Genome-scale CRISPR screen reveals neddylation to contribute to cisplatin resistance of testicular germ cell tumors

Type II testicular germ cell tumors (TGCT) are the most prevalent tumors in young men. Patients suffering from cisplatin resistant TGCTs are facing very poor prognosis demanding novel therapeutic options. Neddylation is a known posttranslational modification mediating many important biological processes including tumorigenesis. Overactivation of neddylation pathway promotes carcinogenesis and tumor progression in various entities by inducing proteasomal degradation of tumor suppressors (e.g., p21, p27). We used a genome-scale CRISPR/Cas9 activation screen to identify cisplatin resistance factors. TGCT cell lines were treated with the neddylation inhibitor (MLN4924)/cisplatin/combination and investigated for changes in viability (XTT assay), apoptosis/cell cycle (flow cytometry) as well as in the transcriptome (3’mRNA sequencing). NAE1 overexpression was detected in cisplatin resistant colonies from the CRISPR screen. Inhibition of neddylation using MLN4924 increased cisplatin cytotoxicity in TGCT cell lines and sensitized cisplatin resistant cells towards cisplatin. Apoptosis, G2/M-phase cell cycle arrest, gH2A.X/P27 accumulation and mesoderm/endoderm differentiation was observed in TGCT cells while fibroblast cells were unaffected. We identified overactivation of neddylation as a factor for cisplatin resistance in TGCTs and highlighted the additive effect of NAE1 inhibition by MLN4924 in combination with cisplatin as a novel treatment option for TGCTs. Total RNA obtained from cell lines treated for 48 hours with MLN4924 only/cisplatin only/combination (MLN4924 and cisplatin) compared to DMSO/DMF/combination (DMSO and DMF) treated control cells. Total 36 samples with n=3 for each condition.