Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs

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

GFI1(36N) as a therapeutic and prognostic marker for myelodysplastic syndrome (MDS)

Inherited gene variants play an important role in malignant diseases. The transcriptional repressor Growth factor independence 1 (GFI1) regulates hematopoietic stem cell (HSC) self-renewal and differentiation. A single nucleotide polymorphism of GFI1 (rs34631763) generates a protein with an asparagine (N) instead of a serine (S) at position 36 (GFI1(36N)) and has a prevalence of 3-5% among Caucasians. Since GFI1 regulates myeloid development, we examined the role of GFI1(36N) on MDS disease course. To this end, we determined allele frequencies of GFI1(36N) in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium and the USA. The GFI1(36N) allele frequency in the 723 MDS patients genotyped ranged between 9-12%. GFI1(36N) was an independent adverse prognostic factor for overall survival, AML-free survival and event-free survival in an univariate analysis. After adjusting for age, bone marrow blast percentage, IPSS score, mutational status and cytogenetic findings, GFI1(36N) remained an independent adverse prognostic marker. With regard to therapy, whereas GFI1(36S) homozygous patients showed sustained response to treatment with hypomethylating agents, GFI1(36N) patients show poor sustained response to this therapy. Since allele status of GFI1(36N) is readily determined using basic molecular techniques, we propose to include GFI1(36N) status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions