FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

PURPOSE: Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. METHODS: In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. RESULTS: In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CONCLUSIONS: CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

A murine model of targeted infusion for intracranial tumors.

Historically, intra-arterial (IA) drug administration for malignant brain tumors including glioblastoma multiforme (GBM) was performed as an attempt to improve drug delivery. With the advent of percutaneous neuorovascular techniques and modern microcatheters, intracranial drug delivery is readily feasible; however, the question remains whether IA administration is safe and more effective compared to other delivery modalities such as intravenous (IV) or oral administrations. Preclinical large animal models allow for comparisons between treatment routes and to test novel agents, but can be expensive and difficult to generate large numbers and rapid results. Accordingly, we developed a murine model of IA drug delivery for GBM that is reproducible with clear readouts of tumor response and neurotoxicities. Herein, we describe a novel mouse model of IA drug delivery accessing the internal carotid artery to treat ipsilateral implanted GBM tumors that is consistent and reproducible with minimal experience. The intent of establishing this unique platform is to efficiently interrogate targeted anti-tumor agents that may be designed to take advantage of a directed, regional therapy approach for brain tumors.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Purpose: Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. Methods: In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. Results: In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. Conclusions: CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

Estrogen increases survival in an orthotopic model of glioblastoma.

Despite the male preponderance for developing glial tumors and a body of published literature that suggests a female gender advantage for long term survival in both human and animal studies, there have been relatively few rigorous investigations into the hormonal effects on glial tumor growth. In a previous study, we concluded that estrogen played a major role in the female survival bias seen in an intracerebral nude rat model of glioblastoma multiforme. Here we explore the potential therapeutic effect of exogenous estradiol delivery in nude rats with orthotopic glioblastoma tumors and examine the mechanism of action of estradiol on reducing tumor growth in this animal model. We administered estradiol, in several dosing regimens, to male, female and ovariectomized nude rats in a survival study. Brain sections, taken at various time points in tumor progression, were analyzed for estrogen receptor protein, proliferative index and apoptotic index. Estradiol increased survival of male, female and ovariectomized nude rats with intracerebral U87MG tumors, in a gender specific manner. The estradiol mediated effect occurred early in tumor progression, and appeared to be caused in-part by an increase in apoptotic activity. It remains unclear if estradiol's effect is direct or indirect and if it is estrogen receptor mediated. Estradiol-based or adjunctive therapy may be beneficial in treating GBM and further study is clearly warranted.

Do brain mets grow while you wait? A volumetric natural history assessment of brain metastases from time of diagnosis to gamma knife treatment.

Brain metastasis (BM) is a common neurologic complication of cancers such as lung, breast, and melanoma. Recently, there has been a shift in treatment of BM from whole brain radiation therapy to stereotactic radiosurgery (SRS) and the success is dependent on tumor volume. While most metastases grow over time, data on growth rate is lacking. Therefore, we document volume changes of metastases before treatment. We retrospectively reviewed MRI imaging records of 82 patients with a total of 294 BMs, treated in our cancer center by one neurosurgeon and one radiation oncologist with Gamma Knife SRS over a three-year period. We measured tumor volume at the time of diagnosis and compared with tumor volume on the day of treatment. Volumes were compared using the Wilcoxon signed-rank test. Lung, melanoma and breast made up the majority of metastases diagnosed. More than 75% of tumors grew and these changes in volume and percent changes in volume were statistically significant. Thirty percent of tumors doubled in size before treatment. Patients with the largest mean pretreatment tumor size were urgently treated within 6 days, yet still demonstrated the largest change in volume. This study is one of the first to document volume changes of brain metastases from the time of diagnosis to SRS treatment. Our results indicate that brain metastases can grow rapidly and it is imperative that we streamline patient management processes to minimize delays in treating patients with SRS, since outcomes are dependent on tumor size.

Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo.

Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in many cancers and represents an attractive molecule for targeted cancer therapy. Although primarily regarded as an intracellular protein with diverse actions, survivin has also been identified in association with circulating tumor exosomes.Experimental Design: We have reported that active, specific vaccination with a long peptide survivin immunogen leads to the development of survivin-specific CD8-mediated tumor cell lysis and prolongation of survival in tumor-bearing mice. In addition to cellular antitumor responses, circulating anti-survivin antibodies are detected in the serum of mice and human glioblastoma patients following vaccination with the survivin immunogen.Results: Here we demonstrate that survivin is present on the outer cell membrane of a wide variety of cancer cell types, including both murine and human glioma cells. In addition, antibodies to survivin that are derived from the immunogen display antitumor activity against murine GL261 gliomas in both flank and intracranial tumor models and against B16 melanoma as well.Conclusions: In addition to immunogen-induced, CD8-mediated tumor cell lysis, antibodies to the survivin immunogen have antitumor activity in vivo Cell-surface survivin could provide a specific target for antibody-mediated tumor immunotherapeutic approaches. Clin Cancer Res; 24(11); 2642-52. ©2018 AACR.

Anticancer drug candidate CBL0137, which inhibits histone chaperone FACT, is efficacious in preclinical orthotopic models of temozolomide-responsive and -resistant glioblastoma.

