Genome-scale CRISPR-Cas9 knockout screening for identification of genes which regulate CD38 expression in RPMI 8226 cells
We showed that interleukin-6 (IL-6) from bone marrow stromal cells downregulated CD38 expression in multiple myeloma (MM) cells. Then we performed a genome-scale CRISPR-Cas9 knockout screening to identify key molecules mediating IL-6-induced CD38 downregulation. Although IL-6 triggered downregulation of CD38 expression on the majority of RPMI 8226 cells, a small fraction maintained relatively high CD38 expression. We therefore sorted those top 5% cells with CD38-high expression and compared these cells with non-sorted control cells. The gene rank based on p-value identified Janus kinase (JAK) 1 and signal transducer-activator of transcription (STAT) 3 as highly positively enriched genes as potential mediators of IL-6-induced CD38 downregulation. We validated STAT3 and found CD38 expression was regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of JAK-STAT3 pathway represents  a novel therapeutic option to enhance CD38 expression and anti-CD38 monoclonal antibody-mediated MM cytotoxicity. Overall design: CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 microRNAs were infected into myeloma cell line (RPMI 8226) cells. After 72 h incubation with interleukin-6, top 5 % cells with CD38-high expression were sorted and compared these cells with non-sorted control cells. Then deep sequencing of embedded sgRNA barcodes was performed. The detailed procedures were conducted according to the article of Nature Protocol (Nat Protoc. 2017;12(4):828-863.).