Whole-genome CRISPR screening identifies <i>N-</i>glycosylation as a genetic and therapeutic vulnerability in <i>CALR</i>-mutant MPNs
Jonas S. Jutzi, Anna E. Marneth, Michele Ciboddo, Ángel Guerra-Moreno, María José Jiménez‐Santos, Anastasia Kosmidou, James W. Dressman, Hongyan Liang, Rebecca Hamel, Patricia Lozano, Elisa Rumi, John G. Doench, Jason Gotlib, Anandi Krishnan, Shannon Elf, Fátima Al‐Shahrour, Ann Mullally
Abstract
Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro 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, through a reduction in MPL cell surface expression. 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 with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.