Search

TTField (Tumor-treating field) therapy utilizes low intensity intermediate frequency AC electric fields to reduce the spread of cancer. While it has attained FDA approval for the treatment of recurrent glioblastoma multiforme, the exact molecular targets of TTField therapy are not well understood. Microtubules are pipe-like polymers of the highly charged (–31 e) and strongly dipolar (dipole moment 1666 D) protein, α, β- tubulin. Studies on the electrical properties of microtubules have recently gained interest, with them being modelled as molecular targets of TTFields. Here, we experimentally show that while tubulin polymerized into microtubules leads to an increase in solution capacitance, unpolymerized tubulin has no appreciable effect. To the best of our knowledge, we present the first experimental quantification of the capacitance of a 20 μm-long microtubule. Using these results, we calculate the resonant frequency of a microtubule meshwork in a cell-like environment to be in the TTField regime. Our results utilize high ionic strength solutions and cell-like concentrations of tubulin to show the potential of microtubules as the targets of TTField action and as intracellular charge-storage devices. We conclude with a hypothesis of an electrically-tunable cell, where the dielectric properties of the cytoskeleton alter local and global charge storage and transport.

BACKGROUND Investigation of mitochondrial DNA (mtDNA) content in glioblastoma has gathered speed in recent years. Our study focusses on the effect of mtDNA content depletion on treatment response in glioblastoma. MATERIALS AND METHODS The study consists of a survival cohort (n=130), a recurrent cohort (32 pairs) and 35 non neoplastic control brain tissues. Malignant glioma cell lines, U87 and LN229 were utilized for radiation biology experiments. Patient tumor tissue was molecularly characterized for IDH, ATRX and TERT promoter mutations, MGMT promoter methylation and EGFR amplification using established methods. Relative quantification of mtDNA content was performed using quantitative real time PCR. Ethidium bromide was used to deplete mtDNA in U87 and LN229 cells. Clonogenic assay and MTT assay were performed to assess sensitivity to radiation and chemotherapy. RESULTS mtDNA content was lower in glioblastoma than in controls. Lower range of mtDNA copy number was associated with poor overall survival (p=0.033) and with markers of tumor aggressiveness like wild type IDH (p=0.02), unmethylated MGMT promoter, amplified EFGR and mutated TERT promoter. Out of 32 recurrent GBM patients, only 19 had received radiation treatment (RT) between the two surgeries and in these patients, mtDNA content was significantly higher in the recurrent tumor when compared to the primary tumor(p=0.01). U87 and LN229 cells showed a higher mtDNA copy number post irradiation (p=0.002). mtDNA depleted U87 and LN229 cells showed higher survival fraction post radiation exposure when compared to parent lines. The IC50 of TMZ was also higher for mtDNA depleted U87 and LN229 cells. CONCLUSION Low mtDNA content is associated with poor survival and aggressive tumor markers in glioblastoma. Lowering the mtDNA content in GBM cells induces radiation resistance as well as resistance to chemotherapy. Irradiation leads to increase in mitochondrial DNA content in both patient samples as well as malignant glioma cell lines.

BACKGROUND Despite best current therapies, median survival for glioblastoma remains little more than a year. There remains an urgent need for improved treatment strategies. Current therapies, including radiation and temozolomide, are known to induce senescence in tumor cells. Advances in aging research have identified drugs with “senolytic” properties to kill senescent cells. This work tests the hypothesis that induction of senescence pathways following radiation can provide a window of opportunity to eliminate residual glioblastoma cells using drugs that block senescent cell-associated pathways (SCAPs). METHODS Human glioblastoma cells were radiated with 10, 15, or 20 Gy and maintained in culture for 3 to 4 weeks to facilitate maturation of a senescent phenotype. Given existence of multiple SCAPs, we evaluated the IC50 for several senolytic candidates including Dasatinib, Quercetin, Fisetin, AMG-232, Onalespib, A1331852, and Navitoclax in radiated vs non-radiated cells. RESULTS Among these, the Bcl-XL inhibitors A1331852 and Navitoclax both selectively killed multiple lines of radiated, senescent tumor cells at lower concentrations as compared to unirradiated, non-senescent tumor cells. Across multiple GBM cell line, the average IC50 for senescent cells was 12.88 nM (95%CI= 9.068–18.58 nM), as compared to the IC50 of non-senescent cells of 103.2 nM (95%CI= 74.24–143.9 nM; p< 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation was not observed in the non-GBM control cells evaluated to date. CONCLUSION Our findings suggest that blocking the anti-apoptotic pathway Bcl-XL in GBM cells rendered at least transiently senescent following radiation may provide an avenue to selectively ablate residual glioblastoma cells following radiation. Our data provide proof of principle that senolytic therapy may represent a previously untapped window of opportunity to increase extent of tumor ablation following completion of standard cytotoxic therapies.

