MS1943

Amino Acid Sequence

3D Structure (/structures/AF-Q15910-F1-model_v6.cif)

Function

Catalytic subunit of the PRC2/EED-EZH2 complex, a Polycomb group (PcG) complex that methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3, leading to transcriptional repression of the affected target gene. Able to mono-, di- and trimethylate 'Lys-27' of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively. Displays a preference for substrates with less methylation, loses activity when progressively more methyl groups are incorporated into H3K27, H3K27me0 > H3K27me1 > H3K27me2. Compared to EZH1-containing complexes, it is more abundant in embryonic stem cells and plays a major role in forming H3K27me3, which is required for embryonic stem cell identity and proper differentiation. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1, CDKN2A and retinoic acid target genes. EZH2 can also methylate non-histone proteins such as the transcription factor GATA4 and the nuclear receptor RORA. Regulates the circadian clock via histone methylation at the promoter of the circadian genes. Essential for the CRY1/2-mediated repression of the transcriptional activation of PER1/2 by the CLOCK-BMAL1 heterodimer; involved in the di and trimethylation of 'Lys-27' of histone H3 on PER1/2 promoters which is necessary for the CRY1/2 proteins to inhibit transcription (By similarity).

Mechanism

The unfolded protein response (UPR) is a quality-control mechanism triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. Under ER stress, the chaperone BiP/GRP78 dissociates from three ER transmembrane sensors—IRE1α, PERK, and ATF6—activating three distinct UPR branches. Activated IRE1α splices XBP1 mRNA to generate XBP1s. PERK phosphorylates eIF2α, attenuating global translation while selectively inducing ATF4. ATF6 translocates to the Golgi apparatus and is cleaved to release its active nuclear form, ATF6(N). These transcription factors coordinately upregulate chaperones, folding enzymes, ER-associated degradation (ERAD), and autophagy to restore cellular proteostasis. However, if the stress persists or remains unresolved, prolonged UPR signaling triggers apoptosis through the CHOP and BAX/BAK-caspase pathways.

Description

We confirmed through quantitative real-time PCR that Xbp1 and its downstream effectors Chop and Bip were upregulated in response to treatment with MS1943 in MDA-MB-468 cells starting at 4 h of treatment and that induction was sustained for at least two days (Supplementary Fig. 14). Of note, the processed/spliced Xbp1 transcript (Xbp1–207) that can result in active XBP139 was the only transcript that was significantly upregulated after treatment with the degrader as evidenced by our RNA-seq data (Fig. 6c). Taken together, these data suggest that EZH2 degradation could result in sustained overactivation of the UPR pathway in MS1943-sensitive cells due to prolonged ER stress, which in turn could be deleterious and lead to apoptosis. Collectively, these results suggest that MS1943 mediates its cytotoxic effects through ER stress and UPR induction in cells that are dependent for their growth on EZH2.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Collectively, these data show that MS1943 was able to effectively reduce EZH2 protein levels in various types of cell. To further characterize MS1943, we assessed its effects on EZH2 protein levels in MDA-MB-468 and HCC70 cells with or without a functional proteasome. Of note, co-treatment with the proteasome inhibitor MG132 was able to rescue EZH2 levels, at least in part, in a concentration-dependent manner in both MDA-MB-468 and HCC70 cells (Supplementary Fig. 6a,b), suggesting that the reduction of EZH2 protein levels resulted from the MS1943 treatment is mediated by a proteasome-related pathway.

Description

MS1943 (concentration for 50% of maximal inhibition of cell growth (GI50) = 2.2 μM) but not EZH2 inhibitors effectively inhibited cell proliferation (Fig. 2a, b).

Description

MS1943 maintained high potency (IC50 = 120 nM) for inhibiting EZH2 methyltransferase activity (Fig. 1b) and was highly selective for EZH2 over a wide range of methyltransferases including EZH1 (Fig. 1b and Supplementary Fig. 2).