**2. Ewing sarcoma**

Ewing sarcoma is a rare, aggressive bone or soft tissue tumor that primarily affects children, adolescents, and young adults (AYAs) with ~1.5 cases per million children and AYAs worldwide. The average age at diagnosis is 15 years. Approximately 20–25% of patients have metastatic disease at diagnosis, which is often unresponsive to intensive therapy [22]. Standard therapy for Ewing sarcoma consists of a multimodality treatment regimen that includes surgical resection and/or local radiation therapy, as well as intensive five-drug chemotherapy and the administration of compressed interval cycles [23].

Most Ewing sarcomas have a chromosomal rearrangement at 22q12 [10]. This led to the identification of the EWSR1 gene, which can be fused to one of several partner genes: FLI1 t(11;22), ERG t(21;22), ETV1 t(7,22), ETV4 t(17,22), or FEV t(2,22). The most common fusion is EWSR1-FLI1, which occurs in ~85% of tumors [24]. In a recent comprehensive study, it was found that in 42% of Ewing sarcomas, the fusion gene results from a loop-like rearrangement, a process known as chromoplexia. These loops always contained the disease-defining fusion at the center, but they interrupted several additional genes and appear to be associated with an aggressive form of Ewing sarcoma [25].

Ewing sarcomas have few other infrequently recurring mutations besides an EWSR1/ETS translocation, including TP53 (5–10%), CDKN2A (10%), and STAG2 (15–20%) [16, 26]. The loss of P53 and STAG2 suggests a rare group of tumors that, together with the translocation, form an aggressive subset of Ewing sarcoma [15, 18]. Furthermore, very little is known about the genetic heterogeneity within the tumor in Ewing sarcoma, its subclonal genetic architecture, and the relationship between these

### *Drug Targeting of Chromosomal Translocations in Fusion-Positive Sarcoma DOI: http://dx.doi.org/10.5772/intechopen.106671*

factors and clinical outcome. The majority of pediatric solid tumors, including Ewing sarcoma, express an active DNA transposase, PGBD5, that can promote site-specific genomic rearrangements in human cells and may promote resistance to therapy [27, 28]. However, whether the genomic landscape of Ewing sarcomas differs in relapse from primary disease is unknown [24]. Recent analyses of DNA methylation status in Ewing sarcoma showed that primary tumors from patients with metastatic disease were more heterogeneous than those with localized disease [29]. However, most Ewing sarcomas have very few additional genetic alterations, suggesting that the fusion is likely the primary cause of disease development. Previous findings suggest that either mesenchymal stem cells or neural crest-derived stem cells are the cell of origin of Ewing sarcoma, although this is still a matter of debate [30, 31].

EWSR1 encodes a protein with a function in RNA binding and transcriptional regulation. The amino terminus of the EWSR1 protein functions as a strong transcriptional activator [32]. All Ewing sarcoma fusion partner genes encode related transcription factors, with conserved DNA-binding ETS domain. These ETS domain transcription factors play an important role in biological development [33]. During each fusion, the amino-terminal transactivation domain of EWSR1 is fused to the ETS domaincontaining carboxyl terminus of the corresponding fusion partner. The resulting fusion gene functions primarily as an aberrant transcription factor. The dominant EWSR1/ETS translocation EWSR1-FLI1 results in heterogeneous expression profiles that have different biological implications. Therefore, variable expression of EWSR1-FLI1 has recently been proposed as a source of heterogeneity in these tumors. Cells with high EWSR1- FLI1 expression (EWSR1-FLI1high) are highly proliferative, whereas EWSR1-FLI1low cells have a strong propensity to migrate, invade, and metastasize [34].

EWSR1-FLI1 can act as both a transcriptional activator and a transcriptional repressor, depending on the sequence of DNA binding sites and the presence of additional co-factors [35, 36]. EWSR1-FLI1 acts directly or indirectly on many important cellular processes such as cell cycle, apoptosis, angiogenesis, metabolism, and cell migration by binding to these sites [24]. EWSR1-FLI1 binds to DNA either at ETS-like consensus sites with a GGAA core motif or at GGAA microsatellites (GGAA-mSats). EWSR1-FLI1 multimers directly induce open chromatin at GGAA-mSats by recruiting the nucleosome remodeling BRG1/BRM-associated factor complex (BAF) and establishing de novo enhancers that interact with promoters to drive gene expression [35, 37]. Fusion multimers physically interact with BAF complexes, which appear to be critical for EWS-FLI1 function, as BAF complexes are required for activation of EWS-FLI1 target genes. The variable length of GGAA-mSats in the germline may lead to differential activity of these enhancers and is an important determinant of tumor progression [38].

Conversely, EWSR1-FLI binds to canonical ETS recognition sites without repeats and represses wild-type ETS factors, which can lead to suppression of enhancers and downregulation of nearby genes [35]. The chimeric transcription factor can directly repress certain genes such as LOX and TGFBR2 through direct interaction and recruitment of the nucleosome remodeling and deacetylase repressor complex (NuRD), which includes histone deacetylases and the histone demethylase LSD1 [39].

Interestingly, EWSR1-ETS fusion proteins also bind to DEAD/DEADH box RNA helicases and modulate their activity, thus also affecting the transcription and splicing machinery of tumor cells and causing changes in overall transcriptome processing [40, 41].

These data demonstrate that EWSR1-FLI1 utilizes distinct chromatin regulatory mechanisms whose interplay at the right time and in the right cellular context leads to the transformed phenotype of Ewing sarcoma.
