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Karolak et al. J Cancer Metastasis Treat 2021;7:15 https://dx.doi.org/10.20517/2394-4722.2021.05 Page 7 of 16

and reduction of cell survival [36-38] . Surprisingly, the EZH2 depletion led to expression of pro-apoptotic genes
[37]
instead of differentiation genes, resulting in initiation of cellular death . The authors demonstrate that the
mechanism of apoptosis in these cells is likely due to derepression of pro-apoptotic gene including the
tumour suppressor FBXO32 after EZH2 knockdown, as well as in reduction of myogenic proteins such as
Myogenin and MyoD, indicating an induction of apoptosis over differentiation . A separate study also
[37]
demonstrated apoptosis in RH30 cells after siRNA silencing of EZH2. However, they also showed that
modest reduction of EZH2 in both ERMS (RD) and ARMS (RH30) cells led to differentiation and
expression of the terminal differentiation marker myosin heavy chain (MHC) in the presence of
differentiation conditions . It is worth noting that EZH2 is involved in a feedback loop with other
[36]
regulatory components such as miR-101, which may serve as a tumour suppressor, and through repression
of this molecule is able to promote tumorigenesis in ERMS, as was shown in a study conducted by Vella and
colleagues . Silencing of EZH2 through siRNAs in ERMS cell lines increased the expression of several
[2]
miRNAs such as miR-29b, miR-214 and miR-101. The latter caused down-regulation of both mRNA and
protein levels of EZH2 and thus, reduction of the tumorigenic potential of ERMS cells in vitro .
[2]
Rhabdoid Tumours (MRTs and ATRTs)
Silencing of EZH2 by shRNAs in SMARCB1-deficient paediatric ATRT cell lines (BT12, BT16 and patient-
derived UPN 737) has been shown to reduce cell proliferation and concomitantly induce cellular senescence
and apoptosis, with the biggest effects seen with combination EZH2-silencing by shRNA and
administration of DZNep. It was also demonstrated that inhibition of EZH2 affected crucial pathways and
molecules involved in cell cycle regulation with downregulation of E2F and c-Myc . Furthermore, in an
[39]
MRT cell line model, sustained knockdown of EZH2 through siRNA manifested as reduction of cellular
[40]
growth and SMARCB1 restoration .
Other STS Subtypes (MPNST, SS, LS)
Stable knockdown of EZH2 using shRNAs in both non-NF1 related and NF-1 related MPNST cell lines
resulted in increased apoptosis, inhibited growth and decreased viability of malignant cells in vitro. By using
MPNST immunodeficient mouse xenografts it was demonstrated that injected EZH2-targeted shRNAs were
also able to inhibit tumorigenicity in vivo . In the SS cell lines possessing either SS18-SSX1 (Aska-SS,
[5]
Yamato-SS) or SS18-SSX2 translocation (Fuji, SYO-1) the silencing of EZH2 by siRNA and shRNA led to
dose-dependent inhibition of cell proliferation in all four cell lines and decreased levels of H3K27me3 .
[3]
Conversely, depletion of EZH2 through RNAi in well-differentiated and dedifferentiated liposarcoma cell
lines with addition of steroids and insulin, led to cell proliferation inhibition, followed by induction of
[41]
differentiation in vitro .

EZH2 modulation through use of small molecular inhibitors
Recently, EZH2 has been the focus of numerous drug discovery efforts, resulting in a number of tool
compounds and clinical candidates targeting EZH2 being developed. The first to be developed was 3-
deazaneplanocin A (DZNep), whose mechanism of action is as an S-adenosyl-L-homocysteine (SAH)
hydrolase inhibitor. DZNep represses S-adenosyl-L-methionine-dependent histone methyltransferase
activity and thus is not a specific EZH2 inhibitor. A number of S-adenosyl-methionine competitive EZH2-
specific inhibitors have since been developed [Table 1], a number of which have been taken into clinical
trials for blood cancers and solid tumours (Tazemetostat and GSK126) [14,42-45] .

Synovial sarcoma
In SS18-SSX1 (HS-SY-II) and SS18-SSX2 (Fuji) translocation-positive and SMARCB1-deficient synovial
sarcoma cell lines, treatment with tazemetostat led to a concentration-dependent inhibition of cell growth
and apoptosis in vitro. Administration of tazemetostat or EPZ011989 in xenograft models of SS, carrying