Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma.

Background: Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods: The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results: GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions: Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Oncolytic Viruses in Cancer Treatment: A Review.

IMPORTANCE: Oncolytic viruses (OVs) are emerging as important agents in cancer treatment. Oncolytic viruses offer the attractive therapeutic combination of tumor-specific cell lysis together with immune stimulation, therefore acting as potential in situ tumor vaccines. Moreover, OVs can be engineered for optimization of tumor selectivity and enhanced immune stimulation and can be readily combined with other agents. The effectiveness of OVs has been demonstrated in many preclinical studies and recently in humans, with US Food and Drug Administration approval of the oncolytic herpesvirus talimogene laherparepvec in advanced melanoma, a major breakthrough for the field. Thus, the OV approach to cancer therapy is becoming more interesting for scientists, clinicians, and the public. The main purpose of this review is to give a basic overview of OVs in clinical development and provide a description of the current status of clinical trials. OBSERVATIONS: In 2016 approximately 40 clinical trials are recruiting patients, using a range of OVs in multiple cancer types. There are also many more trials in the planning stages. Therefore, we are now in the most active period of clinical OV studies in the history of the field. There are several OVs currently being tested with many additional engineered derivatives. In OV clinical trials, there are a number of specific areas that should be considered, including viral pharmacokinetics and pharmacodynamics, potential toxic effects, and monitoring of the patients' immune status. Clinical development of OVs is increasingly focused on their immune stimulatory properties, which may work synergistically with immune checkpoint inhibitors and other strategies in the treatment of human cancer. CONCLUSIONS AND RELEVANCE: Oncolytic viruses are an active area of clinical research. The ability of these agents to harness antitumor immunity appears to be key for their success. Combinatorial studies with immune checkpoint blockade have started and the results are awaited with great interest.

Preclinical Mouse Models for Analysis of the Therapeutic Potential of Engineered Oncolytic Herpes Viruses.

After more than two decades of research and development, oncolytic herpes viruses (oHSVs) are moving into the spotlight due to recent encouraging clinical trial data. oHSV and other oncolytic viruses function through direct oncolytic cancer cell-killing mechanisms and by stimulating antitumor immunity. As further viruses are developed and optimized for the treatment of various types of cancer, appropriate predictive preclinical models will be of great utility. This review will discuss existing data in this area, focusing on the mouse tumor models that are commonly used.

Design of a Microfluidic Chip for Magnetic-Activated Sorting of One-Bead-One-Compound Libraries.

Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized "hit" beads are tedious and lack accuracy, and existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to conventional manual sorting approaches. This chip offers a very convenient yet inexpensive alternative for screening OBOC libraries.

Iron overload enhances human mesenchymal stromal cell growth and hampers matrix calcification.

BACKGROUND: Iron overload syndromes include a wide range of diseases frequently associated with increased morbidity and mortality. Several organs are affected in patients with iron overload including liver, heart, joints, endocrine glands, and pancreas. Moreover, severe bone and hemopoietic tissue alterations are observed. Because of the role of bone marrow mesenchymal stromal cells (BM-MSCs) in bone turnover and hematopoiesis, iron effects on primary BM-MSCs cultures were evaluated. METHODS: Primary human BM-MSCs cultures were prepared and the effects of iron on their proliferation and differentiation were characterized by biochemical analyses and functional approaches. RESULTS: Addition of iron to the culture medium strongly increased BM-MSCs proliferation and induced their accelerated S phase entry. Iron enters BM-MSCs through both transferrin-dependent and transferrin-independent mechanisms, inducing the accumulation of cyclins E and A, the decrease of p27(Kip1), and the activation of MAPK pathway. Conversely, neither apoptotic signs nor up-regulation of reactive oxygen species were observed. Iron inhibited both differentiation of BM-MSCs into osteoblasts and in vitro matrix calcification. These effects result from the merging of inhibitory activities on BM-MSCs osteoblastic commitment and on the ordered matrix calcification process. CONCLUSIONS: We demonstrated that BM-MSCs are a target of iron overload. Iron accelerates BM-MSCs proliferation and affects BM-MSCs osteoblastic commitment, hampering matrix calcification. GENERAL SIGNIFICANCE: Our study reports, for the first time, that iron, at concentration found in overloaded patient sera, stimulates the growth of BM-MSCs, the BM multipotent stromal cell component. Moreover, iron modulates the physiological differentiation of these cells, affecting bone turnover and remodeling.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Histone Deacetylase Inhibitors Increase p27(Kip1) by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation.

Histone deacetylase inhibitors (HDACIs) represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27(Kip1), a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27(Kip1) accumulation due to a reduced protein degradation. p27(Kip1) removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27(Kip1) stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27(Kip1) by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma.

Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells.

Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells.

