Identification Of Oncogenic PRC2 Mutation And Pharmacological Mechanism Of PRC2 Inhibitor In Cancer Treatment

The histone methyltransferase Polycomb Repressive Complex 2(PRC2),composed of EZH1/2,SUZ12,EED and RBAP46/8 subunits,is responsible for tri-methylation of histone H3 on lysine 27 residue(H3K27me3),which is associated with transcriptional repression of target genes.The aberrant expression or mutations of EZH2 have been identified in multiple cancers.Owing to its role in multiple human cancers,the development of small-molecule inhibitors targeting PRC2 as cancer therapeutics is actively ongoing.Tazemetostat,a small-molecule inhibitor competing with the cofactor SAM,have been approved by the FDA for the clinical treatment of epithelioid sarcoma.Gain-of-function(Go F)mutations of EZH2 within the SET domain include Y641F/H/C/S/N,A677 G and A687 V,which have been associated with enhanced H3K27me3 and B-cell lymphomagenesis.Recent progresses on PRC2 complex structure suggested an important role of the N-terminal SAL(SET-Activation Loop),which extends to the back of the SET domain.To tackle the critical residues outside SET domain for this enzyme,we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel Go F mutation W113 C,which increases H3K27me3 in vitro and in vivo and promotes CDKN2 A silencing and tumorigenesis.Different from other Go F mutations,this mutation is located in the SAL motif at EZH2 N-terminus,which stabilizes the SET domain and facilitates substrate binding.W113 C mutation may increases PRC2 activity by stabilizing the SAM-binding pocket in catalytic center.Intriguingly,W113 C mutation leads to Tazemetostat-resistance in cell and in vivo both at H3K27 methylation and tumor proliferation.Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance,effectively decreases H3K27me3 and blocks tumor proliferation in cells expressing EZH2 W113 C.As this mutation is originally identified from lymphoma samples,our results demonstrated its activating characteristic and the deleterious consequence,brought insights on PRC2 regulation,and support to continue explore opportunity of treatment optimization for lymphoma patients.EED inhibitors targeting the H3K27me3-binding pocket and inhibiting PRC2 allosterically can effectively block PRC2 with EZH2 inhibitor-resistant mutations.However,their pharmacological mechanisms are poorly understood.MAK683 is a potent EED inhibitor in clinical trial.In sensitive tumor cells with SWI/SNF deficiency,MAK683 treatment caused a dose-and time-dependent proliferation blockade and resulted in cellular senescence induction in vitro and in vivo.We further performed an integrated epigenomic and transcriptomic analysis and identified a group of genes derepressed by PRC2 inhibition with high H3K27me3 signature.Multiple senescenceassociated secretory phenotype(SASP)genes,such as GATA4,MMP2/10,ITGA2 and GBP1,are in this group besides previously identified CDKN2A/p16.To clarify if p16 is required in SASP induction,we constructed CDKN2A/p16 knockout(KO)cells using CRISPR-Cas9 method and demonstrated that upon PRC2 inhibition the de-repression of SASP genes is detected in multiple sensitive models.Upregulation of these SASPs associated with extracellular matrix(ECM)reorganization and immune infiltration could repress tumorigenesis even in CDKN2A/p16 knockout tumor.Indeed,the combination of PRC2 inhibitor with CDK4/6 inhibitor showed better proliferation inhibition in vitro and result in better anti-tumor effect in vivo.In summary,we characterized a unique EZH2 Go F mutation W113 C outside SET domain from lymphoma patient database.We demonstrated that EZH2 W113 C exhibited high H3K27 trimethylating activity and enhanced proliferation through stabilization of SET domain.Interestingly,the W113 C mutation enables the resistance to SAMcompetitive EZH2 inhibitors but retains the sensitivity to EED inhibitors.Furthermore,we focused on the molecular pharmacological mechanism of PRC2 inhibitors and dissected the critical target genes induced by EED inhibitor MAK683 in sensitive cancer cells deficient in SWI/SNF complex.PRC2 inhibition by MAK683 induces cellular senescence and de-repression of SASP genes,which contributes to ECM reorganization and immune infiltration.