BACKGROUND Radiation therapy is the standard of care for both primary and secondary brain tumors. The majority of patients experience radiation induced hypersomnia (RIH) to varying extents which may impact life quality and treatment tolerance. Previously, we demonstrated that risk is enhanced by presence of select clock gene polymorphisms. The purpose of this study is to establish a mouse model of RIH to test dose effects of cranial irradiation on behavioral sleep and activity prior to introducing tumor in transgenic mice. METHODS Twenty-four non-tumor bearing C57BL/6 mice received whole brain radiation at one of 4 doses (sham, 2Gy, 5Gy, and 15Gy) using a single fraction. Behavior, total activity (distance) and sleep quantity (seconds) were monitored using Phenotyper cages and Ethovision Software with data collected at baseline (10 days) and post-radiation (11 days); percentage of activity was calculated as post-radiation/baseline activity. Blood and tissue samples were collected. RESULTS Baseline activity was not significantly different between groups (F(3.23) = 0.649, p=0.593). Shams and 2Gy mice were not affected by the procedure and activity levels were unchanged, 99.76±1.3% and 103.3±3.5%, respectively. The higher doses, 5Gy and 15Gy, had suppression of general activity compared to baseline with 90.8±2.7% and 88.9±1.3%, respectively. Between the two groups, suppression of activity started after 2 days in 15Gy and 5 days in 5Gy mice. Across three days (8–10), 5Gy and 15Gy (19,872±577sec) had more sleep during their active phase (t=2.27, p=0.03) than sham or 2Gy (17,618±803sec). CONCLUSION Activity and sleep are affected by cranial irradiation in our RIH mouse model in dose dependent manner. This model closely resembles the effects seen in patients and provides a critical tool for interrogating the physiological drivers of RIH. In future studies, we will examine other behaviors and the association between circadian gene variants and RIH susceptibility as previously reported in patients.

BACKGROUND Chronotherapy is the optimally timed administration of treatment based on the circadian clock. In non-CNS cancers, circadian rhythms (CR) drive the expression of mechanisms responsible for radiation sensitivity and associated toxicity. Our previous work has shown that cranial irradiation in GBM patients produces a high incidence of hypersomnolence and susceptibility is linked to circadian clock gene polymorphisms. This study determined if CR can be demonstrated using in vitro GBM and CNS cells and then if sensitivity to radiation is circadian time and tissue dependent. METHODS Rat GBM, fibroblasts, and suprachiasmatic nucleus (SCN) cells were used and compared to human U251 GBM cells. Cells were synchronized using the serum shook method; protein levels for PER2 expression over 24 hr in 4 hr intervals were quantified to confirm CR. Colony formation and 3D invasion assays after radiation (0-8Gy or 8Gy, respectively) 8 and 20 hrs after synchronization were evaluated. RESULTS All cell types demonstrated CR by PER2 expression (trough ~8hr post (CT8); peak ~20hrs post (CT20)). In rat, GBM (LD50 = 4Gy) and SCN cells (LD50 =5 Gy) showed lower sensitivity than fibroblasts (LD50= 2 Gy). Human U251 GBM cells had similar radiation sensitivity to rat GBM. Time-of-day effects were dependent on tissue type with both rat and human GBM cell lines more affected by radiation at CT20, with less invasion (CT20:-35488.5±2622.1 vs CT8:-23160.5±3273.4µm) and survival in the colony assay (F(1,71)=4.105,p< 0.05). Conversely, SCN cells which drive sleep-wake, showed the converse with greater effect at the 8hr compared with 20hr (F(1, 35)=19.93,p< 0.001). CONCLUSIONS These data suggest that timing of radiation could be optimized to improve detrimental effects on healthy tissue, while still providing effective intra-tumor doses. In future studies, we will interrogate the mechanisms driving time-of-day effects in cells and test radiation chronotherapy in our radiation-induced hypersomnolence mouse model.

