The Presence and Potential Role of ALDH1A2 in the Glioblastoma Microenvironment.

Glioblastoma (GBM) is the most aggressive malignant glioma. Therapeutic targeting of GBM is made more difficult due to its heterogeneity, resistance to treatment, and diffuse infiltration into the brain parenchyma. Better understanding of the tumor microenvironment should aid in finding more effective management of GBM. GBM-associated macrophages (GAM) comprise up to 30% of the GBM microenvironment. Therefore, exploration of GAM activity/function and their specific markers are important for developing new therapeutic agents. In this study, we identified and evaluated the expression of ALDH1A2 in the GBM microenvironment, and especially in M2 GAM, though it is also expressed in reactive astrocytes and multinucleated tumor cells. We demonstrated that M2 GAM highly express ALDH1A2 when compared to other ALDH1 family proteins. Additionally, GBM samples showed higher expression of ALDH1A2 when compared to low-grade gliomas (LGG), and this expression was increased upon tumor recurrence both at the gene and protein levels. We demonstrated that the enzymatic product of ALDH1A2, retinoic acid (RA), modulated the expression and activity of MMP-2 and MMP-9 in macrophages, but not in GBM tumor cells. Thus, the expression of ALDH1A2 may promote the progressive phenotype of GBM.

Diversity in responses to oncolytic Lassa-vesicular stomatitis virus in patient-derived glioblastoma cells.

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.

Phase I trial of convection-enhanced delivery of IL13RA2 and EPHA2 receptor targeted cytotoxins in dogs with spontaneous intracranial gliomas.

BACKGROUND: The interleukin-13 receptor alpha 2 (IL13RA2) and ephrin type A receptor 2 (EPHA2) are attractive therapeutic targets, being expressed in ~90% of canine and human gliomas, and absent in normal brain. Clinical trials using an earlier generation IL-13 based cytotoxin showed encouraging clinical effects in human glioma, but met with technical barriers associated with the convection-enhanced delivery (CED) method. In this study, IL-13 mutant and ephrin A1 (EFNA1)-based bacterial cytotoxins targeted to IL13RA2 and EPHA2 receptors, respectively, were administered locoregionally by CED to dogs with intracranial gliomas to evaluate their safety and preliminary efficacy. METHODS: In this phase I, 3 + 3 dose escalation trial, cytotoxins were infused by CED in 17 dogs with gliomas expressing IL13RA2 or EPHA2 receptors. CED was performed using a shape-fitting therapeutic planning algorithm, reflux-preventing catheters, and real-time intraoperative MRI monitoring. The primary endpoint was to determine the maximum tolerated dose of the cytotoxic cocktail in dogs with gliomas. RESULTS: Consistent intratumoral delivery of the cytotoxic cocktail was achieved, with a median target coverage of 70% (range, 40-94%). Cytotoxins were well tolerated over a dose range of 0.012-1.278 µg/mL delivered to the target volume (median, 0.099 µg/mL), with no dose limiting toxicities observed. Objective tumor responses, up to 94% tumor volume reduction, were observed in 50% (8/16) of dogs, including at least one dog in each dosing cohort >0.05 µg/mL. CONCLUSIONS: This study provides preclinical data fundamental to the translation of this multireceptor targeted therapeutic approach to the human clinic.

Multireceptor targeting of glioblastoma.

BACKGROUND: Treatment for glioblastoma (GBM) remains an unmet need in medicine. Novel therapies that address GBM complexity and heterogeneity in particular are warranted. To this end, we target 4 tumor-associated receptors at a time that span virtually all of the GBM microenvironment including bulk tumor cells, infiltrating tumor cells, neovasculature, and tumor-infiltrating cells with one pharmaceutical agent delivering a cytotoxic load. METHODS: We engineered multivalent ligand-based vector proteins termed QUAD with an ability to bind to 4 of the following GBM-associated receptors: IL-13RA2, EphA2, EphA3, and EphB2. We conjugated QUAD with a modified bacterial toxin PE38QQR and tested it in vitro and in vivo. RESULTS: The QUAD variants preserved functional characteristics of the respective ligands for the 4 receptors. The QUAD 3.0 variant conjugate was highly cytotoxic to GBM cells, but it was nontoxic in mice, and the conjugate exhibited strong antitumor effect in a dog with spontaneous GBM. CONCLUSION: The QUAD addresses, to a large extent, the issues of intra- and intertumoral heterogeneity and, at the same time, it targets several pathophysiologically important tumor compartments in GBM through multiple receptors overexpressed in tumors allowing for what we call "molecular resection." QUAD-based targeted agents warrant further pre- and clinical development.

