Candelaria Gomez‐Manzano

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8+ and T-bet+ cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

The eradication of brain tumor stem cells is essential for long-term brain tumor remission after treatment. In this study, we examined the therapeutic potential of an oncolytic adenovirus, Delta-24-RGD, targeted to the abnormal p16INK4/Rb pathway in brain tumor stem cells. Four brain tumor stem cell lines from surgical glioblastoma specimens expressed high levels of adenoviral receptors and allowed for efficient viral infection, replication, and oncolysis in an Rb-dependent manner. Delta-24-RGD induced autophagic cell death, as indicated by accumulation of Atg5 and LC3-II protein and autophagic vacuoles. Treatment of xenografts derived from brain tumor stem cells with Delta-24-RGD statistically significantly improved the survival of glioma-bearing mice (means: 38.5 versus 66.3 days, difference = 27.8 days, 95% confidence interval = 19.5 to 35.9 days, P <.001). Analyses of treated tumors showed that Atg5 expression colocalized with viral fiber protein and delineated a wave front of autophagic cells that circumscribed areas of virally induced necrosis. Our results show for the first time that brain tumor stem cells are susceptible to adenovirus-mediated cell death via autophagy in vitro and in vivo.

Oncolytic viruses selectively lyse tumor cells, disrupt immunosuppression within the tumor, and reactivate antitumor immunity, but they have yet to live up to their therapeutic potential. Immune checkpoint modulation has been efficacious in a variety of cancer with an immunogenic microenvironment, but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies would likely result in both effective and specific cancer therapy. To test the hypothesis, we first constructed oncolytic adenovirus Delta-24-RGDOX expressing the immune costimulator OX40 ligand (OX40L). Like its predecessor Delta-24-RGD, Delta-24-RGDOX induced immunogenic cell death and recruit lymphocytes to the tumor site. Compared with Delta-24-RGD, Delta-24-RGDOX exhibited superior tumor-specific activation of lymphocytes and proliferation of CD8+ T cells specific to tumor-associated antigens, resulting in cancer-specific immunity. Delta-24-RGDOX mediated more potent antiglioma activity in immunocompetent C57BL/6 but not immunodeficient athymic mice, leading to specific immune memory against the tumor. To further overcome the immune suppression mediated by programmed death-ligand 1 (PD-L1) expression on cancer cells accompanied with virotherapy, intratumoral injection of Delta-24-RGDOX and an anti-PD-L1 antibody showed synergistic inhibition of gliomas and significantly increased survival in mice. Our data demonstrate that combining an oncolytic virus with tumor-targeting immune checkpoint modulators elicits potent in situ autologous cancer vaccination, resulting in an efficacious, tumor-specific, and long-lasting therapeutic effect. Cancer Res; 77(14); 3894-907. ©2017 AACR.

Abstract Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2–20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1–69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7–13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05–0.87). A total of 56.2% (95% CI 41.1–70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Oncolytic adenoviruses are promising therapies for the treatment of gliomas. However, untargeted viral replication and the paucity of coxsackie-adenovirus receptors (CARs) on tumor cells are major stumbling blocks for adenovirus-based treatment. We studied the antiglioma activity of the tumor-selective Delta-24 adenovirus, which encompasses an early 1 A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, and of the Delta-24-RGD adenovirus. Delta-24-RGD has an RGD-4C peptide motif inserted into the adenoviral fiber, which allows the adenovirus to anchor directly to integrins.CAR and integrin expression were examined by flow cytometry in six glioma cell lines and in normal human astrocytes (NHAs). Adenoviral vectors containing green fluorescent protein (GFP) (AdGFP and AdGFP-RGD) were used to infect glioma cell lines with high or low CAR expression. Viability of glioma cells infected with different adenoviruses was assessed by trypan blue staining. Adenovirus replication was quantified with the infection-dose replication assay. Athymic mice carrying glioma xenografts received intratumoral injections of Delta-24-RGD or Delta-24 and were followed for survival, which was analyzed by the Kaplan-Meier method and the log-rank test. All statistical tests were two-sided.Half the glioma cell lines expressed low levels of CAR (defined as <50% of cells expressing detectable CAR); all lines expressed integrins in more than 50% of cells. Infection of U-87 MG cells (a low-CAR-expressing line) with AdGFP-RGD resulted in approximately six times more GFP-positive cells than infection with AdGFP. Delta-24-RGD was more cytopathic to both low- and high-CAR-expressing glioma lines than Delta-24, and it replicated more efficiently in both cell lines. In the xenografted mice, intratumoral injection of Delta-24-RGD was associated with longer survival than intratumoral injection of Delta-24 (P<.001, log-rank test). Furthermore, 60% of Delta-24-RGD-treated mice but only 15% of Delta-24-treated mice survived more than 4 months (difference = 45%, 95% CI = 21% to 68%).The antitumor activity of Delta-24-RGD suggests that it has the potential to be an effective agent in the treatment of gliomas.

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.

Abstract Currently, the most efficacious treatment for malignant gliomas is temozolomide; however, gliomas expressing the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) are resistant to this drug. Strong clinical evidence shows that gliomas with methylation and subsequent silencing of the MGMT promoter are sensitive to temozolomide. Based on the fact that adenoviral proteins directly target and inactivate key DNA repair genes, we hypothesized that the oncolytic adenovirus Δ-24-RGD could be successfully combined with temozolomide to overcome the reported MGMT-mediated resistance. Our studies showed that the combination of Δ-24-RGD and temozolomide induces a profound therapeutic synergy in glioma cells. We observed that Δ-24-RGD treatment overrides the temozolomide-mediated G2-M arrest. Furthermore, Δ-24-RGD infection was followed by down-modulation of the RNA levels of MGMT. Chromatin immunoprecipitation assays showed that Δ-24-RGD prevented the recruitment of p300 to the MGMT promoter. Importantly, using mutant adenoviruses and wild-type and dominant-negative forms of the p300 protein, we showed that Δ-24-RGD interaction with p300 was required to induce silencing of the MGMT gene. Of further clinical relevance, the combination of Δ-24-RGD and temozolomide significantly improved the survival of glioma-bearing mice. Collectively, our data provide a strong mechanistic rationale for the combination of oncolytic adenoviruses and temozolomide, and should propel the clinical testing of this therapy approach in patients with malignant gliomas. [Cancer Res 2007;67(24):11499–504]

Oncolytic adenoviruses, such as Delta-24-RGD, are promising therapies for patients with brain tumor. Clinical trials have shown that the potency of these cancer-selective adenoviruses should be increased to optimize therapeutic efficacy. One potential strategy is to increase the efficiency of adenovirus-induced cell lysis, a mechanism that has not been clearly described. In this study, for the first time, we report that autophagy plays a role in adenovirus-induced cell lysis. At the late stage after adenovirus infection, numerous autophagic vacuoles accompany the disruption of cellular structure, leading to cell lysis. The virus induces a complete autophagic process from autophagosome initiation to its turnover through fusion with the lysosome although the formation of the autophagosome is sufficient for virally induced cell lysis. Importantly, downmodulation of autophagy genes (ATG5 or ATG10) rescues the infected cells from being lysed by the virus. Moreover, autophagy triggers caspase activity via the extrinsic FADD/caspase 8 pathway, which also contributes to adenovirus-mediated cell lysis. Therefore, our study implicates autophagy and caspase activation as part of the mechanism for cell lysis induced by adenovirus and suggests that manipulation of the process is a potential strategy to optimize clinical efficacy of oncolytic adenoviruses.

Current evidence indicates that a stem cell-like sub-population within malignant glioblastomas, that overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters, is responsible for multidrug resistance and tumour relapse. Eradication of the brain tumour stem cell (BTSC) compartment is therefore essential to achieve a stable and long-lasting remission.Melatonin actions were analysed by viability cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein expression and quantitative and qualitative promoter methylation methods.Combinations of melatonin and chemotherapeutic drugs (including temozolomide, current treatment for malignant gliomas) have a synergistic toxic effect on BTSCs and A172 malignant glioma cells. This effect is correlated with a downregulation of the expression and function of the ABC transporter ABCG2/BCRP. Melatonin increased the methylation levels of the ABCG2/BCRP promoter and the effects on ABCG2/BCRP expression and function were prevented by preincubation with a DNA methyltransferase inhibitor.Our results point out a possible relationship between the downregulation of ABCG2/BCRP function and the synergistic toxic effect of melatonin and chemotherapeutic drugs. Melatonin could be a promising candidate to overcome multidrug resistance in the treatment of glioblastomas, and thus improve the efficiency of current therapies.

Emerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity.Mouse GL261-glioma model was set up in immunocompetent C57BL/6 mouse for Delta-24-RGD treatment. The changes of the immune cell populations were analyzed by immunohistochemistry and flow cytometry. The anti-glioma immunity was evaluated with functional study of the splenocytes isolated from the mice. The efficacy of the virotherapy was assessed with animal survival analysis. The direct effect of the virus on the tumor-associated antigen presentation to CD8+ T cells was analyzed with an in vitro ovalbumin (OVA) modeling system.Delta-24-RGD induced cytotoxic effect in mouse glioma cells. Viral treatment in GL261-glioma bearing mice caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, viral infection and IFNγ increased the presentation of OVA antigen in OVA-expressing cells to CD8+ T-cell hybridoma B3Z cells, which is blocked by brefeldin A and proteasome inhibitors, indicating the activity is through the biosynthesis and proteasome pathway.Our results demonstrate that Delta-24-RGD induces anti-glioma immunity and offers the first evidence that viral infection directly enhances presentation of tumor-associated antigens to immune cells.

Abstract Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032).

Background: Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors. Purpose: We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis. Methods: Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replicationdeficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. Results: p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G 2 phase before undergoing cell death. Transfer of p21 induced a G 2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G 2 phase, or apoptosis. Conclusions and Implications: Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that overexpression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.

Novel therapies are clearly needed for the treatment of gliomas, and strategies that involve combining oncolytic vectors with chemotherapy hold out significant hope for a more effective treatment of this malignancy. Whether chemotherapy acts directly on tumor cells by inducing cell arrest or cell death, or indirectly by blocking tumor angiogenesis, the resulting delay in tumor growth may provide the oncolytic virus with a wider window of opportunity to overcome the challenge imposed by the growth kinetics of the tumor. In this study we sought to determine whether the oncolytic adenovirus Delta-24-RGD, in combination with everolimus (RAD001), would result in an enhanced anti-glioma effect in vivo. Viability assays showed that Delta-24-RGD antitumoral activity is synergistically enhanced by combination with RAD001. Interestingly, combination treatment of Delta-24-RGD with RAD001 induced autophagy in vitro. We showed that Delta-24-RGD improved survival of tumor-bearing animals in a dose-dependent manner. A significant finding was that RAD001 enhanced the anti-glioma effect of Delta-24-RGD and resulted in the long-term survival of 80% of the experimental animals. Immunostaining of the treated tumors showed upregulation of Atg5, thereby indicating the therapeutic induction of autophagy. This is the first report showing that Delta-24-RGD plus RAD001 causes autophagic cell death, and dramatically increases long-term survival rates of glioma-bearing animals.

Abstract Autophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidence suggests that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dependent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumor suppressor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggers autophagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent manner. RB binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizes RB-induced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels of autophagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that this newly discovered aspect of RB function is relevant to cancer development and therapy. Cancer Res; 70(20); 7882–93. ©2010 AACR.