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent cells capable of differentiating toward osteoblatic and adipocytic phenotypes. BM-MSCs play several key roles including bone remodeling, establishment of hematopoietic niche and immune tolerance induction. Here, we investigated the effect of resveratrol (RSV), a therapeutically promising natural polyphenol, on the commitment of human BM-MSCs primary cultures. Cell differentiation was evaluated by means of morphological analysis, specific staining and expression of osteogenic and adipocytic master genes (Runx-2, PPARγ). To maintain BM-MSC multipotency, all experiments were performed on cells at very early passages. At any concentration RSV, added to standard medium, did not affect the phenotype of confluent BM-MSCs, while, when added to osteogenic or adipogenic medium, 1 µM RSV enhances the differentiation toward osteoblasts or adipocytes, respectively. Conversely, the addition of higher RSV concentration (25 µM) to both differentiation media resulted exclusively in BM-MSCs adipogenesis. Surprisingly, the analysis of RSV molecular effects demonstrated that the compound completely substitutes insulin, a key component of adipogenic medium. We also observed that RSV treatment is associated to enhanced phosphorylation of CREB, a critical effector of insulin adipogenic activity. Finally, our observations contribute to the mechanistic elucidation of the well-known RSV positive effect on insulin sensitivity and type 2 diabetes mellitus.

NEDD9, a novel target of miR-145, increases the invasiveness of glioblastoma.

miR-145 is an important repressor of pluripotency in embryonic stem cells and a tumor suppressor in different cancers. Here, we found that miR-145 is strongly down-regulated in glioblastoma (GB) specimens and corresponding glioblastomaneurospheres (GB-NS, containing GB stem-like cells) compared to normal brain (NB) and to low-grade gliomas (LGG). We observed a direct correlation between miR-145 expression and the progression-free survival (PFS) in LGG patients and overall survival (OS) in GB patients. Using microarray analysis, we identified relevant differences in gene expression profiles between GB-NS over-expressing miR-145 (miRover-NS) and GB-NS Empty (Empty-NS). We focused our attention on HEF1/Cas-L/NEDD9, a scaffold protein involved in invasion in several types of cancer. We confirmed a significant down-regulation of NEDD9 in miRover-NS and we found a higher expression in GB and GB-NS compared to NB. Approximately 50% of LGG patients expressed higher levels of NEDD9 than NB, and the PFS of such patients was shorter than in patients expressing lower levels of NEDD9. We observed that intracranial injection of GB-NS over-expressing miR-145 delays significantly tumor development :deriving tumors showed a significant down-regulation of NEDD9. In addition, we demonstrated a significant inhibition of invasion in silencing experiments with GB-NS shNEDD9 (shNEDD9), and an up-regulation of miR-145 in shNEDD9, suggesting a doublenegative feedback loop between miR-145 and NEDD9. Our results demonstrate the critical role of miR-145 and NEDD9 in regulating glioblastoma invasion and suggest a potential role of NEDD9 as a biomarker for glioma progression.

A radial glia gene marker, fatty acid binding protein 7 (FABP7), is involved in proliferation and invasion of glioblastoma cells.

Glioblastoma multiforme (GBM) is among the most deadly cancers. A number of studies suggest that a fraction of tumor cells with stem cell features (Glioma Stem-like Cells, GSC) might be responsible for GBM recurrence and aggressiveness. GSC similarly to normal neural stem cells, can form neurospheres (NS) in vitro, and seem to mirror the genetic features of the original tumor better than glioma cells growing adherently in the presence of serum. Using cDNA microarray analysis we identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor. Fatty acid-binding protein 7 (FABP7) was identified as one of the most highly expressed genes in NS compared to their adherent counterpart. We found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration. We also evaluated the potential involvement of FABP7 in response to radiotherapy, as this treatment may cause increased tumor infiltration. Migration of irradiated NS was associated to increased expression of FABP7. In agreement with this, in vivo reduced tumorigenicity of GBM cells with down-regulated expression of FABP7 was associated to decreased expression of the migration marker doublecortin. Notably, we observed that PPAR antagonists affect FABP7 expression and decrease the migration capability of NS after irradiation. As a whole, the data emphasize the role of FABP7 expression in GBM migration and provide translational hints on the timing of treatment with anti-FABP7 agents like PPAR antagonists during GBM evolution.

FOXP3, a novel glioblastoma oncosuppressor, affects proliferation and migration.

The transcription factor FOXP3 plays an essential role in regulatory T cell development and function. In addition, it has recently been identified as a tumor suppressor in different cancers. Here, we report that FOXP3 is expressed in normal brain but strongly down-regulated in glioblastoma (GB) and in corresponding GB stem-like cells growing in culture as neurospheres (GB-NS), as evaluated by real time-PCR and confirmed by immunohistochemistry on an independent set of GB. FOXP3 expression was higher in low-grade gliomas than in GB. Interestingly, we also found that neurosphere generation, a feature present in 58% of the GB that we examined, correlated with lower expression of FOXP3 and shorter patient survival. FOXP3 silencing in one GB-NS expressing measurable levels of the gene caused a significant increase in proliferation and migration as well as highly aggressive growth in xenografts. Conversely, FOXP3 over-expression impaired GB-NS migration and proliferation in vitro. We also demonstrated using ChiP that FOXP3 is a transcriptional regulator of p21 and c-MYC supporting the idea that dysregulated expression of these factors is a major mechanism of tumorigenesis driven by the loss of FOXP3 expression in gliomas. These findings support the assertion that FOXP3 exhibits tumor suppressor activity in glioblastomas.