Abstract A23: Novel approaches to Ewing sarcoma therapy

Abstract

Abstract Ewing family of sarcomas (EwS) comprises the fourth most common highly malignant childhood cancer. The driver translocation in these tumors, EWSR1-FLI1, introduces DNA damage and replication stress. These dysregulations render EwS cells reliant on the ATR/CHK1 axis to maintain genome stability. High sensitivity of EwS cells to PARP1 inhibition (PARPi) was reported and attributed to a positive PARP1/EWSR1-FLI1 feedback loop. Further, it has been shown that with EWSR1-FLI1 expression, BRCA1 is trapped with the active transcription complex unable to go to DNA breaks. PARPi impairs the repair of single-strand DNA breaks and alkylated bases leading to double-strand DNA breaks, which cannot be repaired efficiently in BRCA-deficient cancers (synthetic lethality) (1). Several groups including ours previously identified the combination of PARP1i talazoparib (TLZ) with DNA-damaging agent, temozolomide (TMZ), to cause regressions of ~50% of EwS xenograft models. However, both in mice and in children’s trial, the combination causes toxicity and necessitates reduction of the dose of TMZ to ~13% of its single-agent maximum tolerated dose. Combined inhibition of PARP1 and ATR has been shown to synergize in suppressing growth of BRCA-mutant patient-derived xenografts models (2). The long-term goal of the proposed study is to develop more effective EwS therapy based on EWSR1-FLI1-induced vulnerabilities. EwS cells represent heterogeneity in expressing EWSR1-FLI1 fusion protein. As sequencing is generally performed in “bulk” with results being interpreted as average of gene expression patterns derived from vast populations of cells, the biologically relevant differences between cells may not be represented. Single-cell RNA-seq therefore offers new possibilities of overcoming such challenges. By studying single cells, their transcripts and generating sequencing libraries of separate cells, we are able to study fundamental biologic properties of cell populations and biologic systems at a single-cell level. Our studies demonstrate positive correlation between the expression of EWSR1-FLI1 and DNA replication genes at single-cell resolution. Single-cell studies may provide deep understanding of time-dependent behaviors of individual cells in response to dynamic changes of EWSR1-FLI1 expression. We propose that EWSR1-FLI1-induced DNA replication stress leads to reliance of EwS cells on PARP1 and ATR DNA repair pathways in order to maintain genome stability. We have selected number of metal-conjugated antibodies to identify changes in DNA damage response genes in subpopulations of EwS cells using CyTOF. Latest findings on changes of DNA damage/repair genes upon downregulation of EWSR1-FLI1 at a single-cell level will be presented. Citation Format: Dauren Alimbetov, Yidong Chen, Peter Houghton, Raushan Kurmasheva. Novel approaches to Ewing sarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A23.