Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in generation of the chimeric transcription factor EWS-FLI1.

Recently, ES has been reported to be exquisitely sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). This enzyme uses NAD as substrate and was demonstrated to regulate EWS-FLI1 in a feed-back mechanism. NAD is a key metabolite essential for sustaining cellular energy metabolism. It plays a central role in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. The other major mammalian NAD consumer is the deacetylase SIRT1 which we observed to be specifically highly expressed in ES metastases. PARP1 and SIRT1 are crucial for coupling cellular metabolism to transcriptional gene regulation as well as to stress response. Usually, NAD is regenerated from nicotinamide in the NAMPT-dependent salvage pathway or from the reduction of pyruvate via LDHA (Warburg effect), but can also be synthesized de novo from tryptophan. Interestingly, the knockdown of EWS-FLI1 in ES cells comes along with alterations in the expression of multiple enzymes involved in NAD biosynthesis and regeneration, including TDO2, KMO, NMNAT1, NAPRT, NAMPT, and LDHA. We are interrogating the role of EWS-FLI1 mediated modulation of these enzymes and of specific small molecule inhibitors on cellular tryptophan consumption, kynurenine production and intracellular NAD levels of Ewing sarcoma cells. Additionally, we are investigating the functional consequences thereof on constitutive and stress-induced protein poly(ADP)-ribosylation and acetylation.

Preliminary results suggest that NAMPT inhibition diminishes PARP1 activity due to low NAD, implying a pivotal role for the regenerative salvage pathway in ES cells. TDO2-dependent tryptophan consumption increases in the absence of the oncogene.

These studies aim at a better understanding of factors influencing Ewing sarcoma sensitivity to therapies targeting PARP1 and the NAD metabolome.

Citation Format: Cornelia N. Mutz, Raphaela Schwentner, Maximilian O. Kauer, Jozef Ban, Dave N. T. Aryee, Sophie Erhardt, Dietmar Fuchs, Andreas Heitger, Heinrich Kovar. Investigating the NAD metabolome in Ewing Sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1162. doi:10.1158/1538-7445.AM2015-1162