Oncolytic adenoviruses are emerging as a promising alternative therapy for glioma patients and are currently being tested in clinic. In this review, we summarize our experience with gene-based therapy targeting RB pathway in gliomas. Our study has evolved from the development of RB-expressing adenoviral vectors to the characterization of the oncolytic effects on gliomas of the replication competent adenoviruses Delta-24, Delta-24-RGD and ICOVIR. We also review the successful combination of the viruses with chemotherapies that are routinely used in glioma patients, the efficacy of Delta-24-RGD against brain tumor stem cells, the newly described adenovirus-induced autophagy and the potential for the systemic delivery of the oncolytic viruses with human mesenchymal stem cells. Finally, we comment on the preclinical and clinical studies of p53 expressing adenoviral vector and the lessons learned from the experience of Onyx- 015, the first oncolytic adenovirus tested in clinical setting.

Pathological angiogenesis is a hallmark of cancer, specifically of glioblastomas, the most malignant and common primary brain tumor. Vascular endothelial growth factor (VEGF) is the key protein in the regulation of the hypervascular phenotype of primary malignant brain tumors. In this study, we tested VEGF Trap, a soluble decoy receptor for VEGF, in an intracranial glioma model. VEGF Trap was administered in short or prolonged schedules to animals bearing human gliomas at different stages of disease. Of importance, VEGF Trap treatment was efficacious in both initial and advanced phases of tumor development by significantly increasing overall survival. Furthermore, this effect was enhanced in animals treated with more prolonged regimens. In addition, we observed the emergence of a VEGF Trap-resistant phenotype characterized by tumor growth and increased invasiveness. Our results suggest that VEGF Trap will be effective in treating both patients with recurrent or progressive resectable glioblastoma and patients that have undergone extensive initial surgery. Finally, our results indicate that the clinical success of VEGF Trap may depend on a prolonged treatment in combined therapy aiming to simultaneously inhibit angiogenesis and tumor invasion.

The addition of anti-angiogenic therapy to the few treatments available to patients with malignant gliomas was based on the fact that these tumors are highly vascularized and on encouraging results from preclinical and clinical studies. However, tumors that initially respond to this therapy invariably recur with the acquisition of a highly aggressive and invasive phenotype. Although several myeloid populations have been associated to this pattern of recurrence, a specific targetable population has not been yet identified. Here, we present evidence for the accumulation of Tie2-expressing monocytes/macrophages (TEMs) at the tumor/normal brain interface of mice treated with anti-VEGF therapies in regions with heightened tumoral invasion. Furthermore, we describe the presence of TEMs in malignant glioma surgical specimens that recurred after bevacizumab treatment. Our studies showed that TEMs enhanced the invasive properties of glioma cells and secreted high levels of gelatinase enzymatic proteins. Accordingly, Tie2⁺MMP9⁺ monocytic cells were consistently detected in the invasive tumor edge upon anti-VEGF therapies. Our results suggest the presence of a specific myeloid/monocytic subpopulation that plays a pivotal role in the mechanism of escape of malignant gliomas from anti-VEGF therapies and therefore constitutes a new cellular target for combination therapies in patients selected for anti-angiogenesis treatment.

Glioblastoma is the most frequent malignant brain tumor. Even with aggressive treatment, prognosis for patients is poor. One characteristic of glioblastoma cells is its intrinsic resistance to apoptosis. Therefore, drugs that induce alternative cell deaths could be interesting to evaluate as alternative therapeutic candidates for glioblastoma. Salinomycin (SLM) was identified through a chemical screening as a promising anticancer drug, but its mechanism of cell death remains unclear. In the present work we set out to elucidate how SLM causes cell death in glioblastoma cell lines (both established cell lines and brain tumor stem cell lines), aiming to find a potential antitumor candidate. In addition, we sought to determine the mechanism of action of SLM so that this mechanism can be can be exploited in the fight against cancer. Our data showed that SLM induces a potent endoplasmic reticulum (ER) stress followed by the trigger of the unfolded protein response (UPR) and an aberrant autophagic flux that culminated in necrosis due to mitochondria and lysosomal alterations. Of importance, the aberrant autophagic flux was orchestrated by the production of Reactive Oxygen Species (ROS). Alleviation of ROS production restored the autophagic flux. Altogether our data suggest that in our system the oxidative stress blocks the autophagic flux through lipid oxidation. Importantly, oxidative stress could be instructing the type of cell death in SLM-treated cells, suggesting that cell death modality is a dynamic concept which depends on the cellular stresses and the cellular mechanism activated.

Malignant gliomas extensively infiltrate the surrounding normal brain, and their diffuse invasion is one of the most important barriers to successful therapy. Recent studies indicate that the progression of gliomas from low-grade to high-grade may depend on the acquisition of a new phenotype and the subsequent addition of genetic defects. One of the most frequent abnormalities in the progression of gliomas is the inactivation of tumor-suppressor gene p16, suggesting that loss of p16 is associated with acquisition of malignant characteristics. Consistent with this hypothesis, our previous studies showed that restoring wild-type p16 activity into p16-null malignant glioma cells modified their phenotype. In order to understand whether the biological consequences of p16 inactivation in high-grade gliomas included facilitating invasiveness, we used a recombinant replication-deficient adenovirus carrying the cDNA of the p16/CDKN2 gene to infect and express high levels of p16 protein in p16-null SNB19 glioma cells. Invasion of SNB19 glioma cells was tested into two models: invasion of glioma cells through Matrigel-coated transwell inserts and invasion of tumor-cell spheroids into fetal rat-brain aggregates in a co-culture system. Matrigel invasion assays showed that the SNB19 cells expressing exogenous p16 exhibited significantly reduced invasion. Similarly, invasion of p16-treated SNB19 cells into fetal rat-brain aggregates was reduced during a 72 h time period compared to invasion of the adenovirus-control and mock-infected cells. Expression of matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor-cell invasion, in SNB19 cells expressing p16 was significantly reduced compared to that of parental SNB19 and vector-infected cells. Our results show that restoring wild-type p16 activity into p16-null SNB19 glioma cells significantly inhibits tumor-cell invasion, thus suggesting a novel function of the p16 gene.

Malignant gliomas are the prototype of highly infiltrative tumors and this characteristic is the main factor for the inevitable tumor recurrence and short survival after most aggressive therapies. The aberrant communication between glioma cells and tumor microenvironment represents one of the major factors regulating brain tumor dispersal. Our group has previously reported that the tyrosine kinase receptor Tie2/TEK is expressed in glioma cells and brain tumor stem cells and is associated with the malignant progression of these tumors. In this study, we sought to determine whether the angiopoietin 1 (Ang1)/Tie2 axis regulates crosstalk between glioma cells and endothelial cells. We found that Ang1 enhanced the adhesion of Tie2-expressing glioma and brain tumor stem cells to endothelial cells. Conversely, specific small interfering RNA (siRNA) knockdown of Tie2 expression inhibited the adhesion capability of glioma cells. Tie2 activation induced integrin β1 and N-cadherin upregulation, and neutralizing antibodies against these molecules inhibited the adhesion of Tie2-positive glioma cells to endothelial cells. In 2D and 3D cultures, we observed that Ang1/Tie2 axis activation was related to increased glioma cell invasion, which was inhibited by using Tie2 siRNA. Importantly, intracranial co-implantation of Tie2-positive glioma cells and endothelial cells in a mouse model resulted in diffusely invasive tumors with cell clusters surrounding glomeruloid vessels mimicking a tumoral niche distribution. Collectively, our results provide new information about the Tie2 signaling in glioma cells that regulates the cross-talk between glioma cells and tumor microenvironment, envisioning Tie2 as a multi-compartmental target for glioma therapy.

Oncolytic adenoviruses are modified to exploit the aberrant expression of proteins in cancer cells to obtain cancer-selective replication. Moreover, the natural tropism of oncolytic adenoviruses can be redirected to tumor cells. Clinical trials revealed that oncolytic viruses showed poor replication in the tumor that is due in part to the immune response against the virus. More recent data demonstrated that tumor infection might subvert the tumor immune system and lead to an anti-tumor immune response. In the next few years, combination of adenoviruses with immune checkpoint antibodies and other immune modulators will be tested in clinical trials.

Oncolytic adenoviruses, such as Delta-24-RGD (Δ24RGD), are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, Δ24RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with Δ24RGDundergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/2 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with Δ24RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered Δ24RGD-treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with Δ24RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between Δ24RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates Δ24RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.

Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O6-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma.

Abstract BACKGROUND There are no effective treatments for diffuse intrinsic pontine gliomas (DIPGs); these tumors cannot be surgical resected, and diagnosis is based on magnetic resonance imaging. As a result, tumor tissues for molecular studies and pathologic diagnosis are infrequent. New clinical trials are investigating novel medications and therapeutic techniques in an effort to improve treatment of patients with DIPGs. OBJECTIVE To determine the safety, tolerability, and toxicity of an oncolytic adenovirus, DNX-2401, injected into the cerebellar peduncle in pediatric subjects with DIPG and to collect tumor samples of this type of tumor. METHODS Phase I, single-center, uncontrolled trial. A tumor biopsy will be performed through the cerebellar peduncle, and DNX-2401 will be injected immediately after the biopsy. Standard therapy consisting of radiotherapy and chemotherapy will follow in 2 to 6 wk. EXPECTED OUTCOMES Improvement of overall survival and quality of life in patients with DIPG and collection of tumor specimens to study the molecular profiling of these tumors. DISCUSSION The aims of this trial are to contribute to the sample collection of DIPG and to offer treatment during the tumor tissue biopsy using the virus. If this virus works as expected, it could kill the tumor cells with no damage to healthy tissue, functioning as a targeted therapy. It is important to note that edema has not been observed with this virus in all trials performed to date. The information obtained through this and other similar studies may be useful for developing or improving new therapies in the battle against DIPG.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive glial brain tumors that primarily affect children, for which there is no curative treatment. Median overall survival is only one year. Currently, the scientific focus is on expanding the knowledge base of the molecular biology of DIPG, and identifying effective therapies. Oncolytic adenovirus DNX-2401 is a replication-competent, genetically modified virus capable of infecting and killing glioma cells, and stimulating an anti-tumor immune response. Clinical trials evaluating intratumoral DNX-2401 in adults with recurrent glioblastoma have demonstrated that the virus has a favorable safety profile and can prolong survival. Subsequently, these results have encouraged the transition of this biologically active therapy from adults into the pediatric population. To this aim, we have designed a clinical Phase I trial for newly diagnosed pediatric DIPG to investigate the feasibility, safety, and preliminary efficacy of delivering DNX-2401 into tumors within the pons following biopsy. This case report presents a pediatric patient enrolled in this ongoing Phase I trial for children and adolescents with newly diagnosed DIPG. The case involves an 8-year-old female patient with radiologically diagnosed DIPG who underwent stereotactic tumor biopsy immediately followed by intratumoral DNX-2401 in the same biopsy track. Because there were no safety concerns or new neurological deficits, the patient was discharged 3 days after the procedures. To our knowledge, this is the first report of intratumoral DNX-2401 for a patient with DIPG in a clinical trial. We plan to demonstrate that intratumoral delivery of an oncolytic virus following tumor biopsy for pediatric patients with DIPG is a novel and feasible approach and that DNX-2401 represents an innovative treatment for the disease.

