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Item Open Access Transcriptional CDK inhibitors CYC065 and THZ1 induce apoptosis in glioma stem cells derived from recurrent GBM(2021) Juric, Viktorija; Düssmann, Heiko; Lamfers, Martine L. M.; Prehn, Jochen H. M.; Rehm, Markus; Murphy, Brona M.Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.Item Open Access Multiphasic modelling and computation of metastatic lung-cancer cell proliferation and atrophy in brain tissue based on experimental data(2021) Ehlers, Wolfgang; Rehm, Markus; Schröder, Patrick; Stöhr, Daniela; Wagner, ArndtCancer is one of the most serious diseases for human beings, especially when metastases come into play. In the present article, the example of lung-cancer metastases in the brain is used to discuss the basic problem of cancer growth and atrophy as a result of both nutrients and medication. As the brain itself is a soft tissue that is saturated by blood and interstitial fluid, the biomechanical description of the problem is based on the Theory of Porous Media enhanced by the results of medication tests carried out in in-vitro experiments on cancer-cell cultures. Based on theoretical and experimental results, the consideration of proliferation, necrosis and apoptosis of metastatic cancer cells is included in the description by so-called mass-production terms added to the mass balances of the brain skeleton and the interstitial fluid. Furthermore, the mass interaction of nutrients and medical drugs between the solid and the interstitial fluid and its influence on proliferation, necrosis and apoptosis of cancer cells are considered. As a result, the overall model is appropriate for the description of brain tumour treatment combined with stress and deformation induced by cancer growth in the skull.Item Open Access Low-level endothelial TRAIL-receptor expression obstructs the CNS-delivery of angiopep-2 functionalised TRAIL-receptor agonists for the treatment of glioblastoma(2021) Krishna Moorthy, Nivetha; Seifert, Oliver; Eisler, Stephan; Weirich, Sara; Kontermann, Roland E.; Rehm, Markus; Fullstone, GavinGlioblastoma (GBM) is the most malignant and aggressive form of glioma and is associated with a poor survival rate. Latest generation Tumour Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding to death receptors. However, the blood–brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). We therefore investigated if antibody-based fusion proteins that combine hexavalent TRAIL and angiopep-2 (ANG2) moieties can be developed, with ANG2 promoting receptor-mediated transcytosis (RMT) across the BBB. We demonstrate that these fusion proteins retain the potent apoptosis induction of hexavalent TRAIL-receptor agonists. Importantly, blood–brain barrier cells instead remained highly resistant to this fusion protein. Binding studies indicated that ANG2 is active in these constructs but that TRAIL-ANG2 fusion proteins bind preferentially to BBB endothelial cells via the TRAIL moiety. Consequently, transport studies indicated that TRAIL-ANG2 fusion proteins can, in principle, be shuttled across BBB endothelial cells, but that low TRAIL receptor expression on BBB endothelial cells interferes with efficient transport. Our work therefore demonstrates that TRAIL-ANG2 fusion proteins remain highly potent in inducing apoptosis, but that therapeutic avenues will require combinatorial strategies, such as TRAIL-R masking, to achieve effective CNS transport.Item Open Access Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol(2021) Hellwig, Christian T.; Delgado, M. Eugenia; Skoko, Josip; Dyck, Lydia; Hanna, Carol; Wentges, Alexa; Langlais, Claudia; Hagenlocher, Cathrin; Mack, Alexandra; Dinsdale, David; Cain, Kelvin; MacFarlane, Marion; Rehm, MarkusCancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.Item Open Access Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids(2020) Stöhr, Daniela; Schmid, Jens O.; Beigl, Tobias B.; Mack, Alexandra; Maichl, Daniela S.; Cao, Kai; Budai, Beate; Fullstone, Gavin; Kontermann, Roland E.; Mürdter, Thomas E.; Tait, Stephen W. G.; Hagenlocher, Cathrin; Pollak, Nadine; Scheurich, Peter; Rehm, MarkusThe influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.Item Open Access Applying a GAN-based classifier to improve transcriptome-based prognostication in breast cancer(2023) Guttà, Cristiano; Morhard, Christoph; Rehm, MarkusItem Open Access Implementing patient-derived xenografts to assess the effectiveness of cyclin-dependent kinase inhibitors in glioblastoma(2019) Noonan, Janis J.; Jarzabek, Monika; Lincoln, Frank A.; Cavanagh, Brenton L.; Pariag, Arhona R.; Juric, Viktorija; Young, Leonie S.; Ligon, Keith L.; Jahns, Hanne; Zheleva, Daniella; Prehn, Jochen H. M.; Rehm, Markus; Byrne, Annette T.; Murphy, Brona M.Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM. A reduction in viability and significant levels of apoptosis were observed in vitro in human GBM neurospheres following treatment with seliciclib plus drozitumab. While the co-treatment strategy induced a similar effect in PDX models, the dosing regimen required to observe seliciclib-targeted responses in the brain, resulted in lethal toxicity in 45% of animals. Additional studies showed that the second-generation CDK inhibitor, CYC065, with improved potency in comparison to seliciclib, induced a significant decrease in the size of human GBM neurospheres in vitro and was well tolerated in vivo, upon administration at clinically relevant doses. This study highlights the continued need for robust pre-clinical assessment of promising treatment approaches using clinically relevant models.Item Open Access Linking hyperosmotic stress and apoptotic sensitivity(2020) Stöhr, Daniela; Rehm, MarkusHow hypertonic stress induces or sensitizes to cell death signals is incompletely understood and rarely studied in cancer. Heimer et al. demonstrate that hypertonic environments neutralize the antiapoptotic Bcl‐2 family member Mcl‐1 by upregulating its antagonist Noxa. Consequently, hypertonically stressed head and neck squamous cell carcinoma cells rely solely on Bcl‐xL for survival and succumb to apoptosis when challenged by pharmacological Bcl‐xL inhibition. Similar findings were reported in colorectal cancer cells in related manuscripts, suggesting that a common and conserved mechanistic link might exist between hyperosmotic stress and cellular sensitisation to apoptosis.Item Open Access Glioblastoma, from disease understanding towards optimal cell-based in vitro models(2022) Boccellato, Chiara; Rehm, MarkusBackground: Glioblastoma (GBM) patients are notoriously difficult to treat and ultimately all succumb to disease. This unfortunate scenario motivates research into better characterizing and understanding this disease, and into developing novel research tools by which potential novel therapeutics and treatment options initially can be evaluated pre-clinically. Here, we provide a concise overview of glioblastoma epidemiology, disease classification, the challenges faced in the treatment of glioblastoma and current novel treatment strategies. From this, we lead into a description and assessment of advanced cell-based models that aim to narrow the gap between pre-clinical and clinical studies. Such in vitro models are required to deliver reliable and meaningful data for the development and pre-validation of novel therapeutics and treatments. Conclusions: The toolbox for GBM cell-based models has expanded substantially, with the possibility of 3D printing tumour tissues and thereby replicating in vivo tissue architectures now looming on the horizon. A comparison of experimental cell-based model systems and techniques highlights advantages and drawbacks of the various tools available, based on which cell-based models and experimental approaches best suited to address a diversity of research questions in the glioblastoma research field can be selected.Item Open Access Convergence of pathway analysis and pattern recognition predicts sensitization to latest generation TRAIL therapeutics by IAP antagonism(2020) Vetma, Vesna; Guttà, Cristiano; Peters, Nathalie; Praetorius, Christian; Hutt, Meike; Seifert, Oliver; Meier, Friedegund; Kontermann, Roland; Kulms, Dagmar; Rehm, MarkusSecond generation TRAIL-based therapeutics, combined with sensitising co-treatments, have recently entered clinical trials. However, reliable response predictors for optimal patient selection are not yet available. Here, we demonstrate that a novel and translationally relevant hexavalent TRAIL receptor agonist, IZI1551, in combination with Birinapant, a clinically tested IAP antagonist, efficiently induces cell death in various melanoma models, and that responsiveness can be predicted by combining pathway analysis, data-driven modelling and pattern recognition. Across a panel of 16 melanoma cell lines, responsiveness to IZI1551/Birinapant was heterogeneous, with complete resistance and pronounced synergies observed. Expression patterns of TRAIL pathway regulators allowed us to develop a combinatorial marker that predicts potent cell killing with high accuracy. IZI1551/Birinapant responsiveness could be predicted not only for cell lines, but also for 3D tumour cell spheroids and for cells directly isolated from patient melanoma metastases (80-100% prediction accuracies). Mathematical parameter reduction identified 11 proteins crucial to ensure prediction accuracy, with x-linked inhibitor of apoptosis protein (XIAP) and procaspase-3 scoring highest, and Bcl-2 family members strongly represented. Applied to expression data of a cohort of n = 365 metastatic melanoma patients in a proof of concept in silico trial, the predictor suggested that IZI1551/Birinapant responsiveness could be expected for up to 30% of patient tumours. Overall, response frequencies in melanoma models were very encouraging, and the capability to predict melanoma sensitivity to combinations of latest generation TRAIL-based therapeutics and IAP antagonists can address the need for patient selection strategies in clinical trials based on these novel drugs.