Attenuating hypoxia driven malignant behavior in glioblastoma with a novel hypoxia-inducible factor 2 alpha inhibitor.

Hypoxia inducible factor (HIFs) signaling contributes to malignant cell behavior in glioblastoma (GBM). We investigated a novel HIF2α inhibitor, PT2385, both in vitro, with low-passage patient-derived cell lines, and in vivo, using orthotopic models of glioblastoma. We focused on analysis of HIF2α expression in situ, cell survival/proliferation, and survival in brain tumor-bearing mice treated with PT2385 alone and in combination with standard of care chemoradiotherapy. HIF2α expression increased with glioma grade, with over half of GBM specimens HIF2α positive. Staining clustered in perivascular and perinecrotic tumor regions. Cellular phenotype including proliferation, viability, migration/invasion, and also gene expression were not altered after PT2385 treatment. In the animal model, PT2385 single-agent treatment did improve median overall survival compared to placebo (p = 0.04, n = 21) without a bioluminescence correlate (t = 0.67, p = 0.52). No difference in animal survival was seen in combination treatment with radiation (RT)/temozolomide (TMZ)/PT2385 (p = 0.44, n = 10) or mean tumor bioluminescence (t 1.13, p = 0.32). We conclude that HIF2α is a reasonable novel therapeutic target as expressed in the majority of glioblastomas in our cohort. PT2385 as a single-agent was efficacious in vivo, however, an increase in animal survival was not seen with PT2385 in combination with RT/TMZ. Further study for targeting HIF2α as a therapeutic approach in GBM is warranted.

Drug Conjugates for Targeting Eph Receptors in Glioblastoma.

Glioblastoma (GBM) is a complex and heterogeneous tumor that warrants a comprehensive therapeutic approach for treatment. Tumor-associated antigens offer an opportunity to selectively target various components of the GBM microenvironment while sparing the normal cells within the central nervous system. In this study, we conjugated a multivalent vector protein, QUAD 3.0, that can target four receptors: EphA3, EphA2, EphB2, and also IL-13RA2, spanning virtually 100% of the GBM microenvironment, to doxorubicin derivatives. The conjugates effectively bound to all four receptors, although to varying degrees, and delivered cytotoxic loads to both established and patient-derived GBM cell lines, with IC50 values in the low nM range. The conjugates were also non-toxic to animals. We anticipate that the QUAD 3.0 Dox conjugates will be further used in preclinical models and possibly clinics in the foreseeable future.

miR-30 disrupts senescence and promotes cancer by targeting both p16INK4A and DNA damage pathways.

miR-30 is a microRNA frequently overexpressed in human cancers. However, the biological consequence of miR-30 overexpression in cancer has been unclear. In a genetic screen, miR-30 was found to abrogate oncogenic-induced senescence, a key tumor-suppressing mechanism that involves DNA damage responses, activation of p53 and induction of p16INK4A. In cells and mouse models, miR-30 disrupts senescence and promotes cancer by suppressing 2 targets, CHD7 and TNRC6A. We show that while CHD7 is a transcriptional coactivator essential for induction of p16INK4A in senescent cells, TNRC6A, a miRNA machinery component, is required for expression and functionality of DNA damage response RNAs (DDRNAs) that mediate DNA damage responses and p53 activation by orchestrating histone modifications, chromatin remodeling and recruitment of DNA damage factors at damaged sites. Thus, miR-30 inhibits both p16INK4A and p53, 2 key senescence effectors, leading to efficient senescence disruption. These findings have identified novel signaling pathways mediating oncogene-induced senescence and tumor-suppression, and revealed the molecular and cellular mechanisms underlying the oncogenic activity of miR-30. Thus, the miR-30/CHD7/TNRC6A pathway is potentially a novel diagnostic biomarker and therapeutic target for cancer.

Peptide-based PET imaging of the tumor restricted IL13RA2 biomarker.