SB28 is a new luciferase expressing murine glioblastoma (GBM) cell line whose morphology, growth pattern, mutational burden immune infiltrate more closely resembles human GBM and is unresponsive to immunotherapy (OncoImmunology, 7:12, e1501137). Radiation therapy (RT) has the potential to improve response to combinations by multiple means: cell kill that may activate an immune response, effects on vascular system may improve drug access, induction of DNA damage that can be augmented by targeted or chemotherapy, and modulation of the tumor microenvironment. High transforming growth factor β (TGFβ) activity in most GBM, which increases with radiation, likely opposes effective therapy by endorsing an effective DNA damage response and being immunosuppressive. Here evaluated the response of SB28 to radiation in combination with PD-L1 checkpoint blockade or neutralizing TGFβ. Establishment of intracranial tumors was monitored by bioluminescence. We determined that SB28 endogenously activate TGFβ as evaluated by biomarkers. We used radiolabeled antibodies and PET-CT to demonstrate access to intracranial tumors, indicative of a compromised blood-brain barrier. TGFβ neutralizing antibody, 1D11, had little effect on tumor growth compared to mice receiving IgG control antibody (median survival control: 19 days vs 1D11: 20.5 days). A single dose of radiation of 10 Gy delivered to the tumor site increased median survival to 21 day, which was increased to 31 days in mice treated with 1D11. In a second experiment in which the radiation dose was 6 Gy, 1D11 provided no benefit. In contrast, median survival of mice treated with PD-L1 neutralizing antibodies 3 days after being irradiated with 6 Gy demonstrated increased median survival (Control: 18 day; anti-PD-L1 20.5; RT: 25 days; RT + anti- PD-L1: 28 days). Double treatment resulted in 1/7 long term survival and loss of bioluminescence indicative of tumor elimination. Thus, radiation can prime SB28 GBM for response to biological agents.

Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of glioblastoma.

PURPOSE Radiotherapy (RT) is an essential component in the adjuvant treatment of glioblastoma (GBM); however, despite an initial benefit in tumor control, recurrence is nearly universal. The inflammasome is a multimeric cytosolic complex that has been shown to be activated in immune cells following RT. We investigated the relationship of inflammasome activation and disease progression after RT in GBM. METHODS We performed survival analysis for patients with recurrent GBM using the TCGA dataset, stratifying according to expression of inflammasome pathway molecules Caspase 1 (CASP1), IL-1β, and NLRP3. IL-1β expression and colocalization with CD11b+ myeloid cells were analyzed at different time points following RT in a murine glioma GL261 model. We detected the secreted IL-1β, CASP1 activity, and NLRP3 expression in both tumor and infiltrating immune cells, and the downstream effects of their interplay, including cellular apoptosis, proliferation, and viability. RESULTS High expression of CASP1, IL-1β, and NLRP3 are associated with poor survival in recurrent GBM. In a mouse model, recurrent tumors after treatment with RT display higher levels of the inflammasome effector molecule IL-1β, which colocalizes with infiltrating CD11b+ myeloid cells. Using a co-culture system of tumor cell and M2 macrophages, we found that RT enhances inflammasome activation in both cell types, and this interaction restores the proliferative capacity of surviving tumor cells following treatment. RT induces the inflammasome pathway activation in the setting of an associated immune microenvironment rich in tumor-associated macrophages and myeloid-derived suppressor cells (TAM/MDSCs) and not in tumor cells alone. CONCLUSIONS Inhibition of the oncoprogressive crosstalk between GBM tumor cells and M2 macrophages, possibly through disruption of the inflammasome pathway, might be a potential therapeutic strategy with RT.

