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Melanoma is characterized by high glucose uptake, partially mediated through elevated pyruvate dehydrogenase kinase (PDK), making PDK a potential treatment target in melanoma. We aimed to reduce glucose uptake in melanoma cell lines through PDK inhibitors dichloroacetate (DCA) and AZD7545 and through PDK knockdown, to inhibit cell growth and potentially unveil metabolic co-vulnerabilities resulting from PDK inhibition. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC50 values ranging from 13.3 to 27.0 mM. DCA strongly reduced PDH phosphorylation and increased the oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio up to 6-fold. Knockdown of single PDK isoforms had similar effects on PDH phosphorylation and OCR:ECAR ratio as DCA but did not influence sensitivity to DCA. Growth inhibition by DCA was synergistic with the glutaminase inhibitor CB-839 (2- to 5-fold sensitization) and with diclofenac, known to inhibit monocarboxylate transporters (MCTs) (3- to 8-fold sensitization). CB-839 did not affect the OCR:ECAR response to DCA, whereas diclofenac strongly inhibited ECAR and further increased the OCR:ECAR ratio. We conclude that in melanoma cell lines, DCA reduces proliferation through reprogramming of cellular metabolism and synergizes with other metabolically targeted drugs.

Recent advances in immunotherapy have shown unprecedented efficacy and improved survival of patients with metastatic melanoma. Nevertheless, still, a large population, estimated at around 50% of metastatic melanoma patients, do not benefit from immunotherapy. In the past decades, metabolism has become a hallmark in cancer. In melanoma specifically, it was linked to immunotherapy resistance. A high glucose uptake and lactate production is a commonly shared trait in melanoma. Lactate contributes greatly to the acidification of the tumour microenvironment (TME) and confers an immunosuppressive TME that leads to attenuated immunotherapy responses. Modulating metabolism to lower lactate production and thereby reverting the immunosuppressive TME might be a promising line of study to enhance the efficacy of immunotherapy in metastatic melanoma patients. As a proof-of-concept, a 13C-tracer-based clinical trial is planned with dichloroacetate (DCA) to repurpose and validate its use to lower lactate metabolism in melanoma patients. DCA redirects the lactate precursor into mitochondrial metabolism, thus preventing excessive lactate production. The analysis of 13C-tracer enrichment allows for direct insight into the active metabolic processes, contrary to the quantification of metabolite concentrations alone. It is important to realise that many challenges lie ahead for the implementation of DCA as a standard in melanoma therapy. Amongst these challenges is the unmet need for tools and markers to monitor and predict the response of melanoma metabolism to DCA, and more specifically to determine how patient-specific metabolic phenotypes affect this DCA response. In this thesis, I aimed to meet these challenges and present data and the developed methods that lay the groundwork for interpreting pre-clinical and clinical data, studying melanoma metabolism, and monitoring and predicting drug response in vivo in melanoma.

The apical sodium-dependent bile acid transporter (ASBT) is the primary transporter for the uptake of bile acids in the small intestine. It is localized on the apical membrane of the ileal enterocytes and is known for its high capacity and affinity for taurocholic acid in vitro. However, less is known about its activity towards taurocholic acid and other bile acids in vivo due to the lack of a suitable model. The aim of this study was to validate precision-cut intestinal slices (PCIS) of rat and human intestine as ex vivo model to study the activity of ASBT-mediated transport, and subsequently to study regional and interspecies differences in ASBT function. PCIS maintain the natural cell polarization and communication, expression of transporters and metabolizing enzymes. Therefore this model is expected to be more relevant to the in vivo situation than in vitro cell culture models. PCIS of human and rat ileum were prepared and incubated with 0.04-2.00 mM taurocholic acid, deoxycholic acid and cholic acid, respectively at 4 and 37 degrees C. In this study, the respective contribution of active uptake appeared to be higher for taurocholic acid whereas the passive diffusion was higher for deoxycholic acid and cholic acid. Furthermore, the rank order of calculated (apparent) K-m and V-max for these bile acids is in line with literature reports. The active uptake of taurocholic acid in rat PCIS could be inhibited partly by simvastatin and fluvastatin and was fully inhibited by the specific inhibitor GSK2299027B, supporting the involvement of rat ASBT as uptake transporter. In both species, the ASBT-mediated active uptake of bile acids was observed only in the ileum whereas only passive diffusion was observed in the jejunum and colon. In addition, ASBT activity was higher in the human ileum compared to the rat ileum. In the future rat and human PCIS can be used to study the uptake of new ASBT substrates and as a screening method for potential ASBT inhibitors.

Glioblastoma (GBM), one of the most lethal and heterogeneous primary brain tumors, contains cellular hierarchies with both quiescent and self-renewing highly tumorigenic glioblastoma stem cells (GSCs). Cancer stem cells within the context of the brain likely contribute to a lack of progress in patient survival time as they appear to have an innate resistance to both chemotherapeutic drugs and radiotherapy. The aim of this study is to elucidate how constitutive expression of interferons (IFNs) and their downstream signalling pathways maintain GSCs thereby supporting treatment resistance and tumor reoccurrence. To understand the mechanism of action of a subset of IFN regulated genes, originally characterized as a signature for radiation and chemotherapy resistance (IRDS), a panel of patient derived glioma stem cells was generated and manipulated using CRISPR/Cas9 mediated gene-editing. Phenotypic changes in growth, viability and drug resistance were characterised in the edited cells and linked to molecular level events using transcriptomic and proteomic techniques. Using this approach, in conjunction with patient tissue analysis, signalling under basal as well as IFN-activated conditions was studied in the context of GSC status. Immunohistopathology of a glioma tissue micro-array identified an increased expression of IFN induced transmembrane receptors (IFITMs), which co-localised with Nestin, around the blood vessels suggesting that IFITM positive cells were in the stem cell niche. Further analysis showed the presence of both IFITM1 and IFITM3 in GSCs and established that loss of IFITM function impacted on growth rate and cell cycle progression as well as treatment sensitivity. By using wild-type and gene-edited GSC to focus on the relationship between IFITM1 and IFITM3, we have compelling phenotypic and molecular evidence that IFN-induced changes in the localisation of IFITMs impact on the ability of stem cells to ‘escape' normal growth control mechanisms. Taking into consideration the constitutive expression of interferons in the tumor microenvironment, our study provides evidence that IFITMs regulate glioma stem cell self-renewal and are involved in maintaining a quiescent population which can escape chemotherapy. Citation Format: Erisa Nita, Bernard Evers, Marta Nekulova, Gillian Morrison, Kathryn Ball. The role of IFITMs in maintaining glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3777.