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Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Published on: January 5, 2018
Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant
Jonas S Jutzi1, Anna E Marneth1, Michele Ciboddo1,2,3
1Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
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.
Insights
Targeting N-glycosylation offers a new strategy for myeloproliferative neoplasms (MPNs) driven by Calreticulin (CALR) mutations. Inhibiting this pathway preferentially harms CALR-mutant cells, suggesting a potential therapy for MPNs.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Research
Background:
- Calreticulin (CALR) mutations initiate myeloproliferative neoplasms (MPNs).
- Current treatments lack clonal selectivity for CALR-mutant MPNs.
- The precise genetic vulnerabilities of CALR-mutant cells remain largely unexplored.
Purpose of the Study:
- To identify unique genetic dependencies in CALR-mutant MPNs.
- To explore N-glycosylation as a therapeutic target in CALR-mutant MPNs.
- To evaluate the efficacy of N-glycosylation inhibition in preclinical models and patient-derived cells.
Main Methods:
- Whole-genome CRISPR knockout screen in CALR-mutant hematopoietic cells.
- Pharmacological screening of N-glycosylation inhibitors.
- Treatment of Calr-mutant knockin mice with 2-deoxy-glucose (2-DG).
- Megakaryocyte colony-forming unit (CFU-MK) assays using patient-derived bone marrow.
Main Results:
- CRISPR screen revealed differential depletion of N-glycosylation pathway genes in CALR-mutant cells.
- Chemical inhibition of N-glycosylation impaired growth of CALR-mutant cells by reducing MPL surface expression.
- 2-DG treatment preferentially sensitized CALR-mutant cells, normalizing MPN disease features in mice.
- N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow.
Conclusions:
- N-glycosylation represents a unique vulnerability in CALR-mutant MPNs.
- Inhibition of N-glycosylation demonstrates therapeutic potential for CALR-mutant MPNs.
- These findings pave the way for developing clonally selective treatments for MPNs.
