Use of CRISPR-KO screening to identify novel genes and pathways potentially contributing to cisplatin resistance in testicular germ cell tumors.

Abstract

e17020 Background: Testicular germ cell tumors (TGCT) are the most common malignancy in young adult men, with a globally increasing incidence. While these tumors are highly curable due to their exceptional sensitivity to cisplatin (cDDP), a proportion of patients develops a resistance to this chemotherapy, which results in disease progression and patient death. The cause of cisplatin resistance in TGCT has not been elucidated yet and is supposed to be multifactorial. Methods: We conducted a CRISPR Knockout (KO) screening in a TGCT cell line (NCCIT) using a lentiviral sgRNAs library (Addgene #101926) targeting 3015 genes involved in cell cycle regulation and cancer development. The screening population was cultured with or without cDDP (1.25 uM) for 7 days. Samples of treated and control cells were harvested before and after cDDP exposure, the sgRNA cassettes were sequenced by massive parallel sequencing, and the differences in sgRNA abundance were determined using the MAGeCK/MAGeCK-Flute pipeline. Results: The CRISPR-KO screening data analysis identified 59 gene KOs that were significantly positively selected and 7 gene KOs that were significantly negatively selected after cDDP exposure. Among the top positive hits were genes involved in the regulation of apoptosis ( BMF, CDIP1 and BAX ), epigenetic modifiers ( HDAC8, SSX2 ), G-protein regulators ( ADCY8, GNG10 ), and others (e.g. MAGEA2, ACADM, LAMP1 ) — inactivation of these genes potentially contributes to the development of cDDP resistance. Among the top negative hits were genes implicated in DNA damage repair ( FANCB , ERCC5 ), sphingolipid metabolism ( KDSR ), and cytokinesis ( SPECC1L ) — inactivation of these genes may increase sensitivity to cDDP. Conclusions: The results highlight several potential mechanisms of cDPP resistance in TGCT. Some of these genes and pathways are well known and have been previously described in association with platinum resistance in malignant tumors, while others — such as the G-protein regulators, sphingolipid synthesis, and SPECC1L — are novel in this context and may reflect the unique biology of TGCT progression. SPECC1L is involved in spindle organization and microtubule stabilization; its paralog SPECC1 is highly expressed in testis. Polyenoic sphingolipids are essential for the completion of meiosis in male germ cells and spermatozoa production. ADCY8 controls the production of cAMP, which is a key signaling molecule in spermatogenesis. They have also been described in association with malignant proliferation and progression. The identified hits will be validated in subsequent experiments.