**8. Inhibition of key players of the fusion-positive interactome**

Fusion oncoproteins remodel the transcriptional machinery of cells, silencing genes and activating others by creating new enhancers, remodeling chromatin, and critically altering the epigenetic profile of sarcoma cells. By cooperating with histone deacetylases (HDACs) in transcriptional regulatory complexes, fusion oncoproteins affect histone acetylation and chromatin remodeling. For these chromatin remodeling complexes, they recruit BAF complexes as in the case of Ewing sarcoma [37] or alter their function as in the case of synovial sarcoma [79] to enforce pathogenic transcriptional programs. Binding of EWSR1-FLI1 to GGAA mSATs leads to the binding of histone acetyltransferase p300 at many of these sites and an increase in H3K27ac [35, 36]. On the other hand, the PAX3-FOXO1 fusion oncogene of alveolar rhabdomyosarcoma recruits master transcription factors MYOG, MYOD, and MYCN to activated gene loci and alters their histone acetylation which enables binding and manipulation of reader proteins such as BRD4 [55]. In synovial sarcomas, SS18-SSX fusion oncogenes, cause epigenetic restructuring involving HDACs [127]. Conversely, EWSR1-FLI1 translocation recruits histone deacetylases and histone demethylase LSD1 to specific gene loci through direct interaction with the NuRD complex, thereby suppressing their expression in Ewing sarcoma [39].

However, downstream processes also appear to be important for the epigenetic expression profile in FP sarcomas. For example, EWSR1-FLI1 binds to the promoter of the histone methyltransferase EZH2, upregulating its expression and thereby blocking its endothelial and neuronal differentiation abilities [90, 128]. But, chemical inhibitors of EZH2 activity cannot reproduce the results after RNA interference (unpublished). Yet, recent data show that EZH2-containing PRC2 complexes interact with HDAC1, 2 and this HDAC activity mediates the immature, tumorigenic phenotype of Ewing sarcoma [91].

The involvement of HDACs in key mechanisms of sarcoma cell transformation has paved the way for the investigation of HDACi for therapeutic intervention. Preclinical studies have not found significant therapeutic benefits in solid tumors, including sarcomas. Nevertheless, in combination therapies based on HDACi, sarcomas were represented in most cases as an unclassified group [129]. More recent studies are now specifically examining individual sarcomas and attempting to identify meaningful combination therapies based on known/identified mechanisms: In Ewing sarcomas, we observed that CRISPR/Cas9 knockout of individual HDACs such as HDAC1 and HDAC2 inhibited the invasiveness of Ewing sarcomas and blocked local tumor growth of xenografts. RNA analyses showed that treatment with single HDAC inhibitors (HDACi) blocked an EWSR1-FLI1-specific expression profile, and EwS cells in the presence of HDAC inhibitors (HDACi) such as entinostat and romidepsin had increased susceptibility to treatment with chemotherapeutic agents including doxorubicin. HDACi acted synergistically with the EED inhibitor A-395 and together inhibited tumor growth of Ewing sarcoma xenografts [91]. Similarly, the dual HDAC and phosphatidylinositol 3-kinase (PI3K) inhibitor Fimepinostat can thus also provide simultaneous and sustained inhibition of multiple oncogenic pathways in Ewing sarcoma and reduce EWSR1-FLI1 levels and transcriptional activity [97]. Inhibition of HDAC activity largely affects Ewing sarcoma cell proliferation and survival, alone or in combination with DNA-damaging agents, through a variety of pathways that include induction of apoptosis, cell cycle arrest, and prevention of tumor invasion and metastasis [130–133]. Fimepinostat is currently being tested in children and young adults with relapsed or refractory solid tumors (NCT03893487).

In alveolar RMS, class I HDACs such as HDAC1, 2, and 3 appear to play an essential function in PAX3-FOXO1 driven super-enhancers, as corresponding inhibitors disrupt the activity of these tumor-specific super-enhancers and block transcription and cell proliferation [92]. Recent data show that entinostat affects in vivo growth of FP-RMS and inhibits PAX3-FOXO1 via a multistep and indirect process through an HDAC3-SMARCA4-miR-27a axis [134]. Interestingly, the HDAC inhibitor Entinostat is now being clinically tested in pediatric rhabdomyosarcomas (NCT02780804).

Previous studies have shown that HDAC inhibitors disrupt the oncoprotein complex of synovial sarcoma, leading them to apoptosis. Transcriptome analysis showed that HDAC inhibition blocks the cell cycle, neuronal differentiation promotes polycomb repressor complexes and proapoptotic factors were reactivated. HDAC inhibition resulted in a lower tumor burden in the mouse model [135]. In another study, the response of synovial sarcoma to HDACi was consistently characterized by activation of ERKs, EGR1, and the β-endoglycosidase heparanase. Disruption of HDAC-induced ERK-EGR1-heparanase pathway by concomitant treatment of cells with an MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/Roneparstat) enhanced the antiproliferative and proapoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and heparanase core levels. The combination

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

of SAHA with SST0001 prevented the upregulation of ERK-EGR1 heparanase, induced by HDACi, and promoted caspase-dependent cell death. In the mouse model, combined treatment with SAHA and SST0001 enhanced the antitumor effect compared with single-agent administration [127]. Thus, it seems very reasonable to advance mediators of epigenetic processes as treatment targets for FP sarcomas. Pracinostat (SB939) a potent pan-HDAC inhibitor is now being tested in pediatric patients with refractory solid tumors and leukemias (NCT01184274).
