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Supplementary figure 1 describes how Triamterene requires thymidylate synthase expression to induce selectivity in MMR -deficient cells. Related to Figure 2.

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial.Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivo.Results: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks.Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880–90. ©2016 AACR.

The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL–ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations. Cancer Res; 74(5); 1588–97. ©2014 AACR.

Background Glioblastoma multiforme (GBM) is the most common high-grade malignant brain tumour in adults and arises from the glial cells in the brain. The prognosis of treated GBM remains very poor with 5-year survival rates of 5%, a figure which has not improved over the last few decades. Currently, there is a modest 14-month overall median survival in patients undergoing maximum safe resection plus adjuvant chemoradiotherapy. HOX gene dysregulation is now a widely recognised feature of many malignancies. Methods In this study we have focused on HOX gene dysregulation in GBM as a potential therapeutic target in a disease with high unmet need. Results We show significant dysregulation of these developmentally crucial genes and specifically that HOX genes A9, A10, C4 and D9 are strong candidates for biomarkers and treatment targets for GBM and GBM cancer stem cells. We evaluated a next generation therapeutic peptide, HTL-001, capable of targeting HOX gene over-expression in GBM by disrupting the interaction between HOX proteins and their co-factor, PBX. HTL-001 induced both caspase-dependent and –independent apoptosis in GBM cell lines. Conclusion In vivo biodistribution studies confirmed that the peptide was able to cross the blood brain barrier. Systemic delivery of HTL-001 resulted in improved control of subcutaneous murine and human xenograft tumours and improved survival in a murine orthotopic model.

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The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD‐SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα‐expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c‐Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα‐target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self‐renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Simple Summary High-grade gliomas remain difficult-to-treat cancers. Novel treatment options include targeting glycogen synthase kinase 3 (GSK-3) to induce cell death but the mode of drug activity remains unknown and combination with conventional treatment including radiotherapy has not been explored. Here, we describe the effect of targeting GSK-3 with the inhibitor AZD2858 in in vitro and in vivo models of glioma. We established that AZD2858 exposure induces mitotic defects leading to cell death in patient-derived glioma cell lines and tumor growth delay in glioma xenografts. Co-administration also enhanced the effect of radiotherapy. We therefore propose AZD2858 as an adjuvant to radiotherapy in high-grade glioma. Abstract Background: Previous data on glycogen synthase kinase 3 (GSK-3) inhibition in cancer models support a cytotoxic effect with selectivity for tumor cells compared to normal tissue but the effect of these inhibitors in glioma has not been widely studied. Here, we investigate their potential as cytotoxics in glioma. Methods: We assessed the effect of pharmacologic GSK-3 inhibition on established (U87, U251) and patient-derived (GBM1, GBM4) glioblastoma (GBM) cell lines using cytotoxicity assays as well as undertaking a detailed investigation of the effect on cell cycle, mitosis, and centrosome biology. We also assessed drug uptake and efficacy of GSK-3 inhibition alone and in combination with radiation in xenograft models. Results: Using the selective GSK-3 inhibitor AZD2858, we demonstrated single agent cytotoxicity in two patient-derived glioma cell lines (GBM1, GBM4) and two established cell lines (U251 and U87) with IC50 in the low micromolar range promoting centrosome disruption, failed mitosis, and S-phase arrest. Glioma xenografts exposed to AZD2858 also showed growth delay compared to untreated controls. Combined treatment with radiation increased the cytotoxic effect of clinical radiation doses in vitro and in orthotopic glioma xenografts. Conclusions: These data suggest that GSK-3 inhibition promotes cell death in glioma through disrupting centrosome function and promoting mitotic failure and that AZD2858 is an effective adjuvant to radiation at clinical doses.

