EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

Abstract

29Core regulatory circuitry (CRC)-dependent transcriptional network is critical for 30 developmental tumors in children and young adults carrying few gene mutations. 31 Ewing sarcoma resembles these developmental cancers in terms of having both 87 few genomic alterations and a single epigenomic driver, we postulated that such 88 oncogenic CRC model might exist in Ewing sarcoma, to cooperate with the 89 transcriptional function of EWS-FLI1. The current study was aimed to identify 90 and characterize this CRC apparatus in Ewing sarcoma, and to elucidate its 91 functional significance in this cancer. 92 93 94 5 / 46 Results 95 96 We recently characterized the super-enhancer landscape in Ewing sarcoma and 97 confirmed the essential role of EWS-FLI1 in regulating the epigenome of this 98 cancer (11). As introduced earlier, considering that CRC is particularly important 99 for childhood developmental cancers having few genomic lesions, we postulated 100 that such CRC model might also contribute to regulating Ewing sarcoma 101 transcriptome through either cooperating with or mediating the function of EWS-102 FLI1. To test this hypothesis, we first sought to identify mathematically master 103 TFs with high inter-connectivity through binding to their super-enhancers by 104 motif scanning using our established method (18, 24, 25). Because of the central 105 role of EWS-FLI1 in establishing the enhancer landscape in Ewing sarcoma, we 106 made significant modifications of the method by requiring that all candidate TFs 107 have both EWS-FLI1 binding motif and binding peaks in their assigned super-108 enhancers. We initially focused on the A673 cell line, since it is a well-109 characterized Ewing sarcoma line with available H3K27ac and EWS-FLI1 ChIP-110 Seq results (7). As a result, a small set (n=9) of CRC candidates were identified 111 (Fig. 1A), including NKX2-2 and FOS which are known functional cooperators 112 of EWS-FLI1 (7, 26). Because CRC factors have high and specific expression in 113 their corresponding cell types, we interrogated the Cancer Cell Line Encyclopedia 114 (CCLE) dataset and noted that, compared with other 5 CRC candidates (NFATC2, 115 FOS, IRF2, ZBTB7B and MEF2D, Supplement Fig. 1), the expression of 4 116 6 / 46 candidate TFs (KLF15, NKX2-2, TCF4 and RREB1) showed restricted 117 expression pattern in Ewing sarcoma cell lines (defined as top 5 among all cell 118 types, Fig. 1B). However, it should be noted that the specificity of RREB1 119 expression was overall poor, as most cancer types had comparable mRNA levels. Identification of CRC under the regulation of EWS-FLI1 in Ewing sarcoma 120As a parallel analysis, Pearson correlation coefficient of the mRNA levels of 121 these 9 candidates was determined, considering the co-regulatory relationship 122 between CRC members. Notably, the same set of 4 factors (KLF15, NKX2-2, 123 TCF4 and RREB1) displayed strong positive correlations with each other (Fig. 124 1C). In contrast, this correlation pattern was much weaker in other non-Ewing 125 sarco...

Super-enhancers: a new frontier for epigenetic modifiers in cancer chemoresistance

“…Similarly, in Ewing sarcoma, KLF15, TCF4, and NKX2-2 have been identified as the master TFs containing both EWS-FLI1 binding motif and SE peaks [110]. Importantly, these three CRC TFs co-regulate the PI3K/AKT and MAPK signaling pathways to promote the aggressiveness of Ewing sarcoma [110].…”

“…Zhang was recently the first to study SE-associated CRC transcriptional control in lung adenocarcinoma: master TFs ELF3, EHF, and TGIF1 were found to co-occupy the SE region and promote each other's expression through the formation of CRC, which induces the malignant progression of lung adenocarcinoma [109]. Similarly, in Ewing sarcoma, KLF15, TCF4, and NKX2-2 have been identified as the master TFs containing both EWS-FLI1 binding motif and SE peaks [110]. Importantly, these three CRC TFs co-regulate the PI3K/AKT and MAPK signaling pathways to promote the aggressiveness of Ewing sarcoma [110].…”