Systematic Screening Reveals Synergistic Interactions That Overcome MAPK Inhibitor Resistance In Melanoma Cancer Cells

Background:Although vemurafenib has been widely used as a valid treatment,evolved vemurafenib-resistant cells would eventually relapse in patients carrying BRAFV600E mutant.However,because of complex responses to vemurafenib treatment,it is essential to systematically analyze genetic interactions between gene nodes involved in vemurafenib resistance.Here,we established a CRISPR/Cas9-based platform to systematically screen～3,500 combinations of gene pairs in cultured melanoma cells with BRAFV600E mutation by using pairwise sgRNA library.We identified gene pairs that displayed strong growth phenotypes and genetic interactions when perturbed with pairwise sgRNA combinations,and validated several gene pairs with competition growth assay and/or small molecule inhibition assay.Futhermore,we dissected the potential coordinated sensitizing mechanisms of vemurafenib resistance of the NF2 depleted A375 and the original A375 cell line,by using transcriptomic RNA deep sequencing,quantitative RT-PCR and western blot.This work provided systematic information for understanding vemurafenib resistance mechanisms in melanoma cells,which may inspire the discovery of new combinatorial targets and facilitate efficient diagnostic and prognostic pairwise target indicators for vemurafenib resistance.Objective:1.Establishing the high-throughput,quantitative analysis pipeline for identifying significant genetic interactions involved in the process of melanoma vemurafenib resistance.2.Quantifying the phenotypic behavior and genetic interaction strength of the pairwise targets,which were selected from the hits of pairwise sgRNA screening.3.Dissecting the potential mechanisms of coordinated melanoma sensitizing effects by paired inhibition of selected screening hits under the environment of vemurafenib.Methods:1.Established the pairwise knockout CRISPR/Cas9 screening library by designing the library structure,sgRNA spacers,and validating the balance effect of promoter activation,generating single sgRNA vector by golden gate annealed sgRNA pools with U6 vector or 7SK vector using BsaI site and generating pairwisesgRNA vector by golden gate U6 driven sgRNA vector,7SK driven sgRNA vector with destination vector with ESP3I site.2.Identified the potential useful hits by analyzing the drug resistance value and genetic interaction strength from the library results of CRISPR/Cas9 pairwisesgRNA screening,calculating the resistance phenotypic value by using the illumina PE250 sequencing data,assessing genetic interaction strengh by using linear fitting method,and defining the central hub nodes via quantifying their eigencentrality through the drug resistance value network and the genetic interaction strength network.3.Investigated the drug resistant phenotype of genetic pairs and drug pairs by FACS and HCS quantification.4.Dissected the underlying mechanisms of coordinated effect of sensitizing melanoma cell to vemurafenib treatment by using transcriptomic deep sequencing,small molecule inhibition,RT-qPCR,western blot,immunofluorecense and Co-IP assay.Results:1.We constructed pairwise sgRNA library consisting of 45,504 pairwise sgRNA vectors that targeting 3,500 genetic pairs.2.Vemurafenib resistance and genetic interaction strength were calculated from PE250 sequencing data of library infected,10-day treated Cas9-A375 samples of vemurafenib and DMSO,then screening replicates were compared and the data with low correlation was filtered out.3.The central factors that might significantly influence the vemurafenib resistance were marked by analyzing the vemurafenib resistance and the genetic interaction network with the eigencentrality algorithm.4.Multiple combinatorial targets were found and validated from the high-throughput screening data,the immunostaining of melanoma sample slides and the clinical survival data that could sensitize or protect melanoma cells to vemurafenib,include:(1)Co-inhibiton of EGFR+PLK1,ITGB3+IGF1R,ITGB3+JNK cooperatively sensitized A3 75 cell line from vemurafenib.(2)Co-inhibition of NF2+CCNC cooperatively protected A375 cell line from vemurafenib.(3)Co-inhibition of ITGB3+IGF1R cooperatively sensitized NF2 knockout A375 cell line from vemurafenib.(4)Co-inhibition of ITGB3+JNK cooperatively sensitized SK-MEL-28 cell line from vemurafenib.(5)Co-inhibition of HDGF+LGR5 cooperatively decreased the protein expressions of sternness maintenance factors such as BMI1,CD 133,Nestin,SOX2,and negatively affected the tumorsphere formation parameters of A375,H1299 cell lines from vemurafenib or dabrafenib.5.Orchestrated mechanisms of combinatorial targets were studied,include:(1)E-cadherin mRNA abundance was significantly rescued by the perturbation of vemurafenib+lapatinib+rigosertib,vemurafenib+cilengitide+linsitinib.(2)Vimentin mRNA abundance was significantly down-regulated by theperturbation of vemurafenib+lapatinib+rigosertib,vemurafenib+cilengitide+linsitinib.(3)In A375 cell line,lapatinib+vemurafenib exerted an epistatic effect on the BIRC5 inhibition in vemurafenib+lapatinib+rigosertib combination,where vemurafenib+rigosertib exerted an epistatic effect on the up-regulation of cleaved caspase-3 in the vemurafenib+lapatinib+rigosertib combination.(4)Transcriptomic deep sequencing were used to identify the mechanism difference of vemurafenib resistance on A375 and NF2 depleted A375 cell line,where we found that the gene expressions of ROSl,Wnt10b,Integrin,IGF1R,SMAD,FGF were significantly surged,and the gene expressions of PCDH7,DUSP6,Jun were significantly decreased.(5)In A375 cell line,linsitinib exerted an epistatic role on the γ-catenin inhibition in vemurafenib+cilengitide+linsitinib combination but not in NF2 knockout A375 cell line,where vemurafenib+cilengitide+linsitinib exerted the common effects on cell lines of A375 and NF2 depleted A375 by up-regulation of cleaved caspase-3 and down-regulation of phospho-ERK expression level.(6)In A375 cell line,cilengitide+JNK-IN-8 exhibited cooperation on the inhibition of phospho-ERK 1/2 and activation of cleaved caspase-3.(7)In A375 cell line,cilengitide+JNK-IN-8 cooperatively promoting the formation of HDGF-LGR5 protein complex with the exposure to vemurafenib whereas cilengitide+linsitinib inhibiting the formation of HDGF-LGR5 protein complex with the exposure to vemurafenib.Conclusions:In summary,we established a pooled pairwise sgRNA screening and data analysis pipeline to systematically study genetic interactions underlying the vemurafenib resistance,we constructed two networks that characterize the phenotype of drug resistance and the genetic interaction strength,and to assess the central hub node inside.Our strategy to identify EGFR+PLK1,ITGB3+IGF1R,HDGF+LGR5,NF2+CCNC as sensitizing and protective gene pairs which might be useful diagnostic and prognostic indicators for patients developed vemurafenib resistance.Furthermore,ITGB+IGF1R and ITGB3+JNK were discovered as efficient combinatorial targets to overcome NF2 depleted A375 and SK-MEL-28 vemurafenib resistant cell line.Finally,we studied the synergistic mechanisms of adjuvant vemurafenib sensitization.Particularly,we identified that HDGF and LGR5 could adaptively form as a protein complex to enhance cancer cell stemness for responding the stress of MAPK inhibitors,and combinations of cilengitide+linsitinib,cilengitide+JNK-IN-8 could influence the formation of HDGF-LGR5 complex negatively or positively.These findings may help to better understand the MAPK inhibitor resistance from the perspectives of biological meaningful genetic interactions.