**7. Ways to block oncogenic transcription**

The basic mechanism by which fusion-positive sarcomas promote and maintain tumorigenicity is through the activation of pathogenic transcriptional programs. They mediate this (as described above) through direct regulation of genes at promoters, the establishment of de novo enhancers, and aberrant recruitment of transcription cofactors [111]. Pathogenic transcriptional activity is also achieved through dysregulation of epigenetic programs, including the generation of super-enhancers characterized by extended stretches of acetylation at histone H3 lysine 27 (H3K27ac) [112]. These histone marks are recognized by members of the BET family (BRD2, BRD3, BRD4) [113]. They have an essential role in regulating transcription by interacting with various proteins such as RNA polymerase II [114]. This allows multiple approaches to intervene pharmacologically in this pathogenic transcriptional program. For example, the first published inhibitor of BET proteins, JQ1, has also shown much noted antitumor activity against various tumor cells [115]. We demonstrated that the BET inhibitor JQ1 reverses the EWSR1-FLI1 transcriptional signature of Ewing sarcoma cells and inhibits tumor growth of Ewing sarcoma xenografts [116]. These results have been confirmed or further investigated in other studies [109, 117–119] Thus, Jacques et al. confirmed the effect of JQ1 on Ewing sarcoma xenografts and additionally observed their decreased vascularization [117]. The effect on angiogenesis was confirmed by another study that examined rhabdomyosarcoma in addition to Ewing sarcomas and showed a reduction in the expression of tumor-associated angiogenic factors [118]. Finally, EWSR1-FLI1 or EWSR1-ERG were studied in a functional complex with BRD4, MED1, and RNAPII [109], and impairment of this complex was observed either by RNA interference of BRD4 expression or by BET inhibitors [109]. In alveolar rhabdomyosarcoma, PAX3-FOXO1 was shown to recruit BRD4 to establish de novo

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

super-enhancers at myogenic transcription factors. These FP-RMS cells were highly sensitive to JQ1, as it selectively silenced PAX3-FOXO1-driven transcription [55].

Another way to interfere with the pathogenic transcriptional program of FP sarcomas is to pharmacologically inhibit transcription-dependent cyclin kinases (CDKs) CDK7, 8, 9, 12, and 13. These CDKs have an essential role in transcription by phosphorylating the C-terminal domain of RNA polymerase II, thereby regulating transcription initiation and elongation [120]. Indeed, profiling of cancer cell lines with the covalent CDK7/12/13 inhibitor, THZ1, showed exceptional sensitivity in cancer cell lines dependent on dysregulated transcriptional programs [121]. Using chemical genomics screening, Iniguez et al. 2018 found that Ewing sarcomas are particularly sensitive to THZ1. Further, they observed that the selective CDK12/13 inhibitor THZ531 elicited DNA damage repair in an EWSR1-FLI1-dependent manner. Combining these molecules with the PARP inhibitor Olaparib resulted in tumor volume reduction and prolonged survival in both cell lines and patient-derived xenografts without hematopoietic toxicity [122].

Synergistic effects were also observed with a sequential targeting approach using the histone demethylase inhibitor GSK-J4 and the CDK inhibitor THZ1 [123]. We observed that CDK9 binds to EWSR1-FLI1 via the BET protein BRD4. The combination of the CDK9 inhibitor CDKI-73 with the BET inhibitor JQ1 was more effective in reducing Ewing sarcoma cell proliferation and tumor volume in xenografts than either agent alone [124]. Another study also demonstrated synergy between the EWSR1-FLI inhibitor mithramycin and the CDK9 inhibitor PHA-767491. Importantly, the synergy was observed at clinically relevant concentrations of mithramycin [125]. Finally, in synovial sarcomas, Li et al. 2019 observed that inhibition of CDK9, with either siRNA or the CDK9 inhibitor LDC067, impaired synovial sarcoma cell growth and proliferation in a dose-dependent manner. This was also associated with a decrease in RNA polymerase II phosphorylation and an increase in the expression of anti-apoptotic proteins. In addition, inhibition of CDK9 decreased sarcoma cell spheroid formation and cell motility [126].
