ClonoScreen3D - A Novel 3-Dimensional Clonogenic Screening Platform for Identification of Radiosensitizers for Glioblastoma.

PURPOSE: Glioblastoma (GBM) is a lethal brain tumor. Standard-of-care treatment comprising surgery, radiation, and chemotherapy results in median survival rates of 12 to 15 months. Molecular-targeted agents identified using conventional 2-dimensional (2D) in vitro models of GBM have failed to improve outcome in patients, rendering such models inadequate for therapeutic target identification. A previously developed 3D GBM in vitro model that recapitulates key GBM clinical features and responses to molecular therapies was investigated for utility for screening novel radiation-drug combinations using gold-standard clonogenic survival as readout. METHODS AND MATERIALS: Patient-derived GBM cell lines were optimized for inclusion in a 96-well plate 3D clonogenic screening platform, ClonoScreen3D. Radiation responses of GBM cells in this system were highly reproducible and comparable to those observed in low-throughout 3D assays. The screen methodology provided quantification of candidate drug single agent activity (half maximal effective concentration or EC50) and the interaction between drug and radiation (radiation interaction ratio). RESULTS: The poly(ADP-ribose) polymerase inhibitors talazoparib, rucaparib, and olaparib each showed a significant interaction with radiation by ClonoScreen3D and were subsequently confirmed as true radiosensitizers by full clonogenic assay. Screening a panel of DNA damage response inhibitors revealed the expected propensity of these compounds to interact significantly with radiation (13/15 compounds). A second screen assessed a panel of compounds targeting pathways identified by transcriptomic analysis and demonstrated single agent activity and a previously unreported interaction with radiation of dinaciclib and cytarabine (radiation interaction ratio 1.28 and 1.90, respectively). These compounds were validated as radiosensitizers in full clonogenic assays (sensitizer enhancement ratio 1.47 and 1.35, respectively). CONCLUSIONS: The ClonoScreen3D platform was demonstrated to be a robust method to screen for single agent and radiation-drug combination activity. Using gold-standard clonogenicity, this assay is a tool for identification of radiosensitizers. We anticipate this technology will accelerate identification of novel radiation-drug combinations with genuine translational value.

c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I.

c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc-oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts.

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid, direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.

Radiation Responses of 2D and 3D Glioblastoma Cells: A Novel, 3D-specific Radioprotective Role of VEGF/Akt Signaling through Functional Activation of NHEJ.

Glioblastoma is resistant to conventional treatments and has dismal prognosis. Despite promising in vitro data, molecular targeted agents have failed to improve outcomes in patients, indicating that conventional two-dimensional (2D) in vitro models of GBM do not recapitulate the clinical scenario. Responses of primary glioblastoma stem-like cells (GSC) to radiation in combination with EGFR, VEGF, and Akt inhibition were investigated in conventional 2D cultures and a three-dimensional (3D) in vitro model of GBM that recapitulates key GBM clinical features. VEGF deprivation had no effect on radiation responses of 2D GSCs, but enhanced radiosensitivity of GSC cultures in 3D. The opposite effects were observed for EGFR inhibition. Detailed analysis of VEGF and EGF signaling demonstrated a radioprotective role of Akt that correlates with VEGF in 3D and with EGFR in 2D. In all cases, positive correlations were observed between increased radiosensitivity, markers of unrepaired DNA damage and persistent phospho-DNA-PK nuclear foci. Conversely, increased numbers of Rad51 foci were observed in radioresistant populations, indicating a novel role for VEGF/Akt signaling in influencing radiosensitivity by regulating the balance between nonhomologous end-joining and homologous recombination-mediated DNA repair. Differential activation of tyrosine kinase receptors in 2D and 3D models of GBM explains the well documented discrepancy between preclinical and clinical effects of EGFR inhibitors. Data obtained from our 3D model identify novel determinants and mechanisms of DNA repair and radiosensitivity in GBM, and confirm Akt as a promising therapeutic target in this cancer of unmet need.

Glioblastoma's Next Top Model: Novel Culture Systems for Brain Cancer Radiotherapy Research.

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, remains one of the least treatable cancers. Current standard of care-combining surgical resection, radiation, and alkylating chemotherapy-results in a median survival of only 15 months. Despite decades of investment and research into the development of new therapies, most candidate anti-glioma compounds fail to translate into effective treatments in clinical trials. One key issue underlying this failure of therapies that work in pre-clinical models to generate meaningful improvement in human patients is the profound mismatch between drug discovery systems-cell cultures and mouse models-and the actual tumors they are supposed to imitate. Indeed, current strategies that evaluate the effects of novel treatments on GBM cells in vitro fail to account for a wide range of factors known to influence tumor growth. These include secreted factors, the brain's unique extracellular matrix, circulatory structures, the presence of non-tumor brain cells, and nutrient sources available for tumor metabolism. While mouse models provide a more realistic testing ground for potential therapies, they still fail to account for the full complexity of tumor-microenvironment interactions, as well as the role of the immune system. Based on the limitations of current models, researchers have begun to develop and implement novel culture systems that better recapitulate the complex reality of brain tumors growing in situ. A rise in the use of patient derived cells, creative combinations of added growth factors and supplements, may provide a more effective proving ground for the development of novel therapies. This review will summarize and analyze these exciting developments in 3D culturing systems. Special attention will be paid to how they enhance the design and identification of compounds that increase the efficacy of radiotherapy, a bedrock of GBM treatment.

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells.

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060-71. ©2018 AACR.

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

Background: Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug-radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods: We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results: In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions: Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug-radiation combinations for GBM.

Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus.

RNA polymerase (pol) III transcription is a major determinant of biosynthetic capacity, providing essential products such as tRNA and 5S rRNA. It is controlled directly by the tumour suppressors RB and p53. High-risk types of human papillomavirus (HPV), such as HPV16, express the oncoproteins E6 and E7 that can inactivate p53 and RB, respectively. Accordingly, both E6 and E7 stimulate pol III transcription in cultured cells. HPV16-positive cervical biopsies express elevated levels of tRNA and 5S rRNA when compared to biopsies that test negative for HPV or are infected with the lower risk HPV11. Integration of viral DNA into the host cell genome stimulates expression of E6 and E7 and correlates with induction of tRNA and 5S rRNA. Expression of mRNA encoding the pol III-specific transcription factor Brf1 also correlates with the presence of integrated HPV16. Brf1 levels are limiting for tRNA and 5S rRNA synthesis in cervical cells. Furthermore, pol III-transcribed genes that do not use Brf1 are not induced in HPV16-positive biopsies. Three complementary mechanisms may therefore allow high-risk HPV to stimulate production of tRNA and 5S rRNA: E6-mediated removal of p53; E7-mediated neutralization of RB; and induction of Brf1. The resultant increase in biosynthetic capacity may contribute to deregulated cell growth.

Activation by c-Myc of transcription by RNA polymerases I, II and III.

The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.

Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with Rab25 expression in ovarian cancer.

The small GTPase Rab25 has been functionally linked to tumour progression and aggressiveness in ovarian cancer and promotes invasion in three-dimensional environments. This type of migration has been shown to require the expression of the hypoxia-inducible factor 1 alpha (HIF-1α). In this report we demonstrate that Rab25 regulates HIF-1α protein expression in an oxygen independent manner in a panel of cancer cell lines. Regulation of HIF-1α protein expression by Rab25 did not require transcriptional upregulation, but was dependent on de novo protein synthesis through the Erbb2/ERK1/2 and p70S6K/mTOR pathways. Rab25 expression induced HIF-1 transcriptional activity, increased cisplatin resistance, and conferred intraperitoneal growth to the A2780 cell line in immunocompromised mice. Targeting HIF1 activity by silencing HIF-1ß re-sensitised cells to cisplatin in vitro and reduced tumour formation of A2780-Rab25 expressing cells in vivo in a mouse ovarian peritoneal carcinomatosis model. Similar effects on cisplatin resistance in vitro and intraperitoneal tumourigenesis in vivo were obtained after HIF1b knockdown in the ovarian cancer cell line SKOV3, which expresses endogenous Rab25 and HIF-1α at atmospheric oxygen concentrations. Our results suggest that Rab25 tumourigenic potential and chemoresistance relies on HIF1 activity in aggressive and metastatic ovarian cancer. Targeting HIF-1 activity may potentially be effective either alone or in combination with standard chemotherapy for aggressive metastatic ovarian cancer.

Direct regulation of RNA polymerase III transcription by RB, p53 and c-Myc.

The synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is cell cycle regulated in higher organisms. Overexpression of pol III products is a general feature of transformed cells. These observations may be explained by the fact that a pol III-specific transcription factor, TFIIIB, is strongly regulated by the tumor suppressors RB and p53, as well as the proto-oncogene product c-Myc. RB and p53 repress TFIIIB, but this restraint can be lost in tumors through a variety of mechanisms. In contrast, c-Myc binds and activates TFIIIB, causing potent induction of pol III transcription. Using chromatin immunoprecipitation and RNA interference, we show that c-Myc interacts with tRNA and 5S rRNA genes in transformed cervical cells, stimulating their expression. Availability of pol III products may be an important determinant of a cell's capacity to grow. The ability to regulate pol III output may therefore be integral to the growth control functions of RB, p53 and c-Myc.

Cucurbit [7] uril encapsulated cisplatin overcomes resistance to cisplatin induced by Rab25 overexpression in an intraperitoneal ovarian cancer model.

BACKGROUND: Ovarian cancer is the most fatal of gynaecological malignancies, usually detected at a late stage with intraperitoneal dissemination. Appropriate preclinical models are needed that recapitulate both the histopathological and molecular features of human ovarian cancer for drug-efficacy analysis. METHODS: Longitudinal studies comparing cisplatin performance either alone or in a novel cisplatin-based delivery-system, cucurbit[7]uril-encapsulated cisplatin (cisplatin@CB[7]) were performed on subcutaneous (s.c.) and intraperitoneal (i.p.) xenografts using the human ovarian cancer cell line A2780 stably expressing the small GTPase Rab25, which allows A2780 intraperitoneal growth; and luciferase, to allow tumour load measurement by non-invasive bioluminescent imaging. RESULTS: Rab25 expression induced cisplatin resistance compared to the parental cell line as assessed by the MTT assay in vitro. These findings did not translate in vivo, where cisplatin resistance was determined by the microenvironment. Subcutaneous xenografts of either parental A2780 or cisplatin-resistant Rab25-expressing A2780 cells presented similar responses to cisplatin treatment. In contrast, increased cisplatin resistance was only detected in i.p. tumours. Treatment of the cisplatin-resistant i.p. model with the novel cisplatin@CB[7] delivery system resulted in a substantial reduction of i.p. tumour load and increased necrosis. CONCLUSIONS: Poor clinical performance of novel chemotherapeutics might reflect inappropriate preclinical models. Here we present an ovarian i.p. model that recapitulates the histopathological and chemoresistant features of the clinical disease. In addition, we demonstrate that the novel cisplatin-delivery system, cisplatin@CB[7] may have utility in the treatment of drug-resistant ovarian human cancers.

Abrogation of radioresistance in glioblastoma stem-like cells by inhibition of ATM kinase.

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU-55933. We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC's display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU-55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6-3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU-55933 in GBM CSCs. ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.

Differential sensitivity of Glioma stem cells to Aurora kinase A inhibitors: implications for stem cell mitosis and centrosome dynamics.

Glioma stem-cell-like cells are considered to be responsible for treatment resistance and tumour recurrence following chemo-radiation in glioblastoma patients, but specific targets by which to kill the cancer stem cell population remain elusive. A characteristic feature of stem cells is their ability to undergo both symmetric and asymmetric cell divisions. In this study we have analysed specific features of glioma stem cell mitosis. We found that glioma stem cells appear to be highly prone to undergo aberrant cell division and polyploidization. Moreover, we discovered a pronounced change in the dynamic of mitotic centrosome maturation in these cells. Accordingly, glioma stem cell survival appeared to be strongly dependent on Aurora A activity. Unlike differentiated cells, glioma stem cells responded to moderate Aurora A inhibition with spindle defects, polyploidization and a dramatic increase in cellular senescence, and were selectively sensitive to Aurora A and Plk1 inhibitor treatment. Our study proposes inhibition of centrosomal kinases as a novel strategy to selectively target glioma stem cells.

Cucurbit[7]uril encapsulated cisplatin overcomes cisplatin resistance via a pharmacokinetic effect.

The cucurbit[n]uril (CB[n]) family of macrocycles has been shown to have potential in drug delivery where they are able to provide physical and chemical stability to drugs, improve drug solubility, control drug release and mask the taste of drugs. Cisplatin is a small molecule platinum-based anticancer drug that has severe dose-limiting side-effects. Cisplatin forms a host-guest complex with cucurbit[7]uril (cisplatin@CB[7]) with the platinum atom and both chlorido ligands located inside the macrocycle, with binding stabilised by four hydrogen bonds (2.15-2.44 Å). Whilst CB[7] has no effect on the in vitro cytotoxicity of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant sub-lines A2780/cp70 and MCP1, there is a significant effect on in vivo cytotoxicity using human tumour xenografts. Cisplatin@CB[7] is just as effective on A2780 tumours compared with free cisplatin, and in the cisplatin-resistant A2780/cp70 tumours cisplatin@CB[7] markedly slows tumour growth. The ability of cisplatin@CB[7] to overcome resistance in vivo appears to be a pharmacokinetic effect. Whilst the peak plasma level and tissue distribution are the same for cisplatin@CB[7] and free cisplatin, the total concentration of circulating cisplatin@CB[7] over a period of 24 hours is significantly higher than for free cisplatin when administered at the equivalent dose. The results provide the first example of overcoming drug resistance via a purely pharmacokinetic effect rather than drug design or better tumour targeting, and demonstrate that in vitro assays are no longer as important in screening advanced systems of drug delivery.

Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

BACKGROUND: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. METHODOLOGY/PRINCIPAL FINDINGS: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. CONCLUSIONS/SIGNIFICANCE: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.

TRRAP and GCN5 are used by c-Myc to activate RNA polymerase III transcription.

Activation of RNA polymerase (pol) II transcription by c-Myc generally involves recruitment of histone acetyltransferases and acetylation of histones H3 and H4. Here, we describe the mechanism used by c-Myc to activate pol III transcription of tRNA and 5S rRNA genes. Within 2 h of its induction, c-Myc appears at these genes along with the histone acetyltransferase GCN5 and the cofactor TRRAP. At the same time, occupancy of the pol III-specific factor TFIIIB increases and histone H3 becomes hyperacetylated, but increased histone H4 acetylation is not detected at these genes. The rapid acetylation of histone H3 and promoter assembly of TFIIIB, c-Myc, GCN5, and TRRAP are followed by recruitment of pol III and transcriptional induction. The selective acetylation of histone H3 distinguishes pol III activation by c-Myc from mechanisms observed in other systems.

Direct activation of RNA polymerase III transcription by c-Myc.

The proto-oncogene product c-Myc has a direct role in both metazoan cell growth and division. RNA polymerase III (pol III) is involved in the generation of transfer RNA and 5S ribosomal RNA, and these molecules must be produced in bulk to meet the need for protein synthesis in growing cells. We demonstrate here that c-Myc binds to TFIIIB, a pol III-specific general transcription factor, and directly activates pol III transcription. Chromatin immunoprecipitation reveals that endogenous c-Myc is present at tRNA and 5S rRNA genes in cultured mammalian cells. These results suggest that activation of pol III may have a role in the ability of c-Myc to stimulate cell growth.