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Abstract

Cancer is a major healthcare burden worldwide. Despite the high heterogeneity of malignant diseases, several characteristics, described as cancer hallmarks, are common among most tumours. Therapeutic approaches targeting these common features represent a promising path to universally applicable cancer therapy. One of such common cancer hallmarks is metabolic reprogramming, most prominently reflected by the phenomenon of the Warburg effect. The Warburg effect describes an enhanced metabolisation of glucose via the glycolytic pathway instead of oxidative phosphorylation, which results in extremely high glucose consumption and glucose dependence in the majority of tumours. Attempts to target high glucose consumption of tumour cells by the glucose anti-metabolite 2-deoxy-D-glucose (2DG) were hampered by toxic side effects observed upon effective 2DG dosages. Previous work of the Department has shown a possibility to overcome these side effects by combining 2DG with flavaglines, a compound group with high anti-neoplastic potential. The successful chemical synthesis of flavaglines opened up a realistic perspective for clinical translation of the promising drug combination, previously inhibited by the limitations of the complicated extraction processes of natural flavaglines.

The present thesis aims to analyse the mechanisms of action of synthetic flavaglines and to perform first steps towards their preclinical evaluation. Specifically, the in vitro and in vivo characteristics of two novel synthetic flavagline compounds, IMD-1 and IMD-3, were analysed as well as mechanisms of potential therapy resistance. Investigation of synthetic flavagline efficacy in cancer cell lines from different tumour tissues revealed great growth-inhibitory efficacy of IMD-3 treatment in all analysed cancer cell lines. In the majority of the analysed cell lines, the effect could be further enhanced by the combination of IMD-3 with 2DG. Synthetic flavaglines showed low unspecific cytotoxicity and mainly mediated their anti-cancer activity through proliferation inhibition and apoptosis induction. Furthermore, strong inhibition of glucose uptake, downregulation of glucose transporter 1 (GLUT-1) expression as well as inhibition of pentose phosphate pathway (PPP) activity could be identified as a common characteristic of IMD-3 treatment. Systematic analysis of treatmentrelated effects on the transcript level showed induction of cell cycle arrest and inhibition of glucose metabolism, confirming the in vitro observations. To analyse the effect of flavaglines and 2DG in 3D tissue context, tumour spheroids and a 3D co-culture model of tumour cells and normal keratinocytes were used. 3D culture models generally confirmed the mechanistic findings observed in 2D culture, however indicated a different efficacy range with higher drug concentrations or longer drug exposures required for achieving a similar growth-inhibitory effect as in 2D culture. The co-culture experiments also indicated compound doses allowing selective targeting of tumour cells while sparing normal tissue.

Generation of a haploid cell line showing long-term resistance towards IMD-3 treatment allowed the investigation of mechanisms responsible for synthetic flavagline resistance. Strikingly, in contrast to their sensitive counterpart, resistant cells showed no GLUT-1 downregulation upon IMD-3 treatment, whereas PPP activity was increased, indicating a shift in glucose metabolism of treatment-resistant cells. The transcriptome analysis of IMD-3-resistant cells, showing decreased glycolytic activity compared to sensitive cells, pointed at the possibility of using markers of glycolytic activity for prediction of therapy success. For the in vivo evaluation of the anti-tumour activity of synthetic flavaglines a two-step experimental approach using a xenograft model (HCT116 cells in Rag2-/-Il2rg-/- mice) was followed. After an initial dose finding experiment, a second experiment with larger animal groups did not yield significant effects of flavaglines alone or in combination with 2DG regarding tumour growth and survival of animals. Positron Emission Tomography (PET) demonstrated no change in glucose uptake by tumour cells in any of the treatment groups. The analysis of possible reasons for the observed discordance between in vitro and in vivo efficacy data indicated modifications of the drug preparation for in vivo delivery as a potential reason. Although adapted preparation protocols for the flavagline solution allowed successful delivery of the substance to the tumour tissue, as proven by mass spectrometry, no significant effects on tumour growth and survival could be demonstrated in frame of the thesis. Future experiments shall quantify the substance concentration in tumour tissue and study the role of application routes on the drug delivery.

Taken together, the present thesis has identified downregulation of the glucose transporter GLUT-1 and inhibition of glucose uptake as major mechanisms of action of the examined synthetic flavagline derivatives. This observation together with in vitro data, demonstrating a significant and tumour cell-selective growth-inhibitory effect of flavaglines combined with 2DG, indicate the significant clinical potential of this substance group for cancer treatment. First in vivo data obtained in a murine xenograft model demonstrate that optimising pharmacokinetics, including drug delivery and application schemes, represents a major challenge for further preclinical development of flavaglines. Future studies shall therefore focus on optimising these steps in order to successfully translate the highly promising anti-tumour effects of synthetic flavaglines into the setting of living organisms.