oxadiazoles and Glioblastoma

Human glioblastoma (GBM) is a highly aggressive, invasive and hypervascularised malignant brain cancer. Individual circulating tumour cells (CTCs) are sporadically found in GBM patients, yet it is unclear whether multicellular CTC clusters are generated in this disease and whether they can bypass the physical hurdle of the blood-brain barrier.  Here, we assessed CTC presence and composition at multiple time points in 13 patients with progressing GBM during an open-label phase 1/2a study with the microtubule inhibitor BAL101553. We observe CTC clusters ranging from 2 to 23 cells and present at multiple sampling time points in a GBM patient with pleomorphism and extensive necrosis, throughout disease progression. Exome sequencing of GBM CTC clusters highlights variants in 58 cancer-associated genes including ATM, PMS2, POLE, APC, XPO1, TFRC, JAK2, ERBB4 and ALK. Together, our findings represent the first evidence of the presence of CTC clusters in GBM.

Topics: Animals; Benzimidazoles; Brain Neoplasms; Cell Count; Cluster Analysis; Disease Progression; Exome Sequencing; Female; Gene Regulatory Networks; Genetic Variation; Glioblastoma; Humans; Male; Mice; Mutation; Neoplastic Cells, Circulating; Oxadiazoles; Xenograft Model Antitumor Assays

Glioblastoma multiforme is the most common primary brain tumor and among the most lethal types of cancer. Several mono-target small molecule-inhibitors have been investigated as novel therapeutics, thus far with poor success. In this study we investigated the anticancer effects of SB747651A, a multi-target small-molecule inhibitor, in three well characterized patient-derived glioblastoma spheroid cultures and a murine orthotopic xenograft model. Concentrations of 5-10 µM SB747651A reduced cell proliferation, spheroid formation, migration and chemoresistance, while apoptotic cell death increased. Investigation of oncogenic kinase signaling showed decreased phosphorylation levels of mTOR, CREB, GSK3 and GYS1 leading to altered glycogen metabolism and formation of intracellular reactive oxygen species. Expression levels of cancer stemness marker SOX2 were reduced in treated tumor cells and SB747651A treatment significantly prolonged survival of mice with intracranial glioblastoma xenografts, while no adverse effects were observed in vivo at doses of 25 mg/kg administered 5 days/week for 8 weeks. These findings suggest that SB747651A has anticancer effects in glioblastoma. The cancer-related pathophysiological mechanisms targeted by SB747651A are shared among many types of cancer; however, an in-depth clarification of the mechanisms of action in cancer cells is important before further potential application of SB747651A as an anticancer agent can be considered.

Topics: Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Female; Glioblastoma; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Oxadiazoles; Phosphorylation; Xenograft Model Antitumor Assays

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzimidazoles; Brain Neoplasms; CD40 Antigens; Cell Line, Tumor; Cell Proliferation; CTLA-4 Antigen; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression; Glioblastoma; HMGB1 Protein; Immune Checkpoint Inhibitors; Interferon-gamma; Mice; Mitosis; Neoplasm Transplantation; Oxadiazoles; Programmed Cell Death 1 Receptor; Survival Rate; Temozolomide; Tumor Microenvironment

Topics: Acetates; Animals; Cell Line, Tumor; ErbB Receptors; Female; Fluorine Radioisotopes; Glioblastoma; Heterocyclic Compounds, 1-Ring; Heterografts; Immunoconjugates; Maleimides; Mice; Mice, Nude; Neuroglia; Oxadiazoles; Positron-Emission Tomography; Sulfhydryl Compounds

STAT3 is considered to be a key molecule to mediating tumor-induced immunosuppression in various manners at tumor sites, by acting through immune-regulatory cytokines derived from the tumor cells. Specific anti-STAT3 inhibitors have been developed using nude mouse models transplanted with human tumor cells. However, mouse systems cannot accurately represent the human immune response induced by STAT3 inhibitors, and more humanized therapeutic model based on human immune cells and tumors are needed. In the present study, an immune-deficient NOG mouse with the deletion of both MHC-class I and class II genes, an MHC-double knockout mouse (dKO-NOG), was developed and used to establish humanized immunotherapeutic model. We investigated the immunological effect of the STAT3 inhibitor STX-0119 against TMZ-resistant (TMZ-R) U87 glioma tumors by using humanized dKO-NOG mice. We compared the anti-tumor effects of STX-0119 between the nude and humanized dKO-NOG mouse models. An in vivo study using the nude mouse model showed that STX-0119 inhibited the growth of TMZR U87 tumors, but accumulation of tumor-infiltrating lymphocytes (TILs) were not promoted compared with the control levels. In contrast, STX-0119 inhibited tumor growth more rapidly and strongly in humanized dKO-NOG mice than in nude mice, and a large amount of TILs, mainly consisting of CD8

