Long-read sequencing of 15 Ewing sarcoma cell lines uncovers extensive GGAA microsatellite variation shaping the landscape of EWSR1::FLI1 binding

Ewing sarcoma (EwS) is a rare, aggressive bone and soft tissue tumor occurring primarily in adolescents and young adults. EwS is defined by acquired chromosomal translocations between a member of the FET gene family and an ETS transcription factor (e.g., EWSR1::FLI1 in >85% of cases) that bind GGAA repetitive sequences and rewire local transcriptional activity. Germline variation in GGAA microsatellite allele length and motif purity can alter binding of EWSR1::FLI1 and disrupt the ability to dysregulate core driver genes. Further investigation into the molecular features underlying these germline-somatic interactions may yield new insights into the genetic etiology of Ewing sarcoma. We performed whole-genome PacBio Revio HiFi sequencing on high molecular weight DNA extracted from 15 EwS cell lines positive for EWSR1::FLI1 to characterize germline variation in GGAA microsatellites genome-wide. We implemented a custom TRGT pipeline to call microsatellites containing at least 3 GGAA motifs utilizing a genomic repeat library and hidden Markov models. To investigate genomic features that could enhance EWSR1::FLI1 binding, we utilized linear mixed models incorporating PacBio data on microsatellite allele calls and DNA methylation with chromatin accessibility and ChIP-seq of EWSR1::FLI1 binding from the Ewing Sarcoma Cell Line Atlas (ESCLA). In total, 39,319 GGAA microsatellites were characterized, with notable variation observed in microsatellite length (median=16 bp, interquartile range=13-51) and purity of GGAA motifs (mean=93.4%, standard deviation=9.2%). Allelic variation was present between cell lines with a mean of 4 alleles and a maximum of all 30 haplotypes exhibiting a different allele. Increased microsatellite length was the strongest predictor of EWSR1::FLI1 binding (beta=0.711, 95% CI=0.703,0.719), followed by reduced DNA methylation (beta=-0.023, 95% CI=-0.024,-0.022) and increased H3K27ac measures of open chromatin (beta=0.154, 95% CI=0.138-0.170). Analyses of established core EwS genes dysregulated by EWSR1::FLI1 are underway to identify specific microsatellites and allelic combinations that promote EWSR1::FLI1-altered expression of these genes. Together, germline data from long-read whole-genome sequencing and multi-omic profiles from the ESCLA enable integrative analyses for disentangling germline-somatic interactions that alter EWSR1::FLI1 binding and downstream transcriptional regulation.