Objective: Effective adjuvant therapeutic strategies are urgently needed to overcome MAPK inhibitor (MAPKi) resistance, which is one of the most common forms of resistance that has emerged in many types of cancers. Here, we aimed to systematically identify the genetic interactions underlying MAPKi resistance, and to further investigate the mechanisms that produce the genetic interactions that generate synergistic MAPKi resistance.Methods: We conducted a comprehensive pair-wise sgRNA-based high-throughput screening assay to identify synergistic interactions that sensitized cancer cells to MAPKi, and validated 3 genetic combinations through competitive growth, cell viability, and spheroid formation assays. We next conducted Kaplan-Meier survival analysis based on The Cancer Genome Atlas database and conducted immunohistochemistry to determine the clinical relevance of these synergistic combinations. We also investigated the MAPKi resistance mechanisms of these validated synergistic combinations by using co-immunoprecipitation, Western blot, qRT-PCR, and immunofluorescence assays. Results: We constructed a systematic interaction network of MAPKi resistance and identified 3 novel synergistic combinations that effectively targeted MAPKi resistance (ITGB3 + IGF1R, ITGB3 + JNK, and HDGF + LGR5). We next analyzed their clinical relevance and the mechanisms by which they sensitized cancer cells to MAPKi exposure. Specifically, we discovered a novel protein complex, HDGF-LGR5, that adaptively responded to MAPKi to enhance cancer cell stemness, which was up- or downregulated by the inhibitors of ITGB3 + JNK or ITGB3 + IGF1R. Conclusions: Pair-wise sgRNA library screening provided systematic insights into elucidating MAPKi resistance in cancer cells. ITGB3- + IGF1R-targeting drugs (cilengitide + linsitinib) could be used as an effective therapy for suppressing the adaptive formation of the HDGF-LGR5 protein complex, which enhanced cancer stemness during MAPKi stress.

Yu Yu;Minzhen Tao;Libin Xu;Lei Cao;Baoyu Le;Na An;Jilin Dong;Yajie Xu;Baoxing Yang;Wei Li;Bing Liu;Qiong Wu;Yinying Lu;Zhen Xie;Xiaohua Lian

Department of Cell Biology,Basic Medical College,Army Medical University(Third Military Medical University),Chongqing 400038,China;MOE Key Laboratory of Bioinformatics and Bioinformatics Division,Center for Synthetic and System Biology,Department of Automation,Beijing National Research Center for Information Science and Technology,Tsinghua University,Beijing 100084,China;National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China;Department of Thoracic Surgery,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Beijing 100730,China;Beijing Syngentech Co.,Ltd,Beijing 102206,China;School of Life Sciences,Tsinghua University,Beijing 100084,China;The Comprehensive Liver Cancer Center,The 5th Medical Center of PLA General Hospital,Beijing 100039,China

癌症生物学与医学（英文版）

[1]Yu Yu;Minzhen Tao;Libin Xu;Lei Cao;Baoyu Le;Na An;Jilin Dong;Yajie Xu;Baoxing Yang;Wei Li;Bing Liu;Qiong Wu;Yinying Lu;Zhen Xie;Xiaohua Lian-.Systematic screening reveals synergistic interactions that overcome MAPK inhibitor resistance in cancer cells)[J].癌症生物学与医学（英文版）,2022(02):229-252

MAPKi,cilengitide,linsitinib

Systematic,screening,reveals,synergistic,interactions,that,overcome,resistance,cells,Objective,Effective,adjuvant,therapeutic,strategies,are,urgently,needed,which,one,most,common,forms,has,emerged,many,types,cancers,Here,we,aimed,systematically,identify,genetic,underlying,further,mechanisms,produce,generate,Methods,conducted,comprehensive,pair,wise,sgRNA,high,throughput,sensitized,validated,combinations,competitive,growth,viability,spheroid,formation,assays,next,Kaplan,Meier,survival,analysis,Cancer,Genome,Atlas,database,immunohistochemistry,determine,clinical,relevance,these,also,investigated,by,using,immunoprecipitation,blot,qRT,immunofluorescence,Results,constructed,network,identified,novel,effectively,targeted,ITGB3,IGF1R,JNK,HDGF,LGR5,analyzed,their,they,exposure,Specifically,discovered,protein,complex,adaptively,responded,stemness,was,downregulated,inhibitors,Conclusions,Pair,library,provided,insights,into,elucidating,targeting,drugs,could,be,used,therapy,suppressing,enhanced,during,stress

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