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 growt

The impact of a novel 3D cell culture model of glioblastoma on radiation and drug-radiation responses

Novel 3D cell culture models enable cell growth in a more physiological environment than conventional 2D cell cultures. Most importantly, cells need to be embedded in a composition of extracellular matrix proteins similarly present in situ to guarantee conservation of the phenotype. As shown by comparative analyses between 2D, 3D and tumor xenografts, various processes such as signal transduction and DNA repair share great similarity in 3D and in-vivo but not 2D. Based on our long-standing experience, a large variety of endpoints can be determined and many methods can be conducted in 3D matrix-based cell cultures. While this is sometimes not as easy as in 2D and also requires a bit more financial invest, the generated data reflect cell behavior invivo and thus have a higher clinically relevance. Further, we are able to address specific tumor features in detail. For example, malignant tumors show great genetic/epigenetic and morphological/cell biological heterogeneity. Here, a prime example is the stiffness of a tumor. Although we know that the stiffness greatly varies in different parts of the tumor, the underlying mechanisms and prosurvival consequences on the genetic/epigenetic and morphological/cell biological level are far from being understood. 3D matrix-based cell cultures models can elegantly support our efforts to gain more knowledge in this field. Another important point is the sparing of animal experiments based on our broad knowledge that human (patho)physiology is significantly different from mice (or other species). Many decades of in-vivo research have demonstrated that only a negligible proportion of therapeutic approaches could be translated from rodents to humans. In conclusion, 3D cell cultures are powerful tools to generate more clinically relevant information. A broader implementation of this methodology is likely to underscore our efforts to better understand tumor and normal cell radiation responses and foster identification of most critical cancer target

OS1.4 Induction of mitotic cell death : a novel therapeutic strategy for glioblastoma

Background Glioblastoma (GBM) is the most common and lethal adult brain tumour. Tumours typically contain large numbers of binuclear and multinucleated cells, and a subpopulation of relatively quiescent glioma stem-like cells (GSC) that are thought to be responsible for treatment resistance and tumour recurrence. GSC display robust G2/M arrest following ionising radiation (IR) yet are highly prone to aberrant cell division and are sensitive to mitotic spindle checkpoint inhibitors. The aim of this study was to investigate the therapeutic activity of mitotic inducers in preclinical models of GBM. Material and Methods Bioinformatic analysis of mRNA expression data was used to confirm the relevance of mitotic activity following irradiation of GSC in a 3D patient-derived GBM stem cell model. Immunofluorescence, clonogenic survival and cell viability assays were used to evaluate the therapeutic potential of mitotic inducers (ME-344; Wee1 inhibitor AZ1775) in 2D and 3D U87MGLuc and E2, G1, G7, S2 and R15 patient-derived GSC cell culture models. either alone or in combination with IR. In vivo validation was determined in U87-MGLuc orthotopic GBM mouse model. Results Radiation induced downregulation of mRNA expression of several mitotic genes was observed in G7 and E2 cell lines confirming mitotic relevance. In cell viability and clonogenic survival assays, AZ1775 and ME-344 showed potent cytotoxicity against all GSC cell lines in both 2D and 3D, with EC50 values of 0.2 to 0.4 μM for AZ1775 and 0.003 to 0.02 μM for ME-344. ME-344 and AZ1775 triggered profound morphological and cell cycle effects including mitotic induction and arrest, increased mitotic fraction, reduced nuclear tubulin in mitotic cells and, induced mitotic catastrophe in the most sensitive cell lines U87MGLuc and E2. These events were apoptosis-independent. Combination with IR increased GSC cell death in the two GSC models tested to date. Accumulation of cells in mitosis following ME-344 treatment was recapitulated in orthotopic GBM xenografts in vivo, although few mitotic catastrophe events were observed 24 h after treatment. ME-344 demonstrated therapeutic efficacy as a single agent in U87MGLuc2 orthotopic xenografts by extending mouse survival compared to vehicle (p=0.043). Conclusion Two agents that induce mitosis through different mechanisms have promising single agent activity against all GBM cell lines tested in vitro and in vivo. Further preclinical evaluation in combination with IR and/or temozolomide is underway. Results indicate that this therapeutic strategy for GBM has clinical potential

Co-encapsulation of an antigen and CpG oligonucleotides into PLGA microparticles by TROMS technology.

It seems well established that CpG oligonucleotides Th1 biased adjuvant activity can be improved when closely associated with a variety of antigens in, for example, microparticles. In this context, we prepared 1-μm near non-charged PLGA 502 or PLGA 756 microparticles that loaded with high efficiency an antigen (50% ovalbumin (OVA), approximately) into their matrix and CpG-chitosan complexes (near to 20%) onto their surface maintaining OVA and CpG integrity intact. In the intradermal immunization studies, whereas OVA microencapsulated into PLGA 756 alone induced a strong humoral immune response assisted by a very clear Th1 bias (IgG2a/IgG1=0.875) that was decreased by CpG co-delivery (IgG2a/IgG1=0.55), the co-encapsulation of CpG with OVA in PLGA 502 particles significantly improved the antibody response and isotype shifting (IgG2a/IgG1=0.73) in comparison with mice immunized with OVA loaded PLGA 502 (IgG2a/IgG1=0). This improvement was not correlated with the cellular immune response where the effect of co-encapsulated CpG was rather negative (2030.2 pg/mL and 335.3 pg/mL IFN-g for OVA PLGA 502 for OVA CpG PLGA 502, respectively). These results underscore the critical role of polymer nature and microparticle characteristics to show the benefits of coencapsulating CpG motifs in close proximity with an antigen

P08.36 Radioresistance of glioblastoma stem-like cells is associated with DNA replication stress, which is a promising therapeutic target