Pediatric high grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPGs), are aggressive tumors with a dismal outcome. Radiotherapy (RT) is part of the standard of care of these tumors; however, radiotherapy only leads to a transient clinical improvement. Delta-24-RGD is a genetically engineered tumor-selective adenovirus that has shown safety and clinical efficacy in adults with recurrent gliomas. In this work, we evaluated the feasibility, safety and therapeutic efficacy of Delta-24-RGD in combination with radiotherapy in pHGGs and DIPGs models. Our results showed that the combination of Delta-24-RGD with radiotherapy was feasible and resulted in a synergistic anti-glioma effect in vitro and in vivo in pHGG and DIPG models. Interestingly, Delta-24-RGD treatment led to the downregulation of relevant DNA damage repair proteins, further sensitizing tumors cells to the effect of radiotherapy. Additionally, Delta-24-RGD/radiotherapy treatment significantly increased the trafficking of immune cells (CD3, CD4+ and CD8+) to the tumor niche compared with single treatments. In summary, administration of the Delta-24-RGD/radiotherapy combination to pHGG and DIPG models is safe and significantly increases the overall survival of mice bearing these tumors. Our data offer a rationale for the combination Delta-24-RGD/radiotherapy as a therapeutic option for children with these tumors. SIGNIFICANCE: Delta-24-RGD/radiotherapy administration is safe and significantly increases the survival of treated mice. These positive data underscore the urge to translate this approach to the clinical treatment of children with pHGG and DIPGs.

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms.

Glioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.The in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.Delta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.In summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.

Delta-24-RGD is an oncolytic adenovirus that is capable of replicating in and killing human glioma cells. Although intratumoral delivery of Delta-24-RGD can be effective, systemic delivery would improve its clinical application. Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) obtained from healthy donors have been investigated as virus carriers. However, it is unclear whether BM-hMSCs can be derived from glioma patients previously treated with marrow-toxic chemotherapy or whether such BM-hMSCs can deliver oncolytic viruses effectively. Herein, the authors undertook a prospective clinical trial to determine the feasibility of obtaining BM-hMSCs from patients with recurrent malignant glioma who were previously exposed to marrow-toxic chemotherapy.The authors enrolled 5 consecutive patients who had been treated with radiation therapy and chemotherapy. BM aspirates were obtained from the iliac crest and were cultured to obtain BM-hMSCs.The patient-derived BM-hMSCs (PD-BM-hMSCs) had a morphology similar to that of healthy donor-derived BM-hMSCs (HD-BM-hMSCs). Flow cytometry revealed that all 5 cell lines expressed canonical MSC surface markers. Importantly, these cultures could be made to differentiate into osteocytes, adipocytes, and chondrocytes. In all cases, the PD-BM-hMSCs homed to intracranial glioma xenografts in mice after intracarotid delivery as effectively as HD-BM-hMSCs. The PD-BM-hMSCs loaded with Delta-24-RGD (PD-BM-MSC-D24) effectively eradicated human gliomas in vitro. In in vivo studies, intravascular administration of PD-BM-MSC-D24 increased the survival of mice harboring U87MG gliomas.The authors conclude that BM-hMSCs can be acquired from patients previously treated with marrow-toxic chemotherapy and that these PD-BM-hMSCs are effective carriers for oncolytic viruses.

Oncolytic viruses are a promising treatment for patients with high-grade gliomas, but neutralizing antibodies can limit their efficacy in patients with prior virus exposure or upon repeated virus injections. Data from a previous clinical trial using the oncolytic adenovirus Delta-24-RGD showed that generation of anti-viral neutralizing antibodies may affect the long-term survival of glioma patients. Past studies have examined the effects of neutralizing antibodies during systemic virus injections, but largely overlooked their impact during local virus injections into the brain. We found that immunoglobulins colocalized with viral proteins upon local oncolytic virotherapy of brain tumors, warranting a strategy to prevent virus neutralization and maximize oncolysis. Thus, we generated a chimeric virus, Delta-24-RGD-H43m, by replacing the capsid protein HVRs from the serotype 5-based Delta-24-RGD with those from the rare serotype 43. Delta-24-RGD-H43m evaded neutralizing anti-Ad5 antibodies and conferred a higher rate of long-term survival than Delta-24-RGD in glioma-bearing mice. Importantly, Delta-24-RGD-H43m activity was significantly more resistant to neutralizing antibodies present in sera of glioma patients treated with Delta-24-RGD during a phase 1 clinical trial. These findings provide a framework for a novel treatment of glioma patients that have developed immunity against Delta-24-RGD.

Currently, most of the approved clinical gene therapy protocols involve cancer patients and several of the therapies are designed to treat brain tumors. Two factors promoting the use of gene therapy for gliomas are the failure and toxicity of conventional therapies and the identification of the genetic abnormalities that contribute to the malignancy of gliomas. During the malignant progression of astrocitic tumors several tumor suppressor genes are inactivated, and numerous growth factors and oncogenes are overexpressed progressively. Thus, theoretically, brain tumors could be treated by targeting their fundamental molecular defects, provided the gene-drug can be delivered to a sufficient number of malignant cells. However, gene therapy strategies have not been abundantly successful clinically, in part because the delivery systems are still imperfect. In the first part of this brief review we will discuss the most common targets for gene therapy in brain tumors. In the second part, we will review the evolution of adenoviruses as gene vehicles. In addition, we will examine the role of recombinant mutant oncolytic adenoviruses as anticancer tools. From the results to date it is clear that gene therapy strategies for brain tumors are quite promising but more critical research is required, mainly in the vector field, if the strategies are to achieve their true potential in ameliorating patients with gliomas.

Inactivation of the tumor suppressor gene PTEN and overexpression of VEGF are two of the most common events observed in high-grade malignant gliomas. The purpose of this study was to determine whether PTEN controls VEGF expression in gliomas under normoxic conditions. Transfer of PTEN to human glioma cells resulted in the transduction of a functional PTEN protein as evidenced by the upregulation of p27 and modification of the phosphorylation status of Akt. Under normoxic conditions, enzyme-linked immunosorbent assay and Northern blot analyses showed downregulation of VEGF in PTEN-treated cells. Moreover, conditioned media from PTEN-treated glioma cells significantly diminished the ability of endothelial cells to grow and migrate. Western blot assays demonstrated that, in a normoxic environment, PTEN downregulates HIF-1 alpha. Finally, promoter activity assays showed that the VEGF promoter region containing the HIF-1alpha binding site is necessary and sufficient for PTEN-mediated downregulation of VEGF. Experiments with PI3-K inhibitors and kinase assays suggested that PI3-K is mediating the effect of PTEN on VEGF, and not the p42/p48 or p38 MAP kinases. These results indicate that restoration of PTEN function in gliomas may induce therapeutic effect by downregulating VEGF. Furthermore, this close functional relationship between PTEN and VEGF suggests that a better understanding of the transduction signal regulated by PTEN might enhance the knowledge of the cause and physiology of vascular and inflammatory diseases.

During 2007, approximately 200,000 people in the United States will be diagnosed with brain tumors. Gliomas account for 77% of primary malignant brain tumors, and the prognosis has hardly changed in the past 20 years, with only 30% of patients with malignant glioma surviving 5 years after diagnosis. Oncolytic adenoviruses are promising therapies for the treatment of gliomas. Here, report the antiglioma activity of the tumor-selective ICOVIR-5 adenovirus, which encompasses an early 1A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, substitution of the E1A promoter for E2F-responsive elements, and an RGD-4C peptide motif inserted into the adenoviral fiber to enhance adenoviral tropism. Mechanistic studies showed a dramatic addiction of ICOVIR-5 to the E2F1 oncogene in vitro and in vivo. This addiction was mediated by the occupancy of the ectopic adenoviral E2F1-responsive elements by the endogenous E2F1 protein resulting in high level of E1A expression in cancer cells and potent antiglioma effect. Importantly, we showed for the first time the ability of oncolytic adenoviruses to enhance E2F transcriptional activity in vivo, and we provided direct evidence of the interaction of the E2F1 protein with native and ectopic adenovirus promoters. Restoration of Rb function led to the association of Rb/E2F1 repressor complexes with ICOVIR-5 ectopic E2F1 promoter and subsequent down-modulation of E1A, dramatically impairing adenoviral replication. In xenografted mice, intratumoral injection of ICOVIR-5 resulted in a significant improvement of the median survival (P < 0.0001), and furthermore, led to 37% of long-term survivors free of disease. The antitumor activity of ICOVIR-5 suggests that it has the potential to be an effective agent in the treatment of gliomas.

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.

Novel therapies are clearly needed for gliomas, and the combination of oncolytic vectors with chemotherapy possesses a significant hope for the treatment of this malignancy. In addition, combination with chemotherapy allows for lower virus doses to achieve anticancer effect, thus resulting in lower undesirable toxicities due to viral proteins. In this work, we sought to determine whether combination of an oncolytic adenovirus ICOVIR-5, with RAD001 or temozolomide (TMZ) could result in enhanced anti-glioma effect in vivo. We assessed the in vitro cytotoxic effect and replication properties of ICOVIR-5 in combination with RAD001 or TMZ in U87 MG glioma cell line by MTT and TCID(50), respectively. Our data showed that in vitro treatment with RAD001 or TMZ not only interfered with adenovirus replication but, in addition, enhanced its oncolytic properties. To evaluate the in vivo anticancer effect, athymic mice bearing glioma xenografts (5 x 10(5) U87 MG cells/animal) received a single intratumoral injection of ICOVIR-5 (10(7) PFU/animal). RAD001 was given as a regimen of 5 mg/kg 5 days per week until the end of the experiment and TMZ was administered for 5 days at 7.5 mg/kg/mice. Of significance, combination of ICOVIR-5 with RAD001 or TMZ showed a potent anti-glioma effect in vivo, resulting in a dramatic extension of the median animal survival and in 20-40% animals becoming free of disease beyond 90 days.

Glioma cells are more likely to respond to therapy through autophagy than through apoptosis. The most efficacious cytotoxic drugs employed in glioma therapy, such as temozolomide and rapamycin, induce autophagy. Oncolytic adenoviruses, which will soon be tested in patients with gliomas at the University of Texas M. D. Anderson Cancer Center, also induce autophagy. Autophagy in gliomas thus represents a promising mechanism that may lead to new glioma therapies. In this chapter, we present the methods for studying autophagy in glioma cells, including assessment of in vitro cellular markers acidic vesicle organelles, and green fluorescent protein (GFP)-LC3 punctation; biochemical markers LC3-I/II conversion, p62 degradation, Atg12–Atg5 accumulation, and p70S6K dephosphorylation; and ultrastucture of the autophagosomes. In addition, we will address how LC3B and Atg5 up-regulation during autophagy can be examined through immunostaining in treated tumors and the potential of these proteins for use as surrogate markers to monitor therapeutic effects in clinical trials. Finally, we will discuss the challenges of studying autophagy in gliomas and the future directions of such use.

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.

Membrane-bound enzyme relocates to the cell nucleus to modify chromatin, inducing cancer resistance to radiotherapy.

Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) agent bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective.Our group has previously reported that Tie2-expressing monocytes (TEMs) are aberrantly present at the tumor/normal brain interface after anti-VEGF therapies and their significant role in the invasive outgrowth of these tumors.Here, we aimed to further understand the mechanisms leading to this pro-invasive tumor microenvironment.Examination of a U87MG xenogeneic glioma model and a GL261 murine syngeneic model showed increased tumor expression of angiopoietin 2 (Ang2), a natural ligand of Tie2, after anti-angiogenesis therapies targeting VEGF or VEGF receptor (VEGFR), as assessed by immunohistochemical analysis, immunofluorescence analysis, and enzyme-linked immunosorbent assays of tumor lysates.Migration and gelatinolytic assays showed that Ang2 acts as both a chemoattractant of TEMs and an enhancing signal for their tumor-remodeling properties.Accordingly, in vivo transduction of Ang2 into intracranial gliomas increased recruitment of TEMs into the tumor.To reduce invasive tumor outgrowth after anti-angiogenesis therapy, we targeted the Ang-Tie2 axis using a Tie2 decoy receptor.Using syngeneic models, we observed that overexpression of soluble Tie2 within the tumor prevented the recruitment of TEMs to the tumor and the development of invasion after anti-angiogenesis treatment.Taken together, these data indicate an active role for the Ang2-Tie2 pathway in invasive glioma recurrence after anti-angiogenesis treatment and provide a rationale for testing the combined targeting of VEGF and Ang-Tie2 pathways in patients with glioblastoma.