BACKGROUND Glioblastoma is the most aggressive brain tumor with poor prognosis despite the best available treatment. MicroRNAs (miRNAs) are emerging as promising, novel prognostic biomarkers and therapeutic targets in glioblastoma. In a previous study, we demonstrated that miR-4516 predicts poor prognosis and functions as an oncogene in glioblastoma. Aim of the current study is to examine the role miR-4516 in radiation resistance and identify downstream targets contributing to this phenotype METHODS Radiosensitization was evaluated by cell viability and clonogenic assays. Cell apoptosis was evaluated using flow cytometry and immunoblotting. Potential targets of miR-4516 were identified using bioinformatic analysis (Targetscan and miRDB) and confirmed by luciferase reporter assays. Results were validated using immunoblotting. miR-4516 expression in glioblastoma cell lines after radiation treatment was quantified by qRT-PCR. RESULTS Expression of miR-4516 was increased up to 15 fold following radiation treatment, peaking at around 15min-60 min in primary and established glioblastoma cell lines including GBM 08-387, GBM 30 and U87-MG. Furthermore, inhibition of miR-4516 sensitized GBM 08-387, GBM30 and U87-MG cells to radiation in comparison to control groups as determined by cell viability and clonogenic assays. Further, miR-4516 inhibition induced apoptosis in these cell lines following radiation treatment. While conducting mechanistic studies, we found that the tumor-promoting function of miR-4516 was, in part, mediated by inhibition of p21 and PTPN14, two direct targets of miR-4516 CONCLUSION Our data suggest that radiation induces the expression of miR-4516 in glioblastoma cell lines. This miRNA plays a critical role in radio-resistance and promotes aggressive phenotypes in glioblastoma and therefore, functional analyses of its target pathways may uncover novel therapeutically vulnerable target(s) in glioblastoma. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and OSU-CCC (all to AC). The Ton and Patricia Bohnenn Fund for Neuro_Oncology Research (to PR).

Tumor Treating Fields (TTFields) are approved for the treatment of Glioblastoma Multiforme (GBM) in supratentorium. When treating GBM in the suprtaentorial brain, TTFields are delivered using 2 pairs of transducer arrays positioned in close proximity to the tumor. This results in an increased TTFields dose in the affected tumor region, whilst reducing the dose in other regions, particularly the infratentorium. Brain metastases, occur in 10 to 30 percent of adults with cancer, and can occur throughout the brain, including in infratentorial regions. Therefore, when using TTFields to treat brain metastases, it is desirable to deliver TTFields at therapeutic intensities to the entire brain or to the infratentorium depending on the location of the lesions. We performed computational studies to identify array layouts for delivering TTFields at therapeutic intensities to the infratentorium and to the entire brain. The computer simulations utilized a realistic computerized head model of a 40+ years old human male prepared in-house. TTFields could be delivered effectively to the infratentorium using a layout in which a pair of arrays was placed on the lateral aspects of the neck below the ears, and the second pair on the upper head and lower neck yielded field intensities above 1 V/cm in the infratentorial brain. Other layouts in which one pair of arrays was placed on the right temple and left scapula, and the second pair placed on the left temple and right scapula yielded a uniform intensity distribution within the entire brain. This layout could be useful for treating multiple lesion spread in both the infratentorial and supratentorial regions. This study suggests that careful treatment planning could be used to optimize TTFields treatment targeting multiple lesions spread throughout the brain.