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Dacarbazine; Drug Resistance, Neoplasm; Glioblastoma; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; HLA-A2 Antigen; Humans; Leukocytes, Mononuclear; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Knockout; Mice, Nude; Oxadiazoles; Quinolines; STAT3 Transcription Factor; Temozolomide; Tumor Burden; Xenograft Model Antitumor Assays

Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach to overcome resistance is needed.. The efficacy of a combination treatment of STAT3 inhibitor, STX-0119, with rapamycin was investigated against our established TMZ-resistant U87 cell line.. The growth-inhibitory effect of the combination treatment was significant against the TMZ-R U87 cell line (IC. These results suggest that the STAT3 pathway is associated with the mTOR downstream pathway mediated by YKL-40 protein, and the combination therapy of the STAT3 inhibitor and rapamycin could be worth developing as a novel therapeutic approach against TMZ-resistant relapsed gliomas.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Chitinase-3-Like Protein 1; Dacarbazine; Disease Models, Animal; Drug Resistance, Neoplasm; Exome; Glioblastoma; High-Throughput Nucleotide Sequencing; Humans; Male; Oxadiazoles; Protein Kinase Inhibitors; Quinolines; Signal Transduction; STAT3 Transcription Factor; Temozolomide; TOR Serine-Threonine Kinases

Glioblastoma patients have limited treatment options. Cancer stem-like cells (CSLC) contribute to glioblastoma invasiveness and repopulation; hence, they represent promising targets for novel therapies. BAL101553 is a prodrug of BAL27862, a novel microtubule-destabilizing agent inhibiting tumor cell proliferation through activation of the spindle assembly checkpoint, which is currently in phase I/II clinical development. Broad anticancer activity has been demonstrated against human cancer models, including tumors refractory to conventional treatments. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with glioblastoma progression and poor survival. Here, we show that BAL27862 inhibits the growth of two glioblastoma CSLCs. As EB1 is overexpressed in the CSLC line GBM6, which displays a high tumorigenicity and infiltrative pattern of migration in vivo, we investigated drug activity on GBM6 according to EB1 expression. BAL27862 inhibited migration and colony formation at subcytotoxic concentrations in EB1-expressing control cells (GBM6-sh0) but only at cytotoxic concentrations in EB1-downregulated (GBM-shE1) cells. Three administrations of BAL101553 were sufficient to provoke an EB1-dependent survival benefit in tumor-bearing mice. Patterns of invasion and quantification of tumor cells in brain demonstrated that GBM6-sh0 cells were more invasive than GBM6-shEB1 cells, and that the antiproliferative and anti-invasive effects of BAL101553 were more potent in mice bearing control tumors than in EB1-downregulated tumors. This was associated with inhibition of stem cell properties in the GBM6-sh0 model. Finally, BAL27862 triggered astrocytic differentiation of GBM6 in an EB1-dependent manner. These results support the potential of BAL101553 for glioblastoma treatment, with EB1 expression as a predictive biomarker of response. Mol Cancer Ther; 15(11); 2740-9. ©2016 AACR.

