Transcriptional constraint of EWS/FLI by an ETS transcription factor promotes Ewing sarcoma growth [PEDS0009 CUT&Tag]

Pediatric cancers frequently harbor sentinel mutations involving transcription factors (TFs) that dysregulate normal development. A recurrent mechanism involves the ability of mutant TFs to co-opt cell lineage-specific, activating TFs to promote cancer growth. Ewing sarcoma, the second most common pediatric bone cancer, is defined by the presence of a 11;22 chromosomal translocation fusing the N-terminus of the EWS protein with the C-terminal DNA binding domain of an ETS (E26 Transformation Specific) TF family member, most commonly (85-90% of cases), FLI1. The EWS/FLI fusion exhibits the neomorphic ability to pioneer de novo enhancers at repeating 5’-GGAA-3’ motifs in the cell-of-origin, which has not been identified. To date, efforts to elucidate the key mechanisms by which EWS/FLI promotes oncogenesis have prioritized identifying the genes that are profoundly activated by EWS/FLI and highly expressed in Ewing sarcoma compared to other cancers, with particular focus on transcription factors capable of altering cell state. However, it is not known whether, globally, these genes constitute the most critical drivers of Ewing sarcoma cell growth. Here, we describe the results of an unbiased deletion screen revealing that the wild-type repressive ETS family TF, ETV6 (ETS Variant 6, or TEL), is a novel and most critical TF dependency specific to Ewing sarcoma. We demonstrate that the repressive activity of ETV6 constrains EWS/FLI gene activation at GGAA repeat enhancers to promote Ewing sarcoma cell growth. CUT&Tag (Kaya-Okur, et al., Nature Communications, 2019) was used to evaluate ETV6 binding in Ewing sarcoma using the newly derived CCLF_PEDS_0009_T (PEDS0009) cell line (Guenther et al., Clinical Cancer Research, 2019) in cells transduced with lentivirally-packaged CRISPR/Cas9 constructs. All sgRNA sequences used in the Broad Institute AVANA CRISPR/Cas9 screen are available for download at the DepMap Portal (https://depmap.org). sgRNA sequences used to target ETV6 were taken from this screen. The sgETV6-1 forward sequence was 5’-GCAGCCAATTTACTGGAGCA-3’. The sgETV6-2 forward sequence was 5’-GCAGGGATGACGTAGCCCAG-3’. The sgETV6-3 forward sequence was 5’-GTGTGTGTATAGAGTTTCCA-3’. The sgETV6-4 forward sequence was 5’-GTTATGGTGCACATTATCCA-3’. For control sgRNA, sgChr2.2 was used as a cutting control and targets a gene desert on Chromosome 2, 5’-GGTGTGCGTATGAAGCAGTG-3’. For ligation into the LentiCRISPRv2 plasmid, additional bases were added. 5’-CACCG-3’ was added to the beginning of the forward sequence. 5’-AAAC-3’ and 5’-C-3’ were added at the beginning and end of the reverse sequence, respectively. Duplicate paired-end experiments were used to evaluate ETV6 binding in all cases. All CUT&Tag samples have matching Rabbit anti-mouse reference sample from the same batch and treatment condition.