The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma
Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth. Overall design: For the genome-wide CRISPR/Cas9 knockout screen, the A673/EWSR1::FLI1tdTomato/EGFP/Cas9 reporter cell line was transduced with the Human CRISPR Knockout Pooled Library (Brunello) (Addgene, Cat# 73178)10, consisting of ~76,441 sgRNAs targeting 19,114 genes at multiplicity-of-infection (MOI) of 0.3. Cells were selected with 2 µg/ml puromycin for 12 days, followed by 3 days recovery in the absence of puromycin. At least 1x108 cells were FACS sorted using a BD FACSAria II cell sorter, and the population with tdTomatolow EGFPhigh expression (~7% of the total population) was isolated and expanded. Total genomic DNA was extracted from the sorted and unsorted populations, and 20 µg used to prepare a next-generation sequencing (NGS) library.