Topics: Animals; Astrocytes; Benzimidazoles; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Female; Gene Expression; Glioblastoma; Humans; Mice; Microtubule-Associated Proteins; Neoplastic Stem Cells; Oxadiazoles; Tubulin Modulators

Zinc importer proteins (ZIPs) have been proven as important molecular regulators in different cancers. As a member of the solute carrier family, ZIP9/SLC39A9 is overexpressed in prostate and breast cancer and affects B-cell receptor signaling. Here, we present data indicating that changes in intracellular zinc levels in glioblastoma cells can cause enhanced cell survival and cell migration, both hallmarks of the disease process. In particular, treatment of human glioblastoma cells with sublethal doses of cell-permeable heavy metal (Zn

Topics: Benzofurans; Brain Neoplasms; Cation Transport Proteins; Cell Line, Tumor; Cell Movement; Chelating Agents; Enzyme Inhibitors; Ethylenediamines; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycogen Synthase Kinase 3 beta; Humans; Oxadiazoles; Phosphorylation; Transfection; Tumor Suppressor Protein p53

Glioblastoma (GBM) is poorly responsive to current chemotherapy. The nuclear transporter exportin 1 (XPO1, CRM1) is often highly expressed in GBM, which may portend a poor prognosis. Here, we determine the efficacy of novel selective inhibitors of nuclear export (SINE) specific to XPO1 in preclinical models of GBM.. Seven patient-derived GBM lines were treated with 3 SINE compounds (KPT-251, KPT-276, and Selinexor) in neurosphere culture conditions. KPT-276 and Selinexor were also evaluated in a murine orthotopic patient-derived xenograft (PDX) model of GBM. Cell cycle effects were assayed by flow cytometry in vitro and immunohistochemistry in vivo. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase 3/7 activity assays.. Treatment of GBM neurosphere cultures with KPT-276, Selinexor, and KPT-251 revealed dose-responsive growth inhibition in all 7 GBM lines [range of half-maximal inhibitory concentration (IC50), 6-354 nM]. In an orthotopic PDX model, treatment with KPT-276 and Selinexor demonstrated pharmacodynamic efficacy, significantly suppressed tumor growth, and prolonged animal survival. Cellular proliferation was not altered with SINE treatment. Instead, induction of apoptosis was apparent both in vitro and in vivo with SINE treatment, without overt evidence of neurotoxicity.. SINE compounds show preclinical efficacy utilizing in vitro and in vivo models of GBM, with induction of apoptosis as the mechanism of action. Selinexor is now in early clinical trials in solid and hematological malignancies. Based on these preclinical data and excellent brain penetration, we have initiated clinical trials of Selinexor in patients with relapsed GBM.

Topics: Acrylamides; Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Exportin 1 Protein; Glioblastoma; Humans; Hydrazines; Karyopherins; Macaca fascicularis; Male; Mice; Oxadiazoles; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Thiazoles; Treatment Outcome; Triazoles; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors and has a very poor prognosis, with a median survival time of less than 2 years. Once recurrence develops, there are few therapeutic approaches to control the growth of glioblastoma. In particular, temozolomide (TMZ)-resistant (TMZ-R) GBM is very difficult to treat, and a novel approach to overcome resistance is eagerly awaited. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the current study, the efficacy of STX-0119 was evaluated against our established TMZ-resistant U87 cell line using quantitative PCR-based gene expression analysis, in vitro assay and animal experiments. The growth inhibitory effect of STX-0119 on U87 and TMZ-R U87 cells was moderate (IC₅₀, 34 and 45 µM, respectively). In particular, STX-0119 did not show significant inhibition of U87 tumor growth; however, it suppressed the growth of the TMZ-R U87 tumor in nude mice by more than 50%, and prolonged the median survival time compared to the control group. Quantitative PCR revealed that YKL-40, MAGEC1, MGMT, several EMT genes, mesenchymal genes and STAT3 target genes were upregulated, but most of those genes were downregulated by STX-0119 treatment. Furthermore, the invasive activity of TMZ-R U87 cells was significantly inhibited by STX-0119. YKL-40 levels in TMZ-R U87 cells and their supernatants were significantly decreased by STX-0119 administration. These results suggest that STX-0119 is an efficient therapeutic to overcome TMZ resistance in recurrent GBM tumors, and could be the next promising compound leading to survival prolongation, and YKL-40 may be a possible surrogate marker for STAT3 targeting.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasms, Experimental; Oxadiazoles; Quinolines; STAT3 Transcription Factor; Temozolomide; Xenograft Model Antitumor Assays

Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Topics: Apoptosis; Brain Neoplasms; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Kruppel-Like Factor 4; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Oxadiazoles; Phosphorylation; Quinolines; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays