Abstract B019: BRCA1:NRF2 pathway as a new therapeutic target in ewing sarcoma

Abstract

Abstract Ewing sarcoma (EwS) constitutes a highly aggressive pediatric tumor driven by the oncogenic fusion protein EWSR1-FLI1. A critical clinical concern is EwS metastatic spread, and identification of therapeutic targets have been limited by a general lack of mechanistic understanding. However, we do know that EWSR1-FLI1 expression in EwS displays cell-to-cell heterogeneity, and low EWSR1-FLI1 expression level been proposed to increase metastatic phenotypes. This fits with an inherent program in the cells of origin where without the interference of EWSR1-FLI1 expression, these cells would have differentiated on to a more mesodermal/mesenchymal like cells that includes a migratory program. Therefore, identify this inherent program and prevent its activation may provide the key to block EwS metastatic progression. Recently our group showed that EWSR1-FLI1 traps BRCA1 with active RNAPII, resulting in an overactivation of the RNAPII transcription complex. This prevents BRCA1 to relocate to damage, providing a mechanistic basis for the sensitivity to PARP1 inhibition. Importantly, only a small amount of BRCA1 protein is required for its DNA repair function, and PARP1 inhibition only works in the context of BRCA1 absence. With only small amounts of BRCA1 protein needed to circumvent PARP1 inhibitor efficacy it should not be surprising that PARP1 inhibitors failed in EwS clinical trials. Apart from its role in DNA repair, BRCA1 also supports NRF2 transcriptional activity. Analyzing RNAseq data from different EwS cell lines, we found that, due to BRCA1 unavailability, NRF2 pathway is downregulated in EwS. NRF2 role in response to oxidative stress has been extensively studied, and involves glutathione (GSH) recycling at the expense of intracellular glutamine/glutamate. Analyzing an immunohistochemical assay of a tissue microarray, we found that EwS tumors accumulate high level of glutamine, confirming that NRF2 pathway is partially muted in EwS. GSH is a ubiquitous molecule that fulfills xenobiotic detoxification processes, and is also key to prevent ferroptosis, an iron dependent lipid peroxide induced form of cell death. Combining the clinically approved PARP1 inhibitor Olaparib, and the ferroptosis inducer Sulfasalazine, we found a synergistic effect impairing clonogenic capacity, cell growth and survival. Unfortunately given the limited bioavailability and rapid Sulfasalazine systemic clearance, we were only able to stabilize EwS PDX-tumor growth only for the first weeks. Finally, we found that either depletion of EWSR1-FLI1 or KEAP1 (resulting in high levels of NRF2) increases EwS cell motility and that this occurs in an NRF2 dependent manner. Based on our findings we propose a model where fluctuations in EWSR1-FLI1 expression levels impacts BRCA1 dependent NRF2 activity, impacting the balance of stemness on one hand and migration on the other. Our model suggests that targeting NRF2 dependent functions may provide dual benefit first as a novel therapeutic target when EWSR1-FLI1 expression is high while impairing metastatic spread when EWSR1-FLI1 is low. Citation Format: Nicklas Bassani. BRCA1:NRF2 pathway as a new therapeutic target in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B019.