Indentification Of Combinatorial Targets Of BET Inhibitors Using CRISPR/Cas9 System

Non–small cell lung cancer(NSCLC)remains the leading cause of cancer death worldwide.The 5-year overall survival rate for patients with metastatic NSCLC was less than 5%.Currently,the main treatments of NSCLC are chemotherapy and targeted therapy,and researchers are actively dedicated to identifing new therapeutic targets and combinational therapies.Bromodomain and extraterminal domain(BET)proteins,functioning as epigenetic readers to regulate gene expression,play important roles in the development of human tumors and have become a promising therapeutic drug targets.Small-molecule inhibitors of BET family have been used in clinical trials to treat a variety of tumors,including NSCLC.However,it shows less effective due to drug resistance.Combination therapies can effectively solve the problem of drug resistance caused by single agents in clinical trials.The CRISPR/Cas9 systems have been extensively used in large-scale functional genome studies.In order to systematically identify key factors that were involved in the therapeutic effect of BET inhibitor OTX015 in NSCLC,we performed a CRISPR/Cas9 based synthetic lethality screen to identify kinases whose knockout synergized with OTX015.Our result showed that sgRNAs targeting EPHA2 gene was significantly absent in the OTX015 treatment group,and depletion of EPHA2 gene by the CRISPR/Cas9 system remarkably potentiated the anti-tumor efficacy of OTX015.In a set of NSCLC cells,the combination of OTX015 and EphA2 inhibitor ALW-II-41-27 produced strong synergistic effects(combination index values less than 0.7).Co-treatment of OTX015 and ALW-II-41-27 notably suppressed the abilities of colony formation,and induced cell cycle arrest and apoptosis in NSCLC cells.Mechanistically,OTX015/ALW-II-41-27 synergistically inhibited the PI3K/AKT/m TOR signaling pathway.Based on the broad efficacy of OTX015/ALW-II-41-27 against NSCLC cells in vitro,we assessed the anti-cancer efficacy of this combination in vivo using a patient-derived tumor explant mouse model.The results showed marked tumor growth inhibition(P<0.05 or P<0.001)in the dual inhibition of BET and EphA2 group compared to the vehicle control or single agent alone,without causing systematical toxicity in mice.In summary,our data from the CRISPR/Cas9 kinase-genome synthetic lethality screens revealed that targeting EphA2 improved the anti-tumor efficacy of BET inhibitor OTX015.The combination of OTX015 and EphA2 inhibitor ALW-II-41-27 significantly retarded the tumor growth,providing a novel and promising strategy for the treatment of NSCLC in patients.