Small-Inhibitor-Mediated Disruption Of PRADX-EZH2 And Enhancement Of Temozolomide Sensitivity To Glioblastoma

Temozolomide(TMZ)is a frontline chemotherapeutic agent for glioblastoma multiforme(GBM),inducing DNA O~6-methylguanine(O~6-met G)methylation and subsequent DNA strand breaks,leading to tumor cell death.However,GBM cells develop resistance to TMZ through various mechanisms,including O~6-methylguanine-DNA methyltransferase(MGMT)activity and expression levels of DNA repair factors involved in DNA strand breakage repair.Notably,GBM patients undergoing standard TMZ treatment exhibit a median survival period of merely 12-15 months.Therefore,the exploration of more effective drugs and improvements to existing treatment modalities is crucial for enhancing outcomes in GBM patients.Our research group previously reported a novel long non-coding RNA,PRADX,involved in regulating GBM occurrence and progression through interaction with Polycomb repressive complex 2(PRC2).Through computer-aided drug design,we identified EPIC-0307,a small molecule inhibitor disrupting the PRADX-EZH2 interaction,significantly inhibiting tumor cell growth and enhancing TMZ sensitivity.EPIC-0307 holds promise as a potential therapeutic option for GBM patients.Research methods:(1)Computational simulations were conducted to construct the PRADX-EZH2 complex structure,identify key sites,and screen potential inhibitors in a small molecule database.Cytotoxicity,pharmacokinetics,and safety profiles were assessed.(2)EPIC-0307 was selected as a potential inhibitor of PRADX-EZH2 interaction.Validation was performed through RIP and Ch IRP experiments,confirmation of its impact on the PRC2 complex on the PRC2 complex was conducted through Co-IP,and EPIC-0307’s mechanism of inhibiting tumors was investigated through Ch IP,q PCR,Western Blot,and other methods.Its effects on GBM cell cycle arrest and apoptosis were assessed through flow cytometry,immunofluorescence,etc.(3)An investigation of EPIC-0307’s potential synergy with TMZ in MGMT-negative GBM was carried out.In vitro experiments assessed cell viability,and clonogenicity,and confirmed increased TMZ sensitivity.The impact on DNA damage repair inhibition for enhanced TMZ sensitivity was examined.Validation of EPIC-0307’s anti-tumor activity and increased TMZ sensitivity in GBM stem cells and differentiated cells was conducted.(4)EPIC-0307’s effect on TMZ sensitivity in MGMT-positive GBM was evaluated through in vitro experiments.Its impact on DNA damage repair,ATF3,STAT3 pathways,and MGMT levels were explored through q PCR,Western Blot,etc.The role of MGMT in EPIC-0307 sensitizing TMZ was investigated.GBM cell lines with low ATF3 expression were constructed,and EPIC-0307’s regulation of MGMT expression through ATF3-p-STAT3-HDAC1 epigenetic control was studied.The impact of EPIC-0307 on TMZ sensitivity in MGMT-positive GBM tumors was validated in vivo.Research results:(1)PRADX forms a"U"shape structure with EZH2 within the 340-440nm range.Through high-throughput screening and cytotoxicity studies,EPIC-0307,a small molecule compound,demonstrated significant anti-tumor activity in GBM cells with relative insensitivity in normal cells.EPIC-0307 exhibited oral absorption,bloodstream distribution,and notable accumulation in tumor tissues.(2)EPIC-0307 did not affect the expression of PRADX and core members of the PRC2,nor did it influence the assembly of PRC2.However,it selectively blocked the binding of PRADX and EZH2,resulting in decreased H3K27me3 enrichment at downstream targets P21and PUMA,promoting their transcription,inducing cell cycle arrest,and apoptosis.EPIC-0307also suppressed the Rb signaling pathway and lowered E2F1 expression while upregulating P21.(3)EPIC-0307 inhibited the transcription of DNA repair genes CHK1,CHK2,RAD50,RAD51,and MRE11.In MGMT-negative GBM,EPIC-0307 reversed TMZ-induced DNA damage repair,increasing TMZ sensitivity and demonstrating anti-tumor activity both in vitro and in GBM stem cells and differentiated cells.(4)In MGMT-positive GBM,EPIC-0307 not only regulated DNA damage repair but also enhanced TMZ sensitivity by downregulating MGMT expression.EPIC-0307 upregulated ATF3,suppressed the STAT3 pathway,promoted ATF3-p-STAT3 recruiting more HDAC1,leading to decreased H3K27ac levels at the MGMT promoter,and suppressed MGMT transcription.This finding was further confirmed by O6-met G immunofluorescence.Research conclusion:This study found that EPIC-0307 selectively disrupted the binding of PRADX and EZH2,leading to the upregulation of P21 and PUMA expression through epigenetic regulation,resulting in cell cycle arrest and apoptosis in GBM cells.EPIC-0307 also downregulated the expression of DNA repair-related genes in GBM cells,increasing sensitivity to TMZ treatment.In MGMT-positive GBM,EPIC-0307 induced epigenetic silencing of MGMT expression through the ATF3-p STAT3-HDAC1 complex,enhancing sensitivity to TMZ treatment.Overall,EPIC-0307,as a potential epigenetic drug targeting the PRADX-EZH2 interaction,demonstrated anti-tumor activity both in vitro and in vivo in GBM.Its combination with TMZ could serve as a more effective treatment strategy.