Whole-genome CRISPR screening identifies N-glycosylation as an essential pathway and a potential novel therapeutic target in CALR-mutant MPN (Pooled CRISPR Screen).

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells in vitro. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and a normalization of key MPN disease features. These findings advance the development of clonally selective treatments for CALR-mutant MPN. CRISPR pooled screen, BA/F3-MPL cells, n=4 biological replicates per condition, empty vector cells and CALR DEL52-expressing cells, grown in the presence or absence of interleukin 3 (IL3). Genes of special interest included genes related to N-glycosylation, protein secretion, unfolded protein response, protein arginine methylation and top hits from our whole-genome screen. 1 positive control (EV#2 -_Day 21_without IL3) was included in order to corroborate that most of BA/F3-MPL cells are unviable without IL3.