Peptides that target cancer cell surface receptors are promising platforms to deliver diagnostic and therapeutic payloads specifically to cancer but not normal tissue. IL13RA2 is a tumor-restricted receptor found to be present in several aggressive malignancies, including in the vast majority of high-grade gliomas and malignant melanoma. This receptor has been successfully targeted for diagnostic and therapeutic purposes using modified IL-13 ligand and more recently using a specific peptide, Pep-1L. In the current work, we establish the in vitro and in vivo tumor binding properties of radiolabeled Pep-1L, designed for tumor imaging. We radiolabeled Pep-1L with Copper-64 and demonstrated specific cell uptake in the IL13RA2-over expressing G48 glioblastoma cell line having abundant IL13RA2 expression. [64Cu]Pep-1L binding was blocked by unlabeled ligand, demonstrating specificity. To demonstrate in vivo tumor uptake, we intravenously injected into tumor-bearing mice and demonstrated that [64Cu]Pep-1L specifically bound tumors at 24 hours, which was significantly blocked (3-fold) by pre-injecting unlabeled peptide. To further demonstrate specificity of Pep-1L towards IL13RA2 in vivo, we exploited an IL13RA2-inducible melanoma tumor model that does not express receptor at baseline but expresses abundant receptor after treatment with doxycycline. We injected [64Cu]Pep-1L into mice bearing IL13RA2-inducible melanoma tumors and performed in vivo PET/CT and post-necropsy biodistribution studies and found that tumors that were induced to express IL13RA2 receptor by doxycycline pretreatment bound radiolabeled Pep-1L 3-4 fold greater than uninduced tumors, demonstrating receptor specificity. This work demonstrates that [64Cu]Pep-1L selectively binds hIL13RA2-expressing tumors and validates Pep-1L as an effective platform to deliver diagnostics and therapeutics to IL13RA2-expressing cancers.

Novel Molecular Multilevel Targeted Antitumor Agents.

A multifunctional fusion protein, IL-13.E13K-D2-NLS, effectively recognizes glioblastoma (GBM) cells and delivers its portion to the cell nucleus. IL-13.E13K-D2-NLS is composed of a cancer cell targeting ligand (IL-13.E13K), specialized cytosol translocation bacterial toxin domain 2 of Pseudomonas exotoxin A (D2) and SV40 T antigen nuclear localization signal (NLS). We have now tested whether we can produce proteins that would serve as a delivery vehicle to lysosomes and mitochondria as well. Moreover, we examined whether IL-13.E13K-D2-NLS can deliver anti-cancer drugs like doxorubicin to their nuclear site of action in cancer cells. We have thus constructed two novel proteins: IL-13.E13K-D2-LLS which incorporates lysosomal localization signal (LLS) of a human lysosomal associated membrane protein (LAMP-1) for targeting to lysosomes and IL-13-D2-KK2, which incorporates a pro-apoptotic peptide (KLAKLAK)2 (KK2) exerting its action in mitochondria. Furthermore, we have produced IL-13.E13K-D2-NLS and IL-13.E13K-D2-LLS versions containing a cysteine for site-specific conjugation with a modified doxorubicin, WP936. We found that single-chain recombinant proteins IL-13.E13K-D2-LLS and IL-13-D2-KK2 are internalized and localized mostly to the lysosomal and mitochondrial compartments, respectively, without major trafficking to cells' nuclei. We also determined that IL-13.E13K-D2-NLS-cys[WP936], IL-13.E13K-D2-LAMP-cys[WP936] and IL-13-D2-KK2 were cytotoxic to GBM cells overexpressing IL-13RA2, while much less cytotoxic to GBM cell lines expressing low levels of the receptor. IL-13.E13K-D2-NLS-cys[WP936] was the most potent of the tested anti-tumor agents including free WP936. We believe that our receptor-directed intracellular organelle-targeted proteins can be employed for numerous specific and safer treatment applications when drugs have specific intracellular sites of their action.

IL13RA2 targeted alpha particle therapy against glioblastomas.

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.

Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10-6mol/l) relative to uncoated plates (2.4 × 10-6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10-6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

Simultaneous targeting of Eph receptors in glioblastoma.

Eph tyrosine kinase receptors are frequently overexpressed and functional in many cancers, and they are attractive candidates for targeted therapy. Here, we analyzed the expression of Eph receptor A3, one of the most up-regulated factors in glioblastoma cells cultured under tumorsphere-forming conditions, together with EphA2 and EphB2 receptors. EphA3 was overexpressed in up to 60% of glioblastoma tumors tested, but not in normal brain. EphA3 was localized in scattered areas of the tumor, the invasive ring, and niches near tumor vessels. EphA3 co-localized with macrophage/leukocyte markers, suggesting EphA3 expression on tumor-infiltrating cells of bone marrow origin. We took advantage of the fact that ephrinA5 (eA5) is a ligand that binds EphA3, EphA2 and EphB2 receptors, and used it to construct a novel targeted anti-glioblastoma cytotoxin. The eA5-based cytotoxin potently and specifically killed glioblastoma cells with an IC50 of at least 10-11 M. This and similar cytotoxins will simultaneously target different compartments of glioblastoma tumors while mitigating tumor heterogeneity.