Mutated Isocitrate dehydrogenase 1 (IDH1R132H; mIDH1) is found in 50% of all adult gliomas and in 80% of lower-grade gliomas. IDH1R132H promotes 2-hydroxyglutarate production which inhibits histone and DNA demethylases, inducing epigenetic reprogramming of the tumor transcriptome. We recently demonstrated that epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 glioma, harboring p53 and ATRX loss. Furthermore, several studies indicate that IDH1R132H triggers cellular metabolism reprogramming. In this study, RNA-seq data revealed that mIDH1 glioma neurospheres (mIDH1-NS) have downregulated gene ontologies related to mitochondrial metabolism. Analysis of mitochondrial and glycolytic activity, using a Seahorse Analyzer, showed that mIDH1-NS have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of quiescent state. Nevertheless, mIDH1 glioma cells can grow and generate tumors, suggesting that they might maintain self-renewal via autophagy. To investigate this hypothesis, we studied autophagy in wild-type and mIDH1 glioma cells analyzing LC3 conversion, p62 expression, and ULK1 protein phosphorylation. Mouse and human mIDH1 glioma cells exhibited increased expression of pULK1-S555, enhanced conversion of LC3I to LC3II, and decreased expression of p62, indicating that mIDH1 cells have an augmented autophagy. Also, key proteins involved in autophagy, including MST4 and UVRAG, are upregulated in mIDH1-NS. We also studied autophagy flux using LC3-mCherry-GFP reporter to identify LC3 recruitment at the autophagosome membranes and the autolysosome activity. Our results showed that mIDH1 cells have increased LC3 recruitment and enhanced autolysosome activity. Interestingly, autophagy has been related with glioma radioresistance. In our study, mouse and human mIDH1 glioma cells are sensitive to autophagy inhibition. In addition, autophagy inhibition restored radiosensitivity in mouse and human mIDH1 glioma cells, and in our mIDH1 glioma model, enhancing median survival compared with radiation alone. In conclusion, our results indicate that mIDH1 glioma cells maintain self-renewal via activation of autophagy, which represents a novel therapeutic target.

Treatment resistance in glioblastoma is underpinned by highly interconnected DNA damage response (DDR) processes. The FA-pathway is a fundamental DDR process required for the resolution of replication fork impeding lesions, and we have previously shown that it is inactive in normal brain, but is re-activated in glioblastoma, providing a cancer-specific target for combination DDR therapies. Here, we find that elevated FA-pathway gene expression in gliomas is associated with poor survival (-17.1% 5-year OS, p< 0.0001, n=329–REMBRANT). Furthermore, patient-derived glioblastoma stem cell (GSC) populations, which drive therapeutic resistance, display high FA-pathway expression relative to paired bulk tumour cell populations (mean 2.3-fold higher across genes, p=0.0073). We further show that inhibition of a single DDR process (FA-pathway, PARP, ATR or ATM) increases the susceptibility of glioblastoma cell lines and patient-derived GSCs to current adjuvant therapy. Importantly, clinically approved PARP inhibitor (PARPi) monotherapy stimulates robust FANCD2 mono-ubiquitination, supporting a role of FA-pathway activation in response to current DDR-targeted therapy. In clinically-relevant 3D GSC models, simultaneous inhibition of the FA-pathway (FAPi) and PARP or ATR enhanced temozolomide sensitisation compared to a single DDR inhibitor (DDRi). Furthermore, combined FAPi+PARPi consistently conferred radiosensitisation whilst combined FAPi+ATRi led to a profoundly radiosensitising effect; e.g. sensitizer enhancement ratio (SER0.37) of 3.23 (3.03–3.49, 95% CI). Furthermore, comparison of α/β ratio enhancement suggests dual-DDRi strategies fundamentally alter the response of GSCs, whilst single cell gel electrophoresis & immunofluorescence studies suggest FA-pathway based DDRi combinations profoundly delay the resolution of IR-induced DNA strand breaks at 6 hours post-treatment, with increased persistent DNA double strand breaks at 24 hours. In conclusion, simultaneously targeting the FA-pathway and interconnected DDR processes represents an appealing therapeutic strategy. Additionally, constitutive lack of FA pathway function in some tumours, could serve as a novel predictive biomarker for patient response to PARPi and ATRi currently in clinical trials.