Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic re...Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic responsiveness of os cells is therefore of critical importance.Here,we conducted a kinome-wide clustered regularly interspaced short palindromic repeats(CRISPR)screen,coupled with transcriptome sequencing,to identify regulators of DDP sensitivity.This approach revealed protein kinase membrane-associated tyrosine/threonine 1(PKMYT1)as a key regulator of DDP sensitivity in OS.Subsequent analysis of patient-derived clinical specimens,along with in vitro functional assays,demonstrated that DDP treatment induces the activation of PKMYT1 in OS cells.Importantly,PKMYT1 silencing markedly enhances cellular sensitivity to DDP,indicating its role in promoting chemoresistance.Mechanistically,PKMYT1 induces phosphorylation of nucleophosmin 1(NPM1)at the S260 site,which competitively impairs NPM1 SUMOylation.This modification interferes with the recruitment of essential DNA damage response factors,including breast cancer suppressor gene 1(BRCA1),receptor-associated protein 80(RAP80),and RADiation sensitive protein 51(RAD51),ultimately affecting double-strand break(DSB)repair.Furthermore,the selective PKMYT1 inhibitor RP6306 was found to synergize with DDP,amplifying its cytotoxic effects in OS cells.Collectively,these findings highlight PKMYT1 as a promising therapeutic target and provide a rationale for combinatorial strategies to overcome DDP resistance in OS.展开更多
基金supported by the National Natural Science Foundation of China(82272664,82172500)The Science and Technology Innovation Program of Hunan Province(2023RC3085)+8 种基金The Hunan Provincial Health High-Level Talent Scientific Research Project(R2023054)Key Project of Scientific Research of the Education Department of Hunan Province(24Aoo08)The Hunan Provincial Administration of Traditional Chinese Medicine Project(D2022117)The Scientifc Research Program of Hunan Provincial Health Commission(B202304077077)The Hunan Provincial Natural Science Foundation of China(2023JJ40822,2024JJ6559,2024JJ6572,2025JJ60590)The State-funded postdoctoral researchers plan of China(No.GZC20233188)The Fundamental Research Funds for the Central Universities of Central South University(2024ZZTS0174)The Changsha Municipal Natural Science Foundation(Grant No.kq2403077)The Health Research Project of Hunan Provincial Health Commission(202105012224)。
文摘Cisplatin(DDP)remains a cornerstone therapy for osteosarcoma(OS);however,pervasive resistance severely limits its clinical efficacy and worsens patient outcomes.Developing strategies to enhance the chemotherapeutic responsiveness of os cells is therefore of critical importance.Here,we conducted a kinome-wide clustered regularly interspaced short palindromic repeats(CRISPR)screen,coupled with transcriptome sequencing,to identify regulators of DDP sensitivity.This approach revealed protein kinase membrane-associated tyrosine/threonine 1(PKMYT1)as a key regulator of DDP sensitivity in OS.Subsequent analysis of patient-derived clinical specimens,along with in vitro functional assays,demonstrated that DDP treatment induces the activation of PKMYT1 in OS cells.Importantly,PKMYT1 silencing markedly enhances cellular sensitivity to DDP,indicating its role in promoting chemoresistance.Mechanistically,PKMYT1 induces phosphorylation of nucleophosmin 1(NPM1)at the S260 site,which competitively impairs NPM1 SUMOylation.This modification interferes with the recruitment of essential DNA damage response factors,including breast cancer suppressor gene 1(BRCA1),receptor-associated protein 80(RAP80),and RADiation sensitive protein 51(RAD51),ultimately affecting double-strand break(DSB)repair.Furthermore,the selective PKMYT1 inhibitor RP6306 was found to synergize with DDP,amplifying its cytotoxic effects in OS cells.Collectively,these findings highlight PKMYT1 as a promising therapeutic target and provide a rationale for combinatorial strategies to overcome DDP resistance in OS.