Background: The survival rate for patients with glioblastoma (GBM) remains dismal. New therapies targeting molecular pathways dysregulated in GBM are needed. One such clinical-stage drug candidate, CBL0137, is a curaxin, small molecules which simultaneously downregulate nuclear factor-kappaB (NF-ĸB) and activate p53 by inactivating the chromatin remodeling complex, Facilitates Chromatin Transcription (FACT). Methods: We used publicly available databases to establish levels of FACT subunit expression in GBM. In vitro, we evaluated the toxicity and effect of CBL0137 on FACT, p53, and NF-ĸB on U87MG and A1207 human GBM cells. In vivo, we implanted the cells orthotopically in nude mice and administered CBL0137 in various dosing regimens to assess brain and tumor accumulation of CBL0137, its effect on tumor cell proliferation and apoptosis, and on survival of mice with and without temozolomide (TMZ). Results: FACT subunit expression was elevated in GBM compared with normal brain. CBL0137 induced loss of chromatin-unbound FACT, activated p53, inhibited NF-ĸB-dependent transcription, and was toxic to GBM cells. The drug penetrated the blood-brain barrier and accumulated in orthotopic tumors significantly more than normal brain tissue. It increased apoptosis and suppressed proliferation in both U87MG and A1207 tumors. Intravenous administration of CBL0137 significantly increased survival in models of early- through late-stage TMZ-responsive and -resistant GBM, with a trend toward significantly increasing the effect of TMZ in TMZ-responsive U87MG tumors. Conclusion: CBL0137 targets GBM according to its proposed mechanism of action, crosses the blood-brain barrier, and is efficacious in both TMZ-responsive and -resistant orthotopic models, making it an attractive new therapy for GBM.

Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice.

The mammalian brain contains neural stem cells (NSCs) that allow continued neurogenesis throughout the life of the animal. However, neurogenesis is known to decline during aging and, to the extent that neurogenesis is required for normal CNS function, this may contribute to neurodegenerative disease. Decreased neurogenesis could result from loss of NSCs or dysfunction at some later step, and distinguishing these possibilities is important for understanding the cause of the decline. However, because of the inability to distinguish NSCs from their rapidly dividing progeny in situ, it has not been possible to quantitatively assess the NSC populations in young and old animals. In this report we show that the G1 phase-specific expression of the replication factor Mcm2 is a useful marker for detecting slowly cycling putative NSCs in situ and confirm the identity of these cells using both cytosine beta-D-arabinofuranoside (Ara-C) treatment and a double nucleoside analog-labeling technique. The ability to distinguish NSCs from proliferative progenitors has allowed characterization of the expression of several markers including Nestin, Musashi, and GFAP in these different cell types. Furthermore, comparison of the NSC populations in the subventricular zones of young (2-4 months) and old (24-26 months) mice demonstrates an approximately twofold reduction in the older mice. A similar twofold reduction is also observed in the number of neurospheres recovered in culture from old relative to young animals. The reduction in the neural stem cell population documented here is sufficient to account for the reduced level of neurogenesis in old animals.

2-Methoxyestradiol inhibits proliferation of normal and neoplastic glial cells, and induces cell death, in vitro.

2-Methoxyestradiol (2ME), a metabolite of estradiol (E), inhibits proliferation of various tumor cells. In this study we determined the effect of 2ME on human glioblastoma cell lines, in vitro. We compared these cells with cultured astrocytes obtained from traumatized adult rat striatum. Exposure to 2ME had a strong antiproliferative effect on human glioblastoma and caused an increase in the population of apoptotic cells, detected by flow cytometry, in some of the investigated cell lines. A significant number of cells were blocked in the G2/M phase of the cell cycle. Concurrently, the population of cells in the G1 phase decreased in all glioblastoma cell lines. Staining with Hoechst 33258 revealed abnormal nuclear morphology in the proliferating cells treated with 2ME. Treatment with 2ME induced upregulation of wild type p53 in one of the human glioblastoma cell lines as well as in proliferating adult rat astrocytes. We conclude that 2ME inhibits the growth of human glioblastoma cell lines and induces apoptosis, in vitro. This compound deserves further investigation as a treatment for gliomas.

Expression and localization of different forms of DMT1 in normal and tumor astroglial cells.

Divalent metal transporter 1 (DMT1), expressed in many different tissues, is responsible for the transport of a broad range of divalent metal ions. DMT1 exists in at least, four distinct isoforms which differ in both the C-terminus (termed here (-)IRE and (+)IRE) and the N-terminus (transcription proceeds from two different promoters). In the rat, two of the forms possess an additional 31 amino acids in the N-terminus (termed exon 1A) whereas the shorter forms lack this sequence (termed exon 2). Studies were performed to compare differences in expression and localization of these isoforms in low density and confluent cultures of rat astrocytes obtained from traumatized striatum and in rat C6 astrocytoma and human U87 glioblastoma. Results of these experiments reveal the presence of both the (+/-)IRE forms of DMT1 in all cultured cells examined. Western blots using affinity purified antibodies, which differentially recognize the two C-terminal species of DMT1, indicate a strong upregulation of the (+)IRE form in low density astrocyte cultures when compared to confluent cultures. Previously we reported that the (-)IRE form was present in both the nucleus and cytoplasm in neurons and neuronal like cells whereas the (+)IRE form was exclusively cytoplasmic. Similar results were found with the (-)IRE species in astrocytes and astrocytomas, i.e. nuclear and cytoplasmic distribution. This form of DMT1 also colocalizes with the early endosomal marker, EEA, suggesting that (-)IRE species may function in the transport of divalent metals. In contrast to our previous findings, however, the (+)IRE form was found predominantly localized in nucleus in both the primary and neoplastic glial cells. Interestingly, neither form of DMT1 colocalizes with the transferrin receptor. These data suggest that selective compartmentalization of specific isoforms of DMT1 imparts distinct and specialized functions that meet the changing needs of essential divalent transition metals as cofactors within cells.