MGMT promoter methylation is the only accepted biomarker with prognostic role in GBM but its routine implementation is limited partly response to TMZ is heterogeneous, but also due to lack of effective alternative treatment options. Therefore, additional biomarkers are needed to enable better prediction of survival and to improve individualized treatment of GBM patients. A potential new biomarker is the epidermal growth factor receptor variant III (EGFRvIII). This constitutively activated deletion variant is present in approximately one third of all IDH wildtype GBM, but its relevance to treatment response is poorly understood. The aim of the present study was to analyze the impact of endogenous EGFRvIII expression on chemosensitivity and the mechanisms underlying any differential treatment response. EGFRvIII expression was associated with prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. In line with this, we observed increased TMZ sensitivity of EGFRvIII+ and MGMT promoter methylated cells, which translated into improved survival in xenograft experiments. The increased TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cell lines and EGFRvIII+ GBM patient samples. Subsequently, only a moderate reduction in MMR protein expression resulted in a dramatic TMZ resistance, suggesting that EGFRvIII expression specifically sensitized MGMT deficient cells to TMZ treatment by upregulating MMR. Furthermore, EGFRvIII expression in GBM cell lines was accompanied by increased DNA damage, replication fork slowing, stalling and enhanced origin firing, implying replication stress. Targeting of EGFRvIII-dependent replication stress by irinotecan led to hypersensitivity of EGFRvIII+ cells. Taken together this study illustrates that EGFRvIII-induced upregulation of MMR and replication stress increases chemosensitivity thereby highlighting the vulnerability of EGFRvIII+ GBM to available treatments. These important data may also guide the development of new and more effective personalized strategies.

Despite many clinical efforts, the prognosis of GBM patients has not improved in the last 10 years. Epigenetic silencing of the O6-methylguanine-DNA-methyltransferase (MGMT) gene is the only predictive biomarker that is associated with temozolomide (TMZ) response. The establishment of new biomarkers, for personalized treatment is of significant interest. The epidermal growth factor receptor (EGFR) variant III (EGFRvIII) is expressed in approximately 20–30% of GBM. The aim of this study was to investigate if EGFRvIII is a predictive biomarker for TMZ response by analyzing the TMZ sensitivity of isogenic cell cultures and tumour xenografts with/without endogenous EGFRvIII expression. Using two, unique isogenic pairs of MGMT deficient GBM cells we observed that endogenous EGFRvIII expression was associated with a significant increase in TMZ sensitivity in vitro and in vivo. Knockdown of EGFRvIII restored TMZ resistance, confirming EGFRvIII dependence. We observed prolonged accumulation in S/G2-phase in EGFRvIII+ cells after TMZ treatment, indicating increased replication stress induced by TMZ and a significantly higher number of DNA double strand breaks. Because TMZ sensitivity in MGMT negative cells is predominantly determined by functional mismatch repair (MMR), we next analyzed MMR protein expression. We observed that EGFRvIII expression was associated with upregulated MMR proteins and that conversely, even a moderate decrease in MSH6 and MLH1 expression yielded a pronounced increase in TMZ resistance. In accordance with our findings, analysis of clinical data revealed that EGFRvIII positive GBM patients displaying MGMT promotor methylation have significantly better outcomes than EGFRvIII negative patients. In summary, we have shown that MGMT negative EGFRvIII expressing GBM cells and tumours are significantly more sensitive to TMZ compared to isogenic EGFRvIII negative counterparts due to upregulated MMR. Our data highlight a new mechanism of TMZ sensitivity and we conclude that EGFRvIII is a predictive biomarker for response to TMZ in MGMT methylated GBM patients.

Purpose: The DNA mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations; however, it is becoming increasingly clear that a therapy targeting MMR deficiency in general would be clinically very beneficial. Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivo. Results: The selective tumor cell cytotoxicity of triamterene occurs through its antifolate activity and depends on the activity of the folate synthesis enzyme thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double-strand breaks. Conclusions: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR deficiency in a range of different tumor types and could significantly improve patient survival. Clin Cancer Res; 23(11); 2880–90. ©2016 AACR.

BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with an estimated 5-year survival of less 10%. The current standard of care involves maximal tumor resection followed by radiation and chemotherapy with the alkylating agent temozolomide (TMZ). Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene predicts response to TMZ therapy, but does not explain all of the heterogeneity in responses observed in the clinic. The establishment of additional molecular biomarkers, is therefore of significant interest.

The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL–ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations. Cancer Res; 74(5); 1588–97. ©2014 AACR.

Summary Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs., Highlights • RAD51 is overexpressed in glioma stem cells • RAD51 expression levels fall when GSCs are differentiated • RAD51 inhibitors abrogate DNA repair leading to radiosensitization in GSCs • RAD51 inhibition + XR removes SOX2-expressing cells and abolishes clonogenicity, Short and colleagues show overexpression of RAD51 in a poorly differentiated stem-like population of glioma cells and that the homologous DNA double-strand break-repair pathway may represent a specific therapeutic target. RAD51-dependent repair is essential for recovery from radiation and temozolomide-induced DNA damage. Targeting this pathway may provide a tumor cell-specific effect in the context of chemo-radiotherapy.

INTRODUCTION: HOX genes are essential for embryonic development but are dysregulated in numerous cancers, including glioblastoma. High levels of HOX expression are characteristic of a subset of glioma stem cells (GSC) and have been associated with self-renewal capability and treatment resistance. We aimed to determine whether HOX proteins are a potential therapeutic target in glioblastoma. METHOD: HOX expression profiles were determined using quantitative RT-PCR. Patient-derived GSC, glioma cell lines and primary astrocytes were treated with HXR9, a peptide that disrupts the interaction between HOX proteins and their binding partner PBX. Cell viability was determined using the MTS assay. Combination peptide and radiation treatments were assessed by clonogenic assay. Sub-cutaneous xenografts of U87-MG cells were established in Balb/C Nude mice and tumours were treated with intratumoural injection of peptide and/or CT-guided fractionated radiotherapy. RESULTS: Patient-derived GSC showed variable HOX profiles, similar to previously reported HOX-high and -low signatures. HXR9 was potently cytotoxic in a number of GSC cultures, with IC50 values of 3-9 µM. In contrast, normal astrocytes were much more resistant (IC50 = 70 µM), as were the cell lines T98G and U87MG (70 and 35 µM, respectively). An additive cytotoxic effect was observed for combined HXR9 and radiation treatment in vitro, and HXR9 treatment resulted in delayed tumour growth in vivo, both alone and in combination with radiotherapy. CONCLUSION: The HOX inhibitor HXR9 showed potent cytotoxicity in cultured GSC and reduced the rate of tumour growth in vivo. Targeting HOX proteins is a potential adjuvant treatment for glioblastoma alongside current chemoradiotherapy.

Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3′ untranslated region (region B)are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3′-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

BACKGROUND: Resistance of glioblastoma to standard chemoradiotherapy may be mediated, at least partially, by a population of inherently resistant glioma stem cells (GSCs) which are able to survive treatment and reconstitute the tumour. HOX genes are dysregulated in various cancers and a subset of GSCs show high levels of HOX expression. In addition, a HOX gene signature has been associated with treatment resistance in glioblastoma. Our goal was to determine the potential of targeting HOX function as a novel therapy for glioblastoma. METHODS: HOX expression profiles for patient-derived GSCs and established glioma cell lines were determined by quantitative RT-PCR. In vitro, cells were treated with the HOX inhibitor peptide HXR9 (or control peptide CXR9), with or without radiation two hours later. Subsequently, cells were incubated through two doubling times and the MTS assay was used to determine cell viability. In vivo, sub-cutaneous xenografts of U87-MG cells were established in Balb/c Nude mice. Treatments (peptide and/or radiation) or controls were administered in three doses over consecutive days. RESULTS: Analysis of HOX expression in GSC lines demonstrated up-regulation of genes across all four clusters, although expression patterns varied between cell lines. In contrast, the established glioma cell lines T98G and U87-MG showed lower overall HOX expression. The HOX inhibitor peptide HXR9 had potent cytotoxic activity in a number of GSC lines, with IC50 values between 3 and 5 µM. In contrast, T98G and U87-MG were more resistant (IC50s of 70 and 35 µM, respectively), as was a normal neural progenitor cell line NP1 (IC50 65 µM). In vitro, combinations of sub-lethal doses of HXR9 with radiation demonstrated an additive cytoxic effect on GSCs. In vivo, treatment with HXR9 peptide resulted in tumour growth delay when combined with radiotherapy in a mouse xenograft model. CONCLUSIONS: Knockdown of HOX protein function reduced viability of glioma cells in vitro, with greater potency in GSCs than established cell lines. Combination of HOX-inhibition with radiation or demonstrated an additive cytoxicity in vitro and improved tumour growth delay in a preclinical mouse model. Therefore, targeting HOX proteins shows potential as an adjuvant therapy with current standard of care chemoradiotherapy for glioblastoma.