Abstract Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas. Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry. Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen. Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

Several tumor suppressor pathways have been identified as modulators of telomerase function. We examined the functional role of the retinoblastoma-E2F1 pathway in regulating telomerase activity in malignant gliomas.Adenovirus vectors were used to transfer cDNAs into human glioblastoma and sarcoma cells. Telomerase activity was assessed with a telomere repeat amplification protocol. Promoter activity in cancer cells was assessed with promoter-luciferase reporter constructs. Promoter binding was assessed with the chromatin immunoprecipitation (ChIP) assay. We isolated astrocytes from E2F1 transgenic mice and normal mice for in vivo studies. We evaluated the expression of E2F1 and hTERT (the catalytic subunit of human telomerase) mRNAs by reverse transcriptase-polymerase chain reaction and proteins in human glioblastoma samples by immunoblot analysis. Associations between survival among 61 glioblastoma multiforme patients and expression of E2F1 and hTERT mRNA and protein were examined with Kaplan-Meier analysis, the log-rank test, and Cox proportional hazards regression models. All statistical tests were two-sided.Ectopic E2F1 expression increased hTERT promoter activity in cancer cells. We detected an interaction between E2F1 protein and the hTERT promoter. Transgenic E2F1 astrocytes contained functional telomerase protein. E2F1 mRNA expression and hTERT mRNA expression were statistically significantly correlated in human glioblastoma specimens (R = .8; P < .001). Longer median survival was statistically significantly associated with lower E2F1 mRNA expression in tumors (103.6 weeks) rather than with higher expression (46.1 weeks) (difference = 57.5 weeks; 95% confidence interval [CI] = 14.7 to 159.7; log-rank P = .002). E2F1 mRNA was the only factor that was statistically significantly associated with overall survival in a multivariable model (P = .04). Among 27 patients with glioblastoma multiforme samples, the expression of E2F1 protein was statistically significantly associated with survival (log-rank P < .001).E2F1 may participate in telomerase activity regulation in malignant glioma cells. Its expression appears to be strongly associated with the survival of patients with malignant brain tumors.

Replication-competent adenoviruses could provide an efficient method for delivering therapeutic genes to tumors. The most promising strategies among adenovirus-based oncolytic systems are designed to exploit free E2F-1 activity in cancer cells, which in the absence of pRb activates transcription and regulates the expression of genes involved in differentiation, proliferation, and apoptosis. We previously developed Delta24, an E1A-mutant, conditionally replicative oncolytic adenovirus. Here, we examine the ability of a second-generation Delta24 (Delta24-hyCD) engineered to express a humanized form of the Saccharomyces cerevisiae cytosine deaminase gene (hyCD). Real-time quantitative PCR, Western blotting, thin-layer chromatography, and radioisotope quantitative enzymatic assays confirmed the production of a catalytically active hyCD enzyme in the setting of an oncolytic infection in vitro; other experiments assessing local production of 5-fluorouracil and a concomitant bystander effect showed improved cytotoxicity. The IC50 dose of 5-fluorocytosine (5-FC) required for a complete cytopathic effect by the Delta24-hyCD virus was fivefold lower than with Delta24 alone in U251MG and U87MG malignant glioma (MG) cell lines. Intratumoral treatment of mice bearing intracranial U87MG xenografts with Delta24-hyCD+5-FC significantly improved survival, confirming that Delta24-hyCD with 5-FC is a more efficient anticancer tool than Delta24 alone. Histopathologically, Delta24-hyCD replication was accompanied by progressively augmented oncolysis and drug-induced necrosis. These findings demonstrate that Delta24-hyCD with concomitant systemic 5-FC is a significant improvement over the earlier Delta24 oncolytic tumor-selective strategy for therapy of experimental gliomas.

Abstract Purpose: In this study, we sought to determine whether Delta-24 could sensitize glioma cells to the topoisomerase I inhibitor irinotecan (CPT-11) and to identify the mechanisms underlying this enhanced anticancer effect. Experimental Design: We used human glioblastoma cell lines for the in vitro studies. The expression of topoisomerase I was determined in Western blot analyses, and topoisomerase I activity was determined by measuring the relaxation of a supercoiled DNA. The cell cycle distribution of cells was determined by flow cytometry analysis of the cellular DNA content. Cell viability was quantified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Tissue culture infection dose assays were used to quantitate adenovirus replication. For the in vivo studies, athymic mice received intracranial/intratumoral injections of Delta-24 in combination with CPT-11, after which animal survival was monitored. Results: Delta-24 infection caused human glioma cells to accumulate in the S phase and induced the expression and activity of topoisomerase I as shown by Western blot and in vitro enzymatic activity assays. Further, we showed that the sequential administration of Delta-24 and CPT-11 to human glioma cell cultures potentiated the CPT-11-mediated anticancer effect in vitro without modifying the replicative phenotype of the oncolytic adenovirus. In vivo experiments showed that the single intratumoral administration of Delta-24 to intracranially implanted human glioma xenografts followed by the systemic administration of CPT-11 resulted in significantly prolonged animal survival. Conclusions: The combination of Delta-24 treatment with CPT-11 showed an enhanced anticancer effect, which suggests that the interaction between adenoviral and human proteins can be exploited in rational anticancer therapies comprising replication-competent adenoviruses and conventional chemotherapeutic agents.

Resistance and relapse are still primary causes that result in poor effectiveness of chemotherapy in malignant gliomas. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating chemoresistance. We previously found that abnormal high expression of the Tie2 receptor in gliomas was associated with tumor malignancy. Here, we studied the role of Tie2 activation in drug resistance by testing the cytotoxicity of several chemotherapeutic drugs in a panel of human glioma cell lines and brain tumor stem cells and found that Tie2 activation was significantly related to chemoresistance. The essential role of Tie2 in this phenotype was illustrated by silencing Tie2 using specific siRNA, and the subsequent abrogation of the angiopoietin 1 (Ang1)-mediated chemoresistance. Using quantitative real-time PCR and functional drug efflux studies, we observed that Tie2 activation resulted in increased expression of ATP-binding cassette (ABC) transporters. Consistent with these results, downmodulation of ABCG2 or ABCC2 resulted in the inability of Tie2 activation to induce a chemoresistant phenotype. Our results indicate that Tie2 activation may be important in modifying the evolution of gliomas during conventional chemotherapy regimens, and open new avenues for the search of more effective therapies to avoid the inevitable brain tumor recurrence.

Cancer arises as a result of a stepwise accumulation of genetic changes. One of these changes, deregulation of the Rb/E2F1 pathway resulting from alterations in members of the pathway, is a hallmark of all human cancers. These mutations promote tumor development by deregulating the E2F family of transcription factors, which results in uncontrolled cell cycle progression. The E2F1 protein functions as a transcription factor that enhances cell proliferation by binding to the promoter region of several genes, including those that are involved in cell cycle regulatory activities and DNA replication. It is now becoming clear that the role of E2F1 in regulating transcription and cell growth is also highly dependent on the cellular context. This complexity is also evident from analyses of perturbations in E2F-modulated tumor development. For example, deregulated E2F1 expression can either promote or inhibit tumorigenesis depending on the nature of the other oncogenic mutations that are present. This explains the ability of E2F1 to behave as both an oncogene and tumor suppressor gene. Here we focus on reviewing the most recent evidence supporting the “addiction” of gliomas to this versatile transcription factor. We also consider the clinical relevance of this by examining the role of E2F1 as a prognosis factor and as a target for the development of novel strategies.

BACKGROUND: (blind field). METHODS: Patients with recurrent high grade gliomas were enrolled in one of two arms. Group A patients (clinical assessment group) received a single intratumoral injection of DNX-2401 into biopsy-proven recurrent glioma. Group B patients (biological endpoint group) received an initial intratumoral injection through a permanently implanted catheter (to identify the injection site) followed 14 days later by en bloc tumor resection (to obtain post-treatment specimens) and subsequent injection of DNX-2401 into the post-resection tumor bed. Dose was escalated from 1x10^7 to 3x10^10 viral particles (vp) in 8 cohorts. Patients were followed with clinical exams, MRIs, and laboratory studies. RESULTS: Histological analysis of post-treatment en bloc surgical specimens cut perpendicular to the implanted catheter proved for the first time that DNX-2401 was capable of infecting, replicating in, and killing human glioma tumor cells (Group B; N = 12). The maximum dose achieved was 3 × 10^10 vp as planned (Group A; N = 25). DNX-2401 resulted in no toxicity in each cohort. Ongoing outcome analyses show an overall median survival of 11 months. Remarkably, complete responses were seen in 3 patients (12%) (#12, #33, #37), all of whom are currently alive with no evidence of disease (3.2, 2 and 1.75 years after treatment). Serial MRIs revealed a period of increased enhancement prior to tumor regression, consistent with an inflammatory response. Histological analysis of a resected tumor from a symptomatic patient (#20) in Arm A during this period of increased MRI-enhancement identified primarily inflammatory cells (macrophages/CD8 T-cells) and only rare glioma cells. Analyses of inflammatory cytokines in serum revealed that, compared with all other patients, the 3 responders had 10-fold to 10,000-fold increases in Interleukin-12p70 (which polarizes T(h)0-cells to T(h)1-cells and promotes cell-mediated immunity). CONCLUSIONS: DNX-2401 is a new oncolytic virus with a favorable toxicity profile that is capable of replicating in and killing human glioma cells. Efficacy in this early stage trial was impressive with a 12% complete response-rate that has proven to be durable. Molecular studies suggest that viral-induced, anti-tumor cytotoxic immunity (in situ vaccine) may play a role in the anti-glioma effect of DNX-2401. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

Monocytes/macrophages are an influential component of the glioma microenvironment. However, understanding their diversity and plasticity constitute one of the most challenging areas of research due to the paucity of models to study these cells' inherent complexity. Herein, we analyzed the role of monocytes/macrophages in glioma growth by using a transgenic model that allows for conditional ablation of this cell population. We modeled glioma using intracranial GL261-bearing CSF-1R-GFP(+) macrophage Fas-induced apoptosis (MAFIA) transgenic mice. Conditional macrophage ablation was achieved by exposure to the dimerizer AP20187. Double immunofluorescence was used to characterize M1- and M2-like monocytes/macrophages during tumor growth and after conditional ablation. During glioma growth, the monocyte/macrophage population consisted predominantly of M2 macrophages. Conditional temporal depletion of macrophages reduced the number of GFP(+) cells, targeting mainly the repopulation of M2-polarized cells, and altered the appearance of M1-like monocytes/macrophages, which suggested a shift in the M1/M2 macrophage balance. Of interest, compared with control-treated mice, macrophage-depleted mice had a lower tumor mitotic index, microvascular density, and reduced tumor growth. These results demonstrated the possibility of studying in vivo the role and phenotype of macrophages in gliomas and suggested that transitory depletion of CSF-1R(+) population influences the reconstitutive phenotypic pool of these cells, ultimately suppressing tumor growth. The MAFIA model provides a much needed advance in defining the role of macrophages in gliomas.

