Ewing Sarcoma (ES) is the second most common bone cancer occurring in children and adolescents with a peak incidence at the age of 15. In 85% of tumors, the driving force of the malignancy is the chimeric transcription factor EWS-FLI1, resulting from the specific chromosomal translocation t(11;22)(q24;q12). In this dissertation, I report the investigation of two interconnected pathways, both contributing to the “NAD metabolome” of ES. Nicotinamide adenine dinucleotide (NAD) is a key metabolite essential for sustaining cellular energy metabolism and necessary for glycolytic and mitochondrial function, DNA repair, genomic stability, chromatin remodeling, and signal transduction. NAD can either be synthesized de novo from the essential amino acid tryptophan (TRP), or much more efficiently via salvage pathways starting from derivatives of vitamin B3. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of mammalian NAD salvage synthesis and is highly expressed in ES cells. Targeting tumor cell metabolism has become an attractive anti-cancer approach and given the increased metabolic needs of cancer cells, they are supposed to be hit especially hard by NAD depletion. We used the small molecule compound FK866 for inhibition of NAMPT and could show that upon excessive NAD exhaustion cells enter acute metabolic stress such as glycolytic impairment, mitochondrial dysfunction, adenosine triphosphate (ATP) depletion and finally cell death. Especially cells expressing EWS-FLI1 are exquisitely sensitive to NAMPT inhibition in contrast to cells expressing lower levels of EWS-FLI1 as investigated in RNAi-inducible A673sh cells.
The kynurenine pathway is part of NAD de novo synthesis from TRP which is oxidized by tryptophan 2,3-dioxygenase (TDO2). By analyzing several metabolites of the initial steps of TRP degradation, we observed that A673sh cells with low EWS-FLI1 expression strongly enhanced TRP breakdown in favor of kynurenine (KYN) and kynurenic acid (KYNA) production. Strikingly, the downstream effects of KYN and KYNA accumulation led to activation of the aryl hydrocarbon receptor (AHR), a ligand-activated cytoplasmic transcription factor. AHR binds to dioxin-response elements (DRE) in the promoter region of target genes and up-regulates IL8, IL6, IL1B, CYP1A1, CYP1B1, TUFT1, and FAM65B under conditions with low EWS-FLI1 expression. We therefore suggest an involvement of AHR for an alternative survival strategy of A673sh cells with depleted EWS-FLI1 expression. Our data reveal that EWS-FLI1 usually suppresses autocrine AHR signaling by impairing TDO2-mediated TRP breakdown.