Genetic alterations in Ewing’s sarcoma: What do we know so far?

Chromosomal translocations

On the molecular level, EwS is marked by the fusion hallmark of the EWSR1 gene with one of the ETS families of transcription factors. The most frequent fusion is the EWSR1-FLI1. This fusion is the result of the chromosomal translocation entails the FLI1 gene that encodes a transcription factor associated with cell proliferation, growth, and tumorigenesis, and the EWSR1 gene that encodes a protein involved in several cellular processes such as cell signaling and transcription. Tissues are found to have the EWSR1-FLI1 chromosomal event in over 90% of EwS cases.^[11] Additional translocations involving the EWSR1 gene such as t(21;22) (q22;q12)) result in the formation of the fusion gene EWSR1-ERG, as well as fusions with additional genes such as ETV1, E1AF, and FEV in 5–10% of these cases.^[11] Additional chromosomal translocation among EWS-ERG fusion has been well documented as well but with very low incidences.^[11]

Based on a study done by Akiko et al., which indicated that EWS-ETS fusion proteins activate telomerase activity in tumors through over-activation of TERT gene expression. When the chimeric EWSR1-FLI1 gene is expressed, a protein that blocks the apoptosis pathway is produced, resulting in uncontrolled tumor development, and this protein, in turn, stimulates the transcription of the TERT gene, which favorably regulates the expression of telomerase, thus increasing the immortality of tumor cells in EwS.^[12] A strong transcriptional activation domain is gained by the EWSR1-FLI1 and the binding domain is provided by FLI in the context of this fusion protein. For EWSR1-FLI to operate as an oncogene, both of these domains must be present to back up the hypothesis that the fusion protein works as a transcription factor.^[13]

Interestingly, in EwS cases where EWSR1-FLI1 does not exist, alternative events, mostly translocations, are present that fuse EWSR1 to another member of the ETS family, such as the ERG, ETV1, ETV4, and FEV with very low incidences.^[14] To ascertain further potentially EwS causing mutations, Mitchell et al., at the Center for Cancer Research (CCR) in the National Cancer Institute launched a large-scale study protocol on a cohort of (2000 cases) for children and young adults, enrolled in CCR clinical trials to sequence their tumors DNA to ascertain mutations that underlie EwS carcinogenesis. They identified additional three loci at 6p25.1, 20p11.22, and 20p11.23, with low frequency associated with increased EwS risk. This study finding support the inherited genetic to EwS risk and proposes the prominence of the interaction among germline variation and the acquired EWSR1-FLI1 translocations to the etiologic EwS.^[15]

Additional genetic alterations

Despite the prominence of EWSR1-FLI1 fusion for the initiation of EwS, this fusion alone found not to be enough to transform human cells in vitro, suggesting that the additional secondary alteration or related parallel pathways might be crucial for EwS pathogenesis a high rate of likely pathogenic mutations, accounting for 13.1% of the population.^[16]

EwS has been the subject of various studies, all of which have shown that the tumors have a relatively silent genome, with a scarcity of mutations in pathways that would be susceptible to therapy with innovative targeted treatments. These articles discovered recurrent genomic abnormalities in several genes, including studies that showed EWSR1-FLI1 fusion stimulates tumor cells’ production of a protein known as pappalysin-1 (PAPPA), which is responsible for breaking down proteins known as insulin-like growth factor binding proteins (IGFBPs).^[17] This breakdown results in the production of the hormone-IGF-1, which, in turn, promotes the proliferation of cancer cells. Through utilizing cell and animal models, researchers were able to effectively show that inactivating PAPPA is a feasible therapeutic option for EwS.^[17]

Further mutations in the tumor suppressor genes such as CDKN2A and TP53 have been identified as a secondary genetic alteration in EwS in multiple studies. By utilizing a combination of whole-genome sequencing and targeted sequencing approaches, Hemelin et al. found a homozygous deletion affecting CDKN2A in 13.8% and 50% and mutations of TP53 in 6.2% and 71.9% in both EwS’ clinical tumors and cell lines, respectively.^[18,19] Another study by Brohl et al., the same group examined the sequencing data from the genomes of 175 Ewing patients, discovered a high rate of pathogenic or likely pathogenic mutations in 13.1% of patients.^[16] Some of these patients possess genetic mutations in DNA repair genes. However, their analysis in both studies showed that EwS has a very low mutational burden in general (0.15 mutations/Mb).

EWS-FLI1 tends to activate genes involved in cellular differentiation, proliferation, and survival, such as IGF1, NKX2, TOPK, SOX2, and EZH2. EWS-FLI1 also tends to deactivate genes involved in apoptosis and cell cycle, including the IGFBP3, CDKN1C, CDKN1A, and TGFB2.^[18,19] Furthermore, because the EWSR1–FLI1 fusion is constitutively active, it is found to work as an activator of the MYC gene, which is a proto-oncogene that is essential for embryonic development but that relies on extra external and internal stimuli to become activated its ability to function. Although the MYC gene has decreased activity in differentiated cells, its inappropriate activation results in gene amplification, which results in the expression of proteins that are engaged in the control of the cell cycle, differentiation, and genomic instability, which might promote the tumor initiation.^[20]