This study was conducted to obtain evidence that restoration of the retinoblastoma protein function may have therapeutic application for gliomas.The development of glioblastoma multiforme involves progressive inactivation of several tumor suppressor genes. Abnormalities of the retinoblastoma tumor suppressor gene are found in the majority of cancers, including at least 30% of malignant gliomas. No final evidence has been produced about the role of Rb in suppressing glioma growth.To address this question, the Ad5CMV-Rb adenovirus carrying a 3.2-kb cDNA of the Rb gene was constructed. Expression of the exogenous protein was assessed by immunoblot and immunohistochemistry analyses. Growth curve assays were used to evaluate the effect of the Rb protein on glioma cell growth. Flow-cytometry analyses were used to analyze the phenotype of the cell cycle after the transfer of Rb. Human glioma xenografts implanted subcutaneously in nude mice were used for the tumorigenicity assay.After the transfer of Rb, 80% of the treated cells expressed high levels of the retinoblastoma protein for at least 7 days. Within 5 days of treatment, the cells lost the neoplastic morphology and showed marked growth suppression. The majority of the Rb-expressing cells were arrested in the G1 phase of the cell cycle. In addition, the restoration of the retinoblastoma activity rendered the human glioma cells unable to form tumors in nude mice.These findings provide direct evidence that inactivation of the retinoblastoma protein is a critical event in gliomas, and suggest that the restoration of wild-type retinoblastoma activity in these tumors may have therapeutic utility.

Cancer cells in which the <i>PTEN</i> lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than <i>PTEN</i> gene loss. The role of PI3K in the survival of cancer cells that express wild-type <i>PTEN</i> has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type <i>PTEN</i>, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the <i>PTEN</i> lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85α regulatory subunit of PI3K (Δp85) induced apoptosis. Whereas PTEN and Δ85 both inhibited activation of AKT/protein kinase B, only Δp85 inhibited c-Jun NH₂-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant <i>Rac-1</i> (Val¹²), an upstream activator of JNK, abrogated Δp85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (<i>MKK</i>)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of <i>MKK4</i>-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type <i>PTEN</i>, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.

Systematic analyses of the expression of angiogenic regulators in cancer models should yield useful information for the development of novel therapies for malignant gliomas. In this study, we analyzed tumor growth, vascularization, and angiopoietin-2 (Ang2) expression during the development of U-87 MG xenografts. We found that tumoral angiogenesis in this model follows a multistage process characterized by avascular, prolific peripheral angiogenesis, and late vascular phases. On day 4, we observed an area of central necrosis, a peripheral ring of Ang2-positive glioma cells, and reactive Ang2-positive vascular structures in the tumor/brain interface. When the tumor had developed a vascular network, Ang2 was expressed only in peripheral vascular structures. Because Ang2 expression was downmodulated in the late stages of development, probably to maintain a stable tumoral vasculature, we next studied whether sustained Ang2 expression might impair vascular development and, ultimately, tumor growth. Ang2 prevented the formation of capillary-like structures by and impaired angiogenesis in a chorioallantoic membrane chicken model. Finally, we tested the effect of sustained Ang2 expression on U-87 MG xenograft development. Ang2 significantly prolonged the survival of intracranial U-87 MG tumor-bearing animals. Examination of Ang2-treated xenografts revealed areas of tumor necrosis and vascular damage. We therefore conclude that deregulated Ang2 expression during gliomagenesis hindered successful angiogenesis and that therapies that sustain Ang2 expression might be effective against malignant gliomas.

The tyrosine kinase receptor Tie2 was initially identified as a specific vascular growth factor that governed several properties of endothelial cells under both physiological and pathological conditions. It was subsequently found that angiopoietins, the natural ligands of Tie2, modulate Tie2-dependent signaling, which in turn regulates the survival and apoptosis of endothelial cells, controls vascular permeability, and regulates the capillary sprouting that occurs during normal angiogenesis such as through development and ovarian remodeling. Tie2 also seems to play a crucial role in several vascular abnormalities, such as familial venous malformations. Beyond its critical role in angiogenesis, Tie2 also appears to maintain the long-term population and quiescent status of hematopoietic stem cells in the bone marrow stem cell niche. In cancer, Tie2 was originally found to be overexpressed in tumoral vessels. More recently, our laboratory and others have found that Tie2 is also expressed outside the vascular compartment in several types of cancer, including leukemia and solid neoplasms such as gastric tumors, breast tumors, and gliomas. The role of Tie2 in these tumoral cells is currently being explored. In this regard, our group reported the importance of Tie2-expressing glioma cells in their adhesion to the tumoral microenvironment. Because cancer may be considered as a complex organ with several cellular lineages coexisting in the same tumor, the expression of Tie2 by different tumoral compartments makes this cellular receptor an attractive target for cancer therapy.

The fact that glioblastomas, which are one of the most devastating cancers, frequently express the Delta-EGFR (epithelial growth factor receptor) also called mutant variant III of EGFR (EGFRvIII) suggests that this cancer cell-specific receptor might serve as an ideal target for cancer therapy. To assess its potential as such a target, we constructed an oncolytic adenovirus with Retargeted Infectivity Via EGFR (Delta-24-RIVER) on the backbone of Delta-24. This new oncolytic adenovirus targets, as Delta-24 does, the disrupted Rb pathway in cancer cells; in addition, this adenovirus has also been retargeted through the abrogation of CAR binding (Y477A mutation in adenoviral fiber protein) and insertion of an EGFRvIII-specific binding peptide in the HI loop of the fiber protein. As compared with Delta-24, Delta-24-RIVER induced EGFRvIII-selective cytotoxicity in U-87 MG isogenic cell lines and in tetracycline-inducible EGFRVIII expressing U-251 MG cells. Accordingly, by tittering the viral progeny and examining fiber protein expression in the above cells, we showed that the replication of this new construct also correlated with EGFRvIII expression. Consistently, immunohistochemistry staining of the adenoviral capsid protein hexon in the virus-treated tumors revealed that the virus replicated more efficiently in EGFRvIII-expressing U-87 MG.ΔEGFR xenografts than in the tumors grown from U-87 MG cells. Importantly, treatment with Delta-24-RIVER prolonged the survival of animals with intracranial xenografts derived from U-87 MG.ΔEGFR cells. Therefore, our results constitute the first proof of the direct targeting of a cancer-specific receptor using an oncolytic adenovirus.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24-RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24-RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24-RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24-RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24-RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24-RGD for this devastating disease.

In recent years, we have seen an important progress in our comprehension of the molecular basis of pediatric brain tumors (PBTs). However, they still represent the main cause of death by disease in children. Due to the poor prognosis of some types of PBTs and the long-term adverse effects associated with the traditional treatments, oncolytic viruses (OVs) have emerged as an interesting therapeutic option since they displayed safety and high tolerability in pre-clinical and clinical levels. In this review, we summarize the OVs evaluated in different types of PBTs, mostly in pre-clinical studies, and we discuss the possible future direction of research in this field. In this sense, one important aspect of OVs antitumoral effect is the stimulation of an immune response against the tumor which is necessary for a complete response in preclinical immunocompetent models and in the clinic. The role of the immune system in the response of OVs needs to be evaluated in PBTs and represents an experimental challenge due to the limited immunocompetent models of these diseases available for pre-clinical research.

Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-alphaVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-alphaVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-alphaVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF(165)cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-alphaVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer.

The last stage of the adenovirus replication cycle, lysis, is considered not very efficient and remains poorly understood. Pathogen infection induces autophagy in eukaryotic cells. In the case of viruses, autophagy is a double-edged sword that can either facilitate or impede replication. On one hand, autophagy reduces the replication capability of the herpesviruses. On the other hand, the RNA virus poliovirus uses autophagosomes to form replication complexes. Recently we characterized the autophagy induced by the oncolytic adenovirus Delta-24-RGD in brain tumor stem cells. Late in the adenoviral infectious cycle, we observed remarkable upregulation of the Atg12-Atg5 complex and prominent autophagy. In addition, adenovirus-induced autophagy results in disruption of the cytoplasmic structure and the continuity of the cellular membrane. We speculate that adenoviruses induce autophagy to facilitate the release of viral progeny at the end of the infectious cycle. The substitution of 'autophagy' for 'lysis' is not just semantic. Because autophagy is a genetically programmed process and not a passive phenomenon, it immediately suggests interactions between adenovirus proteins and autophagy regulators. Understanding the mechanism underlying adenovirus-mediated autophagy should propel the development of novel vectors with enhanced capability to release viral progeny and, as a result, morepotent oncolytic effect.

Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

Viroimmunotherapy is evolving as a strong alternative for the standard treatment of malignant gliomas. Promising results from a recent clinical trial testing the anticancer effect of Delta-24-RGD in patients with glioblastoma suggested the induction of antitumoral immunity after viral administration. To further enhance the anti-glioma immune effect, we have armed Delta-24-RGD with the costimulatory ligand GITRL (Delta-24-GREAT [Glucocorticoid Receptor Enhanced Activity of T cells]).We tested the infectivity and replication of Delta-24-GREAT, and the expression of ectopic GITRL in human and murine glioma cell lines. In vivo experiments involved the intracranial implantation of glioma cells into an immunocompetent model to study the anticancer effect, and rechallenging experiments to study long-term protection. Phenotypic and functional characterization of lymphocyte populations were performed by FACS and ELISA for Th1 cytokines expression, respectively.Our results showed that Delta-24-GREAT infects and induces the expression of GITRL. Delta-24-GREAT prolonged the survival of glioma-bearing immunocompetent mice and resulted in both anti-viral and anti-glioma immune responses, including increased frequency of central memory CD8+ T cells. Rechallenging the surviving mice with a second implantation of glioma cells did not lead to tumor growth; however, the surviving mice developed lethal tumors when B16/F10 melanoma cells were implanted intracranially, strongly indicating that the immune response was specific for glioma antigens.GITRL-armed Delta-24-RGD treatment results in an antigen-restricted antitumor memory, an enhanced anti-glioma effect, and the generation of central immune memory. Our results strongly indicate that this strategy represents a vertical advance in virotherapy designed to treat patients with malignant brain tumors.

Oncolytic or tumor-selective adenoviruses are constructed as novel antiglioma therapies. After infection, the invading genetic adenoviral material is activated within the host cell. E1A and E1B adenoviral proteins are expressed immediately. E1A protein interacts with cell cycle regulatory proteins, such as retinoblastoma (Rb), driving the cell into the S phase and ensuing viral replication. The action of E1A stimulates the cellular p53 tumor suppressor system, which results in growth arrest or apoptosis, and halts adenovirus replication. However, adenoviral E1B interacts with p53 protein, preventing the DNA replication process from being abrogated by the induction of p53-mediated apoptosis. It was subsequently hypothesized that mutant adenoviruses that were unable to express wild-type E1A or E1B proteins could not replicate in normal cells with functional Rb or p53 pathways but instead would replicate and kill glioma cells that had defects in the regulation of these tumor suppressor pathways. Mutant E1B adenoviruses have already entered the clinical setting as an experimental treatment for patients with malignant gliomas. Mutant E1A adenoviruses are now in preclinical development as antiglioma therapy. In this review, the authors describe the mechanisms underlying the production of oncolytic adenoviruses, preclinical and clinical experiences with specific oncolytic adenoviruses, and the possibilities of combining mutant oncolytic adenoviruses with gene therapy or conventional therapies for managing malignant gliomas.

