Novel cell membrane cation transporter associated with platinum therapy resistance in pancreatic adenocarcinoma identified through CRISPR/Cas9 loss-of- function screen

Abstract

Background: Platinum agents form the backbone of chemotherapy regimens for pancreatic cancer. However, the biologic mechanisms of platinum resistance remain poorly understood. Utilizing a new biologic approach, we performed a loss of function, unbiased, genome-wide screen to identify genes associated with platinum resistance. Methods: The novel CRISPR/Cas9 technology was used to inactivate 20,000 genes in the MiaPaCa2 pancreatic cancer cell line. Each gene was targeted with 6 guide RNAs (sgRNA) delivered by lentiviral transduction. After puromycin selection, 2 replicates of cells were intermittently treated with 5uM cisplatin for 24 hours over 4 weeks. A second set of replicates served as non-treatment control. Finally, genomic DNA was extracted from surviving cells, guide sequences amplified and sequenced. MAGeCK analysis identified significantly enriched guides in the treated cells. Results: Multiple genes were identified that, when lost, confer resistance to platinum agents (p- value <0.05). Amongst known mediators of chemoresistance such as Keap1 and 4 members of the STAGA chromatin modifying complex: USP22, TADA1, TAF5L and TAF6L, the loss of the cation transporter MAGT1 was associated with platinum resistance. sgRNA knock-out of these genes when compared to cisplatin treated non-target controls validated the screening results. Conclusion: Our chemotherapy investigation platform identified a novel role for the membrane magnesium cation transporter, MAGT1, which has been associated with X-linked immunodeficiency, but never implicated in platinum resistance hitherto. Our findings nominate MAGT1 as a candidate biomarker for response to platinum therapy and open new avenues of mechanistic investigation into platinum action, including the cellular mechanisms of platinum drugs uptake and the interplay between intracellular magnesium level and platinum cytotoxicity.