Introduction: The inevitability of tumour recurrence in glioblastoma (GBM) patients despite multi-modality treatment consisting of surgery, radiotherapy and chemotherapy, is reflected by a median survival of only 14 months. Tumour recurrence is thought to be driven by a small population of glioblastoma stem-like cells (GSCs) that are resistant to conventional therapies. DNA damage response (DDR) pathways have been shown to be up-regulated in GSCs and implicated in radioresistance and treatment failure. However the precise cause of enhanced DDR signalling in GSCs and the extent to which these signalling networks contribute to therapy resistance remains elusive. The objectives of this study were to investigate the underlying cause of DDR upregulation and treatment resistance in GSCs with a view to identifying novel and promising therapeutic targets. Materials and Methods: A panel of primary patient derived GBM cell lines cultured under conditions to enrich for or deplete the tumour stem cell population (GSC vs bulk respectively) were utilised in order to investigate enhanced GSC DDR under basal conditions and in response to ionising radiation. Confirmatory studies were also performed in cells sorted for the putative GSC marker CD133. The effects of a panel of small molecule DDR inhibitor agents on cell survival in GSC and bulk cells were quantified. Results: GSCs exhibited higher levels of total and activated DDR targets ATR, CHK1, ATM and PARP1 under basal conditions and were radioresistant compared to paired bulk populations. This was not due to increased levels of reactive oxygen species (ROS). Instead, we show that RPA is significantly higher in replicating GSCs and confirm by DNA fibre assays that GSCs and CD133+ cells have increased numbers of stalled replication forks, fewer new origins and slower DNA replication compared to bulk or CD133- populations, demonstrating for the first time that replication stress (RS) is a hallmark of GSCs. We identify increased expression of long neural genes as a likely mechanism for RS and DNA double strand breaks (DSBs) in GSCs and show that their radioresistance is reversed by dual inhibition of key RS and DDR proteins ATR and PARP. Conclusions: This study demonstrates the novel finding that replication stress is a hallmark of GSCs and resonates with recently published studies in neural progenitor cells showing that RS preferentially induces DNA DSB in long neural genes. Taken together, we implicate RS as a driver of enhanced DDR in GSCs and identify novel therapeutics with potential to improve clinical outcomes by overcoming the radioresistance of GB

P04.74 Preclinical evaluation of combinations targeting the DNA damage response in 2D and 3D models of glioblastoma stem cells

Background Despite surgical resection followed by DNA-damaging adjuvant therapies, glioblastoma remain incurable. Increasing evidence demonstrates that aberrations within the DNA damage response (DDR) of cancer stem cells contribute to treatment resistance. We have previously shown that the Fanconi Anaemia (FA) pathway, a key DDR process, remains inactive in normal brain but is re-activated in glioblastoma, making it an appealing foundational target for cancer-specific combination therapies. Since intratumoural heterogeneity in glioblastoma and inherent capacity for functional redundancy within DDR networks are established concepts - we aimed to determine whether combined and hypothesis-driven targeting of the FA pathway along with interconnected DDR processes could form a basis for effective multimodal therapies. Material and Methods Bioinformatic analysis of mRNA expression data (REMBRANDT database) was used to confirm the relevance of FA pathway activity in glioma. Subsequently, immunofluorescence and cell viability assays were used to validate and establish the therapeutic potential of novel FA pathway inhibitors (nFAPi) and inhibition of related DDR targets in established cell models. Finally, combinations targeting the DDR were optimised using immunoblotting, and assessed using clonogenic survival in 2D and novel 3D patient-derived glioblastoma stem cell models. Results High expression of downstream FA pathway genes is strongly associated with poor survival (-17.1% 5-year OS, n=329, Log-rank, P Conclusion Simultaneously targeting the FA pathway and interconnected DDR processes in glioblastoma represents a promising therapeutic strategy. Early mechanistic studies suggest this approach augments DNA damage and enhances IR-induced cell cycle arrest in G2/M, however further preclinical evaluation is ongoing

Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies

HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer

Background: HAGE protein is a known immunogenic cancer-specific antigen. Methods: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. Results: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGEþ) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGEþexpression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+ did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+ and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+ residual disease (P=0.0003). Conclusions: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC

A joint physics and radiobiology DREAM team vision - Towards better response prediction models to advance radiotherapy.

Radiotherapy developed empirically through experience balancing tumour control and normal tissue toxicities. Early simple mathematical models formalized this practical knowledge and enabled effective cancer treatment to date. Remarkable advances in technology, computing, and experimental biology now create opportunities to incorporate this knowledge into enhanced computational models. The ESTRO DREAM (Dose Response, Experiment, Analysis, Modelling) workshop brought together experts across disciplines to pursue the vision of personalized radiotherapy for optimal outcomes through advanced modelling. The ultimate vision is leveraging quantitative models dynamically during therapy to ultimately achieve truly adaptive and biologically guided radiotherapy at the population as well as individual patient-based levels. This requires the generation of models that inform response-based adaptations, individually optimized delivery and enable biological monitoring to provide decision support to clinicians. The goal is expanding to models that can drive the realization of personalized therapy for optimal outcomes. This position paper provides their propositions that describe how innovations in biology, physics, mathematics, and data science including AI could inform models and improve predictions. It consolidates the DREAM team's consensus on scientific priorities and organizational requirements. Scientifically, it stresses the need for rigorous, multifaceted model development, comprehensive validation and clinical applicability and significance. Organizationally, it reinforces the prerequisites of interdisciplinary research and collaboration between physicians, medical physicists, radiobiologists, and computational scientists throughout model development. Solely by a shared understanding of clinical needs, biological mechanisms, and computational methods, more informed models can be created. Future research environment and support must facilitate this integrative method of operation across multiple disciplines