Replication-competent oncolytic adenoviruses hold considerable promise for treating malignant gliomas. The toxicity of the clinically tested E1B-55 kDa mutant virus is negligible; however, its full clinical potential is still being evaluated. The purpose of the present study is to compare the antiglioma activity in vitro and in vivo between Delta-24, an E1A mutant adenovirus, and RA55, an E1B-55 kDa mutant adenovirus. We selected human glioma cell lines that were tumorigenic in nude mice and express wild-type p53 (U-87 MG, D54 MG) or mutant p53 (U-251 MG, U-373 MG) protein. Our studies demonstrated that Delta-24 induced a more potent antiglioma effect in vitro than RA55. Moreover, Delta-24 replicated markedly more efficiently than RA55 in both wild-type and mutant p53 scenarios. Importantly, direct intratumoral injection of Delta-24, but not RA55, significantly suppresses tumor growth in intracranial (U-87 MG, U-251 MG) or subcutaneous (D54 MG) animal models. Staining for hexon protein detected replicating adenoviruses in xenografts infected with Delta-24, but not with RA55. Collectively, these data indicate that E1A mutant adenoviruses targeting the Rb pathway are more powerful putative agents for antiglioma therapy than E1B mutant adenoviruses, and suggest that E1A mutant adenoviruses should be tested in the clinical setting for patients with malignant gliomas.

Abstract The Rb/E2F pathway is deregulated in most human brain tumors, and the finding that loss of E2F1 reduced pituitary tumorigenesis in Rb+/− mice suggests that loss of pRb induces brain tumors by activating E2F1. We therefore investigated the role of E2F1 in the development and maintenance of brain cancer using a transgenic mouse model engineered to express E2F1 specifically within glial cells (GFAP-tgE2F1). GFAP-tgE2F1 mice developed a highly penetrant phenotype characterized by neurologic defects, and examination of the brains revealed the presence of brain tumors in 20% of these animals. Importantly, the distribution of tumors according to mouse age suggests the existence of a bimodal pattern of tumor development, forcing a comparison with the human disease. Mice, at an early age, with deregulated E2F1 show the formation of embryonal brain tumors such as medulloblastoma, choroid plexus carcinoma, and primary neuroectodermal tumor. Conversely, at an older age, mice escaping embryonal tumor formation present with malignant gliomas, which are typically identified in the human adult population. Thus, this study offers the first evidence for a global role of E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. [Cancer Res 2007;67(9):4005–9]

In 2004, brain tumor stem cells (BTSCs) were isolated from surgical human malignant gliomas. This cancer cell population has been identified as the root for tumor initiation and resistance to therapies. Thus, it is imperative to develop new therapies that can eradicate this subpopulation to improve the prognosis of patients with brain tumors. Our group previously reported the antiglioma effect of the tumor-selective oncolytic adenovirus Delta-24-RGD that is now being tested in a phase I clinical trial for patients with malignant gliomas. We also showed that Delta-24-RGD infects, replicates in, and induces cell death in BTSCs. Interestingly, we observed that adenoviral-infected cells undergo autophagy and that autophagy-related cytoplasmic vacuolization might be part of the lysis process. Here, we summarize the materials and methods used in our study as follows: establishment of neurosphere cultures from surgical samples of human glioblastoma multiformes; assessment of stem cell markers; examination of adenoviral receptors in BTSCs; evaluation of the cytotoxicity induced by oncolytic adenoviruses; and assessment of autophagy in oncolytic adenovirus-infected BTSCs in vitro, and finally we describe a method to detect upregulation of the autophagy-related protein Atg5 in tumors treated with Delta-24-RGD.

Abstract BACKGROUND Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 10 6 cells/20 mL to 1 × 10 8 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 10 8 cells/10 mL (10 7 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.

Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells.Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3).Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD-treated tumors revealed increased CD8+ T-cell infiltration.Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

Currently, there is a lack of effective therapies for the majority of glioblastomas (GBMs), the most common and malignant primary brain tumor. While immunotherapies have shown promise in treating various types of cancers, they have had limited success in improving the overall survival of GBM patients. Therefore, advancing GBM treatment requires a deeper understanding of the molecular and cellular mechanisms that cause resistance to immunotherapy. Further insights into the innate immune response are crucial for developing more potent treatments for brain tumors. Our review provides a brief overview of innate immunity. In addition, we provide a discussion of current therapies aimed at boosting the innate immunity in gliomas. These approaches encompass strategies to activate Toll-like receptors, induce stress responses, enhance the innate immune response, leverage interferon type-I therapy, therapeutic antibodies, immune checkpoint antibodies, natural killer (NK) cells, and oncolytic virotherapy, and manipulate the microbiome. Both preclinical and clinical studies indicate that a better understanding of the mechanisms governing the innate immune response in GBM could enhance immunotherapy and reinforce the effects of chemotherapy and radiotherapy. Consequently, a more comprehensive understanding of the innate immune response against cancer should lead to better prognoses and increased overall survival for GBM patients.

Abstract Background Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. Methods The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. Results The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. Conclusions The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Oncolytic viruses (OVs) are biological therapeutic agents that selectively destroy cancer cells while sparing normal healthy cells. Besides direct oncolysis, OV infection induces a proinflammatory shift in the tumor microenvironment and the release of tumor‐associated antigens (TAAs) that might induce an anti‐tumor immunity. Due to their immunostimulatory effect, OVs have been explored for cancer vaccination against specific TAAs. However, this approach usually requires genetic modification of the virus and the production of a new viral vector for each target, which is difficult to implement for low prevalent antigens. In a recent study, Chiaro et al. presented an elegant proof of concept on how to implement the PeptiCRAd vaccination platform to overcome this limitation for the treatment of mesothelioma. Authors showed the feasibility of identifying immunogenic TAAs in human mesothelioma and using them to coat oncolytic adenovirus particles. The result was a customized virus‐based cancer vaccine that circumvents time and resource‐consuming steps incurred from genetically engineering viruses. Although some questions remain to be addressed, this interesting approach suggests novel strategies for personalized cancer medicine using oncolytic virotherapy.

The ability to transfer exogenous genes to cancer cells has yielded a wealth of information about the neoplastic processes that occur at molecular and cellular levels. Current research focuses on defining the biochemical factors that govern the interplay between cell growth and cell death in gliomas. The identification of tumor suppressor genes has greatly enhanced our understanding of the molecular mechanism of brain tumors. Accomplishing the transition from basic science to clinical practice is a major challenge for the future of brain tumor research. The concept of tumor suppressor genes is examined, with particular emphasis on the functional studies of the role of the <i>p53, p16, Rb,</i> and <i>PTEN/MMAC1</i> genes in gliomas. Moreover, recent advances linking tumor suppressor genes, apoptosis, and cell-cycle control pathways in brain tumors are reviewed. The ability to detect mutations in tumor suppressor genes plays an important role in cancer diagnosis and prognosis. Perhaps of greatest significance has been the realization that tumor suppressor genes may provide novel targets for development of specific anticancer therapies for brain tumors.

The treatment for malignant gliomas is suboptimal. Oncolytic adenoviruses hold the promise of being effective agents for the treatment of solid tumors. Importantly, the first oncolytic viral therapy has just been approved for use in combination with chemotherapy for late-stage refractory nasopharyngeal cancer by the Chinese State FDA, following a successful Phase III randomized clinical trial. The concept underlying treatment with oncolytic adenoviruses is based on cancer selectivity by confining viral replication and infectivity to cancer cells. For this purpose, the main strategies used currently to modify the viruses include: functional deletions in essential viral genes; tumor- or tissue-specific promoters used to control the expression of these viral genes; and tropism modification to redirect adenovirus to the cancer cell surface. In the near future, oncolytic adenoviruses need to be optimized to fully realize their potential as critical anticancer tools and, thus, improve the prognosis for patients with malignant gliomas.

Osteosarcoma is an aggressive bone tumor occurring primarily in pediatric patients. Despite years of intensive research, the outcomes of patients with metastatic disease or those who do not respond to therapy have remained poor and have not changed in the last 30 years. Oncolytic virotherapy is becoming a reality to treat local and metastatic tumors while maintaining a favorable safety profile. Delta-24-ACT is a replicative oncolytic adenovirus engineered to selectively target cancer cells and to potentiate immune responses through expression of the immune costimulatory ligand 4-1BB. This work aimed to assess the antisarcoma effect of Delta-24-ACT. MTS and replication assays were used to quantify the antitumor effects of Delta-24-ACT in vitro in osteosarcoma human and murine cell lines. Evaluation of the in vivo antitumor effect and immune response to Delta-24-ACT was performed in immunocompetent mice bearing the orthotopic K7M2 cell line. Immunophenotyping of the tumor microenvironment was characterized by immunohistochemistry and flow cytometry. In vitro, Delta-24-ACT killed osteosarcoma cells and triggered the production of danger signals. In vivo, local treatment with Delta-24-ACT led to antitumor effects against both the primary tumor and spontaneous metastases in a murine osteosarcoma model. Viral treatment was safe, with no noted toxicity. Delta-24-ACT significantly increased the median survival time of treated mice. Collectively, our data identify Delta-24-ACT administration as an effective and safe therapeutic strategy for patients with local and metastatic osteosarcoma. These results support clinical translation of this viral immunotherapy approach.

The coronavirus disease 2019 (COVID-19) pandemic has produced a new global challenge for patients with cancer. The disease and the immunosuppression induced by cancer therapies have generated a perfect storm of conditions to increase the severity of the symptoms and worsen the prognosis. However, a few clinical reports showcased the power of viruses to induce remission in some patients suffering from liquid tumors. Here, we reviewed six cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that resulted in cancer remission, simultaneously highlighting the strengths and the unique challenges of oncolytic virotherapy. Virotherapy has become a special case of cancer immunotherapy. This paradigm-shifting concept suggests that oncolytic viruses are not only promising agents to combat particularly immunologically suppressed, immunotherapy-resistant tumors but also that the trigger of local inflammation, such as SARS-CoV-2 infection of the respiratory pathways, may trigger an abscopal effect sufficient to induce the remission of systemic cancer.

ancer is a disease of a series of genes.Thus, theoretically, brain tumors could be treated by targeting their fundamental molecular defects.Currently, most of the approved clinical protocols for gene therapy involve cancer patients.Several of these protocols are designed to improve the treatment of brain tumors.In this brief report, we analyze the rationale, advantages, and disadvantages of a series of gene therapy approaches against brain tumors that include transfer of tumor suppressor genes and cell-cycle modulators; suicide or prodrug strategies; immunogene therapy; antiangiogenesis; and oncolytic virus therapy.In summary, in this review, we highlight the translational advances in molecular medicine that broaden our battery of therapies for patients with brain tumors.

The current standard of care for malignant gliomas is surgical resection and radiotherapy followed by extended adjuvant treatment with the alkylating agent temozolomide. Temozolomide causes DNA damage, which induces cell death. Through changes in the DNA-repair machinery, glioma cells develop resistance to temozolomide, compromising the therapeutic effect of the drug. Oncolytic viruses, such as herpes simplex viruses and adenoviruses, are being introduced into clinical trials as a new treatment for this malignancy. Biological studies have revealed that these viruses use mechanisms to either inactivate (adenovirus) or take advantage of (herpes simplex virus) the cellular DNA-repair machinery to achieve productive replication. Adenoviruses express proteins from the early genes to either downregulate the damage-repair enzyme, O(6)-methylguanine-DNA methyltransferase, or degrade poly (ADP-ribose) polymerase or the Mre11-Rad50-NBS1 complex, which detects DNA strand breaks. Temozolomide enhances herpes simplex virus oncolysis by upregulating the DNA repair-related genes growth arrest DNA damage 34 and ribonucleotide reductase. The interactions between viruses and the DNA-repair machinery suggest that a combined temozolomide and viral therapy will overcome the limitations of a single therapy by diminishing chemoresistance or enhancing oncolysis. This hypothesis has been supported by promising findings from preclinical and clinical studies.