Using in vitro and in vivo assays, we define a network of Her/Hes dimers underlying transcriptional negative feedback within the zebrafish segmentation clock. Some of the dimers do not appear to be DNA-binding, whereas those dimers that do interact with DNA have distinct preferences for cis regulatory sequences. Dimerization is specific, with Hes6 serving as the hub of the network. Her1 binds DNA only as a homodimer but will also dimerize with Hes6. Her12 and Her15 bind DNA both as homodimers and as heterodimers with Hes6. Her7 dimerizes strongly with Hes6 and weakly with Her15. This network structure engenders specific network dynamics and imparts greater influence to the Her7 node. Computational analysis supports the hypothesis that Her7 disproportionately influences the availability of Hes6 to heterodimerize with other Her proteins. Genetic experiments suggest that this regulation is important for operation of the network. Her7 therefore has two functions within the zebrafish segmentation clock. Her7 acts directly within the delayed negative feedback as a DNA-binding heterodimer with Hes6. Her7 also has an emergent function, independent of DNA binding, in which it modulates network topology via sequestration of the network hub.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red (1,2). This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) (3). The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy (3,4). One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein (5). When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

A cell population with stem cell like characteristics is thought to underlie treatment resistance and local recurrence in glioma. In this work we investigate the role of the DNA repair protein RAD51 in these cells and the impact of inhibiting homologous recombination repair on radiation resistance in vitro and in vivo. We used a model of inducible differentiation of patient derived glioma cells to investigate repair protein levels, repair foci formation and kinetics in clonogenic, stem-like populations and their differentiated counterparts. We examined co-expression of stem cell markers with RAD51 protein at whole population level using western blotting, immunocytochemistry and RT-PCR in cultured cells and immunohistochemistry in tumor material. Single cell expression was analysed using the Fluidigm C1 platform. We examined the effect of two specific inhibitors of RAD51 (B02, RI-1) on the same cell pairs in vitro and used the γH2AX assay to assess differences in repair kinetics. We used subcutaneous models of glioma to evaluate the effect of one of these agents (RI-1) on tumour growth delay with and without fractionated radiation doses in vivo. Primary glioma stem cells expressed high levels of RAD51 protein and exhibited high numbers of foci per nucleus following radiation exposure. Levels of both protein and repair foci fell after cells were transferred to differentiating conditions (serum+BMP4), as did expression of stem cell markers (NESTIN, SOX2). Single cell analysis showed a highly significant association between SOX2 and RAD51 expression (P< 0.0001). A similar association between RAD51 expression and stem cell marker expression was demonstrated in a series of glioblastoma specimens. When primary glioma cultures were treated with B02, RI-1 or siRNA targeting RAD51 in vitro, significant radiosensitisation was achieved with dose modifying factors ∼1.4 after single radiation doses. Delayed repair of DNA double strand breaks was also apparent, with a higher proportion of γH2AX foci unresolved in treated cells at 24 hours post irradiation. We also demonstrated significantly enhanced tumor growth delay in vivo when RI-1 was combined with fractionated radiation therapy (5Gy x 3) in a subcutaneous model. These data suggest that RAD51 dependent DNA repair by homologous recombination represents a specific target in the stem-like fraction of glioblastoma and inhibiting this pathway is a promising approach to radiosensitisation. Citation Format: Henry King, Helen Payne, Tim Brend, Anjana Patel, Alex Wright, Teklu Englu, Lucy Stead, Heiko Wurdak, Susan C. Short. Radioresistance in glioma stem cells driven by Rad51 dependent homologous recombination repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3303. doi:10.1158/1538-7445.AM2015-3303