Abstract PR015: Revealing a novel role for YB-1 as metabolic regulator in Ewing sarcoma

Abstract

Metastatic Ewing Sarcoma (EwS) remains a rare but aggressive disease with little to no treatment options available. Therefore, identifying new therapeutic targets is essential. During the establishment of a metastatic niche, sarcoma cells must rapidly adapt to environmental stressors. They achieve this by stalling translation complexes and accumulating mRNA along with RNA-binding proteins in structures known as Stress Granules (SGs). The essential SG protein Y-Box binding protein 1 (YB-1) is highly expressed in EwS and is associated with poor patient outcomes. Previous work from our lab demonstrated that YB-1 contributes to tumor progression and growth. New multi-omics data on EwS cells correlates with these findings and revealed a role for YB-1 in mitochondrial control in these high-risk sarcomas, specifically in regulating electron transport chain (ETC) complexes and possibly mitochondrial translation. We hypothesize that YB-1 interferes with mitochondrial function in EwS. We discovered that under oxidative stress as expected many metabolic pathways are downregulated in a EwS polysome sequencing dataset we generated, when compared with a YB-1 RNA interaction dataset we could identify less binding of ETC complex I and IV transcripts to YB-1 under oxidative stress as well. This was particularly interesting since many of those genes are transcribed and translated in the mitochondria. A transient knockdown of YB-1 increased levels of ROS, as well as mitochondrial superoxides and interfered with the oxygen consumption rate. This indicated a disruption of regular ETC complex activity under YB-1 depletion. To test this we performed a mito-FUNCAT (fluorescent non-canonical amino acid tagging) assay to track mitochondrial protein synthesis. We could confirm less HPG incorporation with lack of YB-1. Furthermore, since YB-1 is mainly known to be a SG regulator in EwS cells, we performed live-cell imaging under oxidative stress, which showed an accumulation of YB-1 around mitochondria. To test whether those were SGs we fixed and stained sodium arsenite treated cells for another SG marker, UBAP2L. Z-stack imaging followed by 3D analysis revealed a co-localization of SGs to mitochondria, supporting our interest in YB-1 and SGs as metabolic regulators. Together these findings indicate that YB-1 and SGs might be contributing to mitochondrial function in EwS cells. This interaction could further lead to prolonged survival of these sarcoma cells under metastasis. Targeting the SG-mito axis could provide a novel therapeutic option for EwS.