Malignant gliomas, the most common subtype of primary brain tumor, are aggressive, highly invasive, and neurologically destructive. First-line treatment of gliomas consists of surgery and radiotherapy, followed by chemotherapy with temozolomide. However, even with this strong regimen, the prognosis of patients with the most malignant variant, glioblastoma multiforme is poor. Because of the lack of effective treatments and the high vascularity that characterizes these tumors, antiangiogenic therapy of gliomas is being studied. This approach is supported by encouraging preclinical data in both in vitro and in vivo models. Clinical studies have shown that these agents do not cause high toxicity; and due to the effect they exert on vessel permeability, patients can avoid the use of corticosteroids and their accompanying adverse. Moreover, in studies of these agents, we have observed improvements in several parameters normally used to measure therapy response. However, whether these parameters are reliable for understanding and measuring the anticancer effect of antiangiogenic molecules is unknown. In addition, resistance to angiogenic therapy is already evident, and in studies performed in animal models, this resistance was associated with the appearance of more invasive phenotypes. These models give us the opportunity to further understand what causes therapy resistance and will allow us to test new combination therapies. Future studies are directed to understand if it is possible to target not only the bulk of the tumor but also the putative tumor niche composed of tumor cells, vessels, and stroma. Keywords: Brain tumor, angiogenesis, cancer stem cell, antibody, tyrosine kinase inhibitor

Introduction Glioblastoma is the most malignant brain tumor in adults and is associated with poor survival despite multimodal treatments. Glioma stem-like cells (GSCs) are cells functionally defined by their self-renewal potential and the ability to reconstitute the original tumor upon orthotopic implantation. They have been postulated to be the culprit of glioma chemo- and radio-resistance ultimately leading to relapse. Understanding the molecular circuits governing the GSC compartment is essential. SOX2, a critical transcription regulator of embryonic and neural stem cell function, is deregulated in GSCs however; the precise molecular pathways regulated by this gene in GSCs remain poorly understood. Results We performed a genome-wide analysis of SOX2-regulated transcripts in GSCs, using a microarray. We identified a total of 2048 differentially expressed coding transcripts and 261 non-coding transcripts. Cell adhesion and cell-cell signaling are among the most enriched terms using Gene Ontology (GO) classification. The pathways altered after SOX2 down-modulation includes multiple cellular processes such as amino-acid metabolism and intercellular signaling cascades. We also defined and classified the set of non-coding transcripts differentially expressed regulated by SOX2 in GSCs, and validated two of them. Conclusions We present a comprehensive analysis of the transcriptome controlled by SOX2 in GSCs, gaining insights in the understanding of the potential roles of SOX2 in glioblastoma.

AbstractCancer arises from a stepwise accumulation of genetic changes. Among these changes,deregulation of the Rb/E2F1 pathway and constitutively active telomerase are pivotalmilestones for the attainment of immortality and maintaining the neoplastic phenotype.We recently showed the Rb/E2F1 pathway to be a direct modulator of telomerase activityin normal and cancer cells, specifically in malignant gliomas. In addition, we reportedthat the correlation between the levels of expression of E2F1 and hTERT -the catalyticsubunit of telomerase- in a subset of patients with glioblastoma multiforme confersclinical relevance to the role of E2F1 in triggering or maintaining hTERT expression.Here we review the evidence supporting the mechanistic linkage between E2F1 andtelomerase activation. We also consider the clinical implications of this association interms of prognostic significance and opportunities for the development of new and morerational therapeutic strategies.

The retinoblastoma protein (RB) is the product encoded by the first tumor suppressor gene identified, and exerts a pleitropic spectrum of functions. Although RB interacts with hundreds of proteins, one of the key factors to understand many of its functions is the interaction with the E2F family of transcription factors. For instance, the physical interaction between RB and E2F1, a pro-apoptotic member of the E2F family, is a master gatekeeper for cell cycle progression. We have recently reported that the RB-E2F1 axis has an important role in the regulation of autophagy. Our studies show that the transfer of Rb to Rb-null cells results in the induction of autophagy, whereas transfer of E2F1 results in the induction of apoptosis. Rb and E2F1 proteins are part of two families of proteins whose members may play agonistic or antagonistic roles. It would be very interesting to ascertain whether other members of the RB family, p107 and p130, are also regulators of autophagy, as it could be logically expected, and whether there is overlapping in the regulation of autophagy by RB and the RB-homologous proteins.

The fusion protein VEGF(121)/rGel composed of the growth factor VEGF(121) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF(121)/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.We investigated the binding, cytotoxicity and internalization profile of VEGF(121)/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with (125)I-VEGF(121)/rGel demonstrated binding specificity that was competed with unlabeled VEGF(121)/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF(121)/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC(50) levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF(121)/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF(121)/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF(121)/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF(121)/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.Taken together, these data confirm the selectivity of VEGF(121)/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.

Angiogenesis is thought to depend on a perfectly coordinated balance between endogenous-positive and negative regulatory factors. Of these factors, the vascular endothelial growth factor (VEGF) and angiopoietins (Angs) seem to play an essential role. Recently, we reported the expression of the Ang-natural receptor, Tie2, in neoplastic astrocytic cells within gliomas. Because of the VEGF/Ang2 functional partnership together with the presence of Tie2 in gliomas, we hypothesized a role of Ang2 on the modulation of VEGF levels in these tumors. We examined the effect of Ang2 on VEGF expression in a panel of glioma cells, which showed that Ang2 inhibited VEGF expression at both mRNA and protein levels in Tie2-expressing cells, but not in Tie2-negative cells. VEGF promoter analysis showed that Ang2 regulated VEGF expression at the transcriptional level in relation to a decrease in HIF-1alpha expression and HIF-DNA-binding activity. Tie2 silencing by siRNA rescued the Ang2-mediated downmodulation of VEGF, suggesting an essential role for Tie2 in this regulatory loop. To our knowledge, this is the first report on the role of Ang2/Tie2 in the regulation of HIF-1alpha/VEGF expression, providing additional evidence of the intrinsic coordination that occurs among these factors during angiogenesis.

Glioblastoma (GBM) is the most aggressive brain tumor, with 12-15 months median survival time despite current treatment efforts. Among the alternative treatment approaches that have gained acceptance over the last decade is the use of replication-competent oncolytic adenoviruses, which are promising due to their relatively low toxicity and tumor-specific targeting. Three-dimensional (3D) tumor models can mimic the physiological microenvironment of GBM tumors and provide valuable information about the interaction between tumor cells and adenoviruses. Therefore, robust in vitro 3D tumor models are critical to investigate the mechanisms underlying tumor progression and explore the cytotoxicity effect of the adenovirus on tumor cells. In this study, we used a hydrogel microwell platform to generate in vitro 3D GBM spheroids and studied their interactions with the Delta-24-RGD adenovirus. The results showed that the cultured 3D spheroids were successfully infected by the Delta-24-RGD. A significant cell lysis was observed. Cell viability was decreased approximately 37%, 54% and 65% with 10, 50, and 100 MOIs, respectively. The infection of the Delta-24-RGD was found more effective on 3D spheroids when compared to 2D monolayer cell culture. These results implicate that our hydrogel microwell platform could provide a promising 3D model to investigate the oncolytic potential of the viruses in vitro.

Abstract Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2–20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12months was 52.7% (95% CI 40.1–69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5months (10.7–13.5months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05–0.87). A total of 56.2% (95% CI 41.1–70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Adenoviral E1A is a versatile protein that can reprogram host cells for efficient viral replication. The nuclear import of E1A is mediated by a nuclear localization signal; however, whether E1A can be actively exported from the nucleus is unknown. We first reported a CRM1-dependent nuclear export signal (NES) in E1A that is conserved in the group C adenoviruses. We showed that CRM1 and E1A coimmunoprecipitated and that blockage of CRM1 function by leptomycin B or small interfering RNA resulted in the nuclear localization of E1A. Through mutational analyses, we identified an active canonical NES element within the E1A protein spanning amino acids 70-80. We further demonstrated that phosphorylation of adjacent serine (S)89 resulted in the cytoplasmic accumulation of E1A. Interestingly, coincident with the accumulation of cells in the S/G2/M phase and histone H1 phosphorylation, E1A was relocated to the cytoplasm at the late stage of the viral cycle, which was blocked by the CDC2/CDK2 inhibitor roscovitine. Importantly, titration of the progenies of the viruses in infected cells showed that the replication efficiency of the NES mutant adenovirus was up to 500-fold lower than that of the wild-type adenovirus. Collectively, our data demonstrate the existence of a NES in E1A that is modulated by the phosphorylation of the S89 residue and the NES plays a role for an efficient viral replication in the host cells.

Three studies have reported the results of phase 1 clinical trials of virotherapy for malignant gliomas, underscoring the momentum in the field. In the first study, 1 Lang FF Conrad C Gomez-Manzano C et al. Phase I study of DNX-2401 (Delta-24-RGD) oncolytic adenovirus: replication and immunotherapeutic effects in recurrent malignant glioma. J Clin Oncol. 2018; 36: 1419-1427 Crossref PubMed Scopus (247) Google Scholar investigators used a replication-competent, tumour-selective adenovirus to treat patients with recurrent glioblastomas. In a group in which patients received virus treatment only (n=25), 20% of the patients survived more than 3 years after treatment, and three patients had tumour volume reductions of 95% or more. In the second study, 2 Desjardins A Gromeier M Herndon JE et al. Recurrent glioblastoma treated with recombinant poliovirus. N Engl J Med. 2018; 379: 150-161 Crossref PubMed Scopus (283) Google Scholar intratumoural infusion of a tumour-selective poliovirus into patients with recurrent malignant glioma improved overall survival, which reached a plateau of 21% at 24 months that was sustained at 36 months. In the most recent report, 3 Friedman GK Johnston JM Bag AK et al. Oncolytic HSV-1 G207 immunovirotherapy for pediatric high-grade gliomas. N Engl J Med. 2021; 384: 1613-1622 Crossref PubMed Scopus (30) Google Scholar investigators treated children with malignant gliomas with intratumoural injections of a herpes simplex virus. The median overall survival time was 12·2 months (8·0–16·4), and four (36%) of 11 patients were still alive 18 months after virotherapy. These trials support the concept of oncolytic virotherapy as a unique approach for cancer immunotherapy. 4 Jiang H Gomez-Manzano C Rivera-Molina Y Lang FF Conrad CA Fueyo J Oncolytic adenovirus research evolution: from cell-cycle checkpoints to immune checkpoints. Curr Opin Virol. 2015; 13: 33-39 Crossref PubMed Scopus (31) Google Scholar Neural stem cell delivery of an oncolytic adenovirus in newly diagnosed malignant glioma: a first-in-human, phase 1, dose-escalation trialNSC-CRAd-S-pk7 treatment was feasible and safe. Our immunological and histopathological findings support continued investigation of NSC-CRAd-S-pk7 in a phase 2/3 clinical trial. Full-Text PDF

The therapeutic efficacy of standard cancer treatments such as chemotherapy may be improved if they are combined with gene-therapy. Less than 30% of patients with glioblastoma multiforme respond to adjuvant chemotherapy. Actively dividing cells are generally more sensitive to chemotherapy than are non-dividing cells. To determine whether forced cell-cycle progression selectively sensitizes tumor cells to alkylating agents, we examined the effects of overexpressing the E2F-1 protein (a positive regulator of cell-cycle progression) on the sensitivity of two malignant human glioma cell lines, U-251 MG and D-54 MG, to BCNU and temozolomide. Treating these cells with 20-35 μM BCNU or 20-30 μM temozolomide resulted in 50% growth inhibition (IC50) within 4 or 6 days, respectively. By contrast, cells that were first induced to overexpress E2F-1 protein by infection with an adenoviral vector had IC50s that were 37-50% lower. Conversely, transferring the cyclin-dependent kinase inhibitors p16 and p21 to the cells, also by adenoviral infection, produced 3 to 4-fold increases in chemoresistance. Cell-cycle analyses showed that the combination of E2F-1 overexpression and treatment with BCNU or temozolomide increased the proportion of cells in S phase, but the combination of p16 or p21 overexpression and drug treatment reduced the proportion of cells in S phase. These observations suggest that overexpression of genes that positively control cell-cycle progression may be useful for increasing the sensitivity of glioma cells to alkylating agents.

Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of ElA protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CRi (Delta-39), CR2 (Delta-24) regions, or both regions (Delta-24/39) of the ElA protein. Delta-39, Delta-24 induced a cytopathic effect with similar efficiency in glioma cells, a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, ElA protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CRi mutant ElA proteins, inhibition of the proteasome activity resulted in the striking rescue of ElA levels. We conclude that the level of ElA protein is a critical determinant of oncolytic phenotype, we propose a completely novel strategy for the design, construction of conditionally replicative adenoviruses.

Current therapy for glioma is suboptimal. The transfer of apoptosis genes to tumors constitutes one of the most promising strategies for cancer gene therapy. We have previously shown that massive apoptosis occurs when wild-type p53 or E2F-1 expression is induced in glioma. However, the mechanism of action and the efficiency in inducing apoptosis of these two proteins are not similar. Adenovirus-mediated p53 gene transfer is ineffective in causing apoptosis in glioma cells that retain wild-type p53 genotype or overexpress the p21 protein. The p16/Rb/E2F pathway is the most frequent target of genetic alterations in gliomas, and therefore constitutes a suitable target for gene therapy strategies. However, the transfer of either the p16 or Rb gene to glioma cells results in cytostatic effect. The E2F-1 protein is able to induce generalized apoptosis in gliomas independently of the p53, p16 or Rb status. In addition, p21- or p16-mediated growth arrest did not protect glioma cells from E2F-1-mediated apoptosis. The apoptotic molecule bax is induced in p53-mediated apoptosis, but bax is not induced in E2F-1-mediated apoptosis in glioma cells. Careful selection of patients may be necessary before designing therapeutic strategies using either p53 or E2F-1 as a therapeutic tools for glioma patients.

Delta-24-RGD is a novel replication-competent, tumor-selective, oncolytic adenovirus with enhanced infectivity. Based on promising preclinical studies, we undertook a first-in-human Phase I clinical trial with biological endpoints in order to assess the capacity of Delta-24-RGD to replicate in human gliomas, and to determine safety and initial efficacy. Patients with recurrent high-grade gliomas were enrolled in one of two arms. Group A (clinical assessment group) received a single intratumoral injection of Delta-24-RGD into biopsy-proven recurrent glioma. Group B (biological endpoint group) received an initial intratumoral injection through an implanted catheter followed 14 days later by en bloc tumor/catheter resection (to obtain post-treatment specimens) and subsequent injection of Delta-24-RGD into the post-resection cavity. Dose was escalated in 8 cohorts (1x107-3x1010vp). Histological analysis of post-treatment en bloc surgical specimens proved for the first time that Delta-24-RGD is capable of infecting, replicating in, and killing/lysing glioma tumor cells (Group B; N = 12). Delta-24-RGD resulted in no toxicity and the MTD was 3 × 1010vp (Group A; N = 25). Outcome analyses show an overall median survival of 11 months. Remarkably, complete responses were seen in 3 patients (12%) all of whom are still alive with no evidence of disease 3.2, 2 and 1.75 years after treatment. Serial MRIs revealed increased enhancement before tumor regression, consistent with inflammatory-mediated responses. Histological analysis of resected tumor from a symptomatic patient in Arm A during the period of increased MRI-enhancement identified macrophages and CD8 T-cells with only rare tumor cells. Compared with all other patients, the 3-responders had 10-fold to 1000-fold increases in Interleukin-12p70 (which induces Th1-responses and cell-mediated immunity). In conclusion, Delta-24-RGD is a new oncolytic virus with a favorable toxicity-profile that is capable of cycles of infection-replication-lysis in human glioma cells, and that can produce durable complete-responses in subsets of patients. Viral-induced anti-tumor-immunity likely plays a role in the anti-glioma effect.

During the last decade, we have witnessed several key advances in the field of neuro-oncology. First, there were conceptual advances in the molecular and cell biology of malignant gliomas including the discovery in 2004 of brain tumor stem cells. Second, the Cancer Genome Atlas project has been extremely useful in the discovery of new molecular markers, including mutations in the IDH1 gene, and has led to a new classification of gliomas based on the differentiation status and mesenchymal transformation. In addition, use of the 1p/19q marker and O6-methylguanine-DNA methyltransferase methylation status have been identified as guides for patient selection for therapies and represent the first steps toward personalized medicine for treating gliomas. Finally, progress has been made in treatment strategies including the establishment of temozolomide as the criterion standard for treating gliomas, the adoption of bevacizumab in the clinical setting, and developments in experimental biological therapies including cancer vaccines and oncolytic adenoviruses.

Gliomas are highly resistant to any kind of treatment. Multiple genetic abnormalities exist in gliomas indicating that effective gene therapy should be directed towards replacement of multiple rather than single genes. Bax is a protein of the Bcl-2 family that promotes apoptosis and functions as a tumor suppressor gene. The E2F family of transcription factors plays a pivotal role in the regulation of cell-cycle and cell-death related genes in gliomas. We examined the therapeutic potential of the simultaneous transfer of Bax and E2F molecules (1, 2 or 4) to gliomas. We used first generation E1A-deleted adenoviral vectors to transduce the E2Fs and Bax cDNAs. The recombinant adenoviral vector encoding bax uses the inducible Cre-loxP system to transduce the protein expression. Western blot analysis and immunofluorescence assays demonstrated high level of expression of the exogenous proteins. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated an additive effect of these molecules to induce cell death via apoptosis. Western blot analysis showed that the ectopic expression of E2F-1 decreased the level of expression of Bax. These results indicate that E2F-1 and Bax have an additive anti-glioma effect when expressed simultaneously at high levels. Our data also suggest that Bax is not involved in the E2F-1-mediated apoptosis.

See the article by Saga et al, on pages 1048–1056. The golden age of biochemistry was characterized by the meticulous description of the main catabolic and anabolic cellular pathways.1 During catabolism, for instance, under unfavorable environmental conditions, glucose and other immediate principles are degraded to produce energy, most significantly in the form of ATP. During anabolism, prior to cell division, energy is consumed to synthesize and assemble carbohydrates, proteins, and lipids.1 The catabolic process of glucose utilization and the production of ATP may occur in normoxic conditions with the efficient production of ATP, or under hypoxic conditions leading to the production of pyruvate and lactic acid and yielding a much lower number of ATP molecules. While these are biochemical features typical of normal cells, the classical studies by Otto Warburg1 suggest that a reprogramming of metabolism occurs in most cancer cells. Warburg found that cancer cells use the anaerobic pathway (i.e., fermentation, resulting in the production of lactate) to metabolize glucose, even in the presence of a sufficient supply of oxygen. Why cancer cells should prefer the fermentation pathway when oxygen is abundantly available was not clear, but it was speculated to be related to the need for an anabolic metabolism to produce enough lipids, proteins, and carbohydrates to allow the safe division of cells during cancer proliferation.2,3 We now know that solid tumors in general and gliomas in particular proliferate under hypoxic conditions,4 so the shift from glucose metabolism to anaerobic conditions is probably required for cell proliferation in tumors. Major progress in understanding the Warburg effect was made during the past decade, when the molecular pathways regulating this re-directed metabolism in cancer were described.5 Importantly, the accumulating evidence suggests that genetic aberrations inherently related to cancer initiation and progression are important factors in the redirection of cancer metabolism in tumors.6 Inactivation of tumor suppressor genes plays an important role in the reprogramming of the metabolism of cancer cells, and some of these genes are partially responsible for the Warburg effect. For instance, p53 decreases glycolysis and increases the use of the tricarboxylic acid cycle and oxidative phosphorylation and, through p21 activation, regulates responses to abnormal redox potentials and high levels of reactive oxygen species.6 Thus, loss of p53 function may lead to the Warburg effect. The loss of PTEN, another tumor suppressor gene in gliomas, and the concurrent increase of AKT-1 phosphorylation also favor the Warburg effect.6 On the other hand, the activation of hypoxia-inducible factors (HIFs)7 and oncogenes such as cMYC8,9 plays major roles in regulating the metabolism of cancer cells.10 HIFs are regulated by the cellular hypoxic response, and among the HIF-regulated genes are 9 of the 10 enzymes that function in glycolysis and several other proteins that promote anabolism.11 cMyc activates the transcription of more than a thousand genes that regulate many aspects of cellular biology, including cell metabolism. cMyc, which enhances the rate of protein synthesis, mitochondrial mass, and cell mass, may control both glycolysis and metabolism under aerobic conditions.8 Therefore, HIFs and cMyc may compete or cooperate for the control of metabolic pathways in cancer cells under hypoxic conditions or with a better oxygen supply.10 Saga and colleagues, in this issue, report that they have identified two clonal populations in a mouse model of gliomas. Clone A displays rapid cell proliferation and relies on glucose uptake and anaerobic glycolysis and, accordingly, shows an abundance of activated hexokinase 2 (HK2), pyruvate kinase isozyme M2 (PKM2), and lactate dehydrogenase A (LDHA). In contrast, clone B is composed of slowly cycling stem cells and depends on mitochondrial respiration. The characteristics of these two populations immediately suggest that the master regulators in the metabolism of clones A and B are different: clone A is driven by HIFs because it adapts to survive under hypoxia, and clone B is driven by cMyc. From a therapeutic standpoint, this hypothesis is important because targeting either of these proteins exclusively would allow, in the best-case scenario, the complete eradication of one of the clones, possibly without affecting the natural history of the tumor due to the persistent growth of the other clone. Furthermore, Saga and collaborators report that even though the predominant feature of metabolism in each clone is different, cells from each clone can easily reverse their control of energy and anabolism by switching the main source of energy from glycolysis to oxygen-based processes and vice versa. Clearly, the plasticity of glioma cells and their potential to adapt to challenging situations are also present at the level of metabolic pathway regulation.12 Perhaps the silver lining of the data is the observation that HK2, PKM2, and LDHA are targets of both cMyc and HIFs13,14 and, therefore, that inhibiting these downstream enzymes might efficiently choke cancer cells by simultaneously blocking—perhaps only partially—the regulation of glioma metabolism by two of these main master proteins.15 In summary, the report by Saga and colleagues offers solid evidence of the variable capability of glioma clones to regulate their metabolism through the mechanism described by Warburg. The authors also suggested that clones could switch from the Warburg effect to mitochondria-based energy production if the mechanisms controlling glycolysis are challenged. They provide evidence that the concept of cell plasticity at the metabolic level should be added to the multifaceted landscape of glioblastomas12 and should always be kept in mind when therapeutic approaches or imaging systems that target glioma metabolism are being designed.