenic treatments. Here, we identify p32/gC1qR/HABP, a mitochondrial protein that is also expressed at the cell surface of activated (angiogenic) endothelial cells and tumor cells, as a receptor for the CGKRK peptide. The results demonstrate the ability of p32 to cause internalization of a payload bound to p32 into the cytoplasm. We also show that nardilysin, a protease capable of cleaving CGKRK, plays a role in the internalization of a p32-bound payload. As p32 is overexpressed and surface displayed in breast cancers, we studied the efficacy of the nanosystem in this cancer. We show highly significant treatment results in an orthotopic model of breast cancer. The specificity of cell surface p32 for tumor-associated cells, its ability to carry payloads to mitochondria, and the efficacy of the system in important types of cancer make the nanosystem a promising candidate for further development.

Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization. DOI: http://dx.doi.org/10.7554/eLife.17681.001

Adoptive Cell Therapy of Systemic Metastases Using erbB-2-Specific T Cells Redirected with a Chimeric Antibody-Based Receptor

Immunotherapy of cancer using adoptive cell transfer combined with the advent of gene-engineering technologies has become an appealing option for a wide spectrum of cancers. In contrast to T cell receptor-based approaches, which are MHC restricted, chimeric antibody-based receptors (CAR), pioneered by our group, allow for a broader application, which are not restricted to individual tissue types. Here, we describe our studies using T cells redirected with CAR specific to the erbB-2 growth factor proto-oncogene as a common tumor target antigen. In a murine model for lung metastasis, we demonstrate that under defined conditions, CAR-expressing T cells (T-bodies) can eliminate systemic lung metastases, which are generally felt to be incurable. The antitumor effect of systemically injected T-bodies was augmented by using increased injected cell doses and repeated administration cycles as well as by pre-vaccination of the tumor-bearing mice. Most importantly, we were able to establish a protocol enabling the use of MHC mismatched T-bodies in a safe and effective manner. We found that a single dose of allogeneic T-bodies under mild immunosuppressive conditions could cure metastases, demonstrating the efficacy of this modality against disseminated disease. These results provide a proof of principle for using allogeneic erbB-2-specific T-bodies as a standard treatment of erbB-2-expressing tumors.

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.