Katrina Stevenson

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, NFκB, 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.

Transcriptional repression of telomerase RNA gene expression by c-Jun-NH2-kinase and Sp1/Sp3.

Telomerase is essential for immortalization of most human cancer cells. Expression of the core telomerase RNA (hTR) and reverse transcriptase (hTERT) subunits is mainly regulated by transcription. However, hTR transcriptional regulation remains poorly understood. We previously showed that the core hTR promoter is activated by Sp1 and is repressed by Sp3. Here, we show that the mitogen-activated protein kinase kinase kinase 1 (MEKK1)/c-Jun-NH(2)-kinase (JNK) pathway represses hTR expression by a mechanism that involves Sp1 and Sp3. Promoter activity was induced by the JNK inhibitor SP600125 and was repressed by activated MEKK1. Repression by MEKK1 was blocked by SP600125 or enhanced by coexpression of wild-type but not phosphoacceptor mutated JNK. SP600125 treatment also increased levels of endogenous hTR. Mutations in the hTR promoter Sp1/Sp3 binding sites attenuated SP600125-mediated promoter induction, whereas coexpression of MEKK1 with Sp3 enhanced hTR promoter repression. Chromatin immunoprecipitation showed that levels of immunoreactive Sp1 associated with the hTR promoter were low in comparison with Sp3 in control cells but increased after JNK inhibition with a reciprocal decrease in Sp3 levels. No corresponding changes in Sp1/Sp3 protein levels were detected. Thus, JNK represses hTR promoter activity and expression, apparently by enhancing repression through Sp3.

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms.

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability.

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

Abstract 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.

A ‘synthetic-sickness’ screen for senescence re-engagement targets in mutant cancer backgrounds

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.

Evaluation of four different small animal radiation plans on tumour and normal tissue dosimetry in a glioblastoma mouse model

OBJECTIVE: Small animal radiotherapy research platforms such as XStrahls SARRP enable more precise irradiation of tumours and normal tissues in pre-clinical models of cancer. Using an orthotopic G7 glioblastoma xenograft model we studied the impact of four different radiotherapy plans on tumour and normal tissue dosimetry. METHODS: Plans were created using four different approaches (single beam, parallel opposed pair, single plane arcs, couch rotation arcs) and dose volume histograms (DVH) for the tumour and the relevant organs at risk (OARs) (mouth, ipsilateral brain, contralateral brain, brain stem) were compared for a sample mouse subject. To evaluate the accuracy of delivery, treatment plans were recreated in solid-water phantoms and delivered to radiochromic film. RESULTS: Favourable tumour dosimetry was achieved by all plans. DVH analysis showed that different plans could be used to spare specific OARs depending on the objectives of the study. The delivery accuracy of the various treatments was better than 2%/2mm (dose difference/distance to agreement) in terms of global γ analysis. CONCLUSION: Small animal radiotherapy research platforms are an exciting addition to the pre-clinical research environment. Such systems improve the conformality of irradiation of tumours and OARs while maintaining a high degree of accuracy and enable investigators to optimise experiments in terms of tumour coverage and inclusion or exclusion of relevant OARs. ADVANCES IN KNOWLEDGE: This study confirms the utility of the SARRP in terms of the accuracy of plan delivery, and informs decisions on treatment planning to optimise the clinical relevance and scientific value of experiments.

Abstract 738: Design and synthesis of novel N10-protected pyrrolobenzodiazepine (PBD) prodrugs for use in nitroreductase-mediated GDEPT therapies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The over-expression of telomerase in cancer cells has been previously exploited for gene therapy strategies. One approach involves the use of a plasmid containing a telomerase promoter to control the expression of an exogenous nitroreductase enzyme capable of activating bioreductively-sensitive prodrugs. CB1954 is the most commonly studied prodrug for use in bioreductive GDEPT approaches, although it has a number of drawbacks including relatively low potency, inherent toxicities and a lack of patent protection. Therefore, we have designed some novel bioreductive prodrugs based on the sequence-selective DNA-interactive pyrrolo\[2,1-c\]\[1,4\]benzodiazepine (PBD) antitumour agents. The PBDs interact covalently with DNA through formation of a covalent aminal bond between their electrophilic N10-C11 position and the nucleophilic C2-NH2 of guanine bases. The prodrug design concept involves the introduction of a bulky bioreductively-sensitive protecting group at the N10-position which effectively blocks interaction with DNA thus reducing potency. However, release of the N10-protecting group under bioreductive conditions restores the ability to interact with DNA along with the original biological activity. As proof-of-principle, we installed a p-nitrobenzylcarbamate group at the N10-position of a PBD. We found that upon reduction to the N10-(p-aminobenzylcarbamate), this grouping self-immolated to afford the biologically-active parent PBD, p-aminobenzyl alcohol and carbon dioxide. Control molecules including non-reducible N10-benzyl- and N10-SEM-protected analogues incapable of self-immolation were also synthesized. Along with the parent N10-unsubstituted PBD, these molecules were all evaluated in matched in vitro panels of human A2780 (ovarian), A549 (lung), C33a (cervical) and 5637 (bladder) tumour cells, one panel (“NTR+”) being transfected with plasmids containing the Nitroreductase (NTR) gene under the control of a CMV promoter, a surrogate for the telomerase promoter. The NTR+ panel was found to be more sensitive to the prodrug than the NTR- panel, with a rank order of sensitisation of 18.4 > 8.1 > 2.6 and 1.5 for the A2780, A549, C33a and 5637 cell lines, respectively. Crucially, the prodrug was significantly less cytotoxic in all cell lines (e.g., IC50 = 0.29 and 0.015μ in NTR- and NTR+ A2780 cells, respectively) compared to the parent non-N10-substituted PBD (e.g., IC50 = 0.000151and 0.00028µM in NTR- and NTR+ A2780 cells, respectively). The N10-benzyl and SEM control molecules were significantly less cytotoxic in both NTR+ and NTR- A2780 cells, with IC50 values ranging from 1.9 to 3.3μM. In preliminary in vivo experiments, the N10-(p-nitrobenzylcarbamate) prodrug was evaluated in a nude mouse human tumour xenograft model implanted with A2780 CMV-NTR cells. A clear tumour response to the prodrug was observed at non-toxic doses. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 738.