Figure 4.
N-glycosylation inhibitors preferentially reduce growth of mutant CALR-transformed cells and reduce MPL surface expression. (A) Overview of N-glycosylation biosynthesis and endoplasmic reticulum (ER)-resident trimming proteins (labeled in black) differentially required for growth of CALRΔ52-transformed cells (compared with EV or CALRΔ52 cells grown in the presence of IL3) as determined by scoring as a “hit” in either of our 2 CRISPR screens (negative log2 fold change > 1). After biosynthesis of the N-glycan tree (dolichol-P-P-GlcNAc2Man9Glc3), it is transferred to a newly synthesized glycoprotein such as MPL (red line) by the OST complex. OST complex members that scored in our screens include OST4, OSTC, STT3A, RPN1, RPN2, and DAD1. CALR then binds and folds the glycoprotein. The graph also depicts the mode of action of N-glycosylation inhibitors (labeled in red) used in B-I. (B) Growth curves of BA/F3-MPL-EV or -CALRΔ52–expressing cells in the presence or absence of IL3, respectively. Cells were treated with DMSO, 10 μM or 50 μM 2-deoxyglucose (2-DG). Data were normalized to DMSO control for each cell line. (C) Normalized geometric mean MPL (CD110) cell surface expression of BA/F3-MPL-EV, -CALRΔ52, or –Jak2V617F–expressing cells following 2-DG treatment for 24 hours. EV control cells were grown in the presence of IL3, CALRΔ52, and Jak2V617F cells in the absence of IL3. (D-I) Growth curves and MPL surface expression as described in panels B-C. DMSO, 1 μM tunicamycin (D-E), DMSO, 1 μM or 10 μM ML414, 5-[(Dimethylamino)sulfonyl]-N-(5-methyl-2-thiazolyl)-2-(1-pyrrolidinyl)-benzamide (NGI1) (F-G), and DMSO, 20, or 50 μM castanospermine (H-I). Statistical significance in panels B, D, F, and H was calculated using 2-sided Student t tests at the 72-hour time point. Growth curves were performed in duplicate 2 to 4 times. Statistical significance in panels C, E, G, and I was determined using 1-way analysis of variance. MPL measurements were performed in duplicate 2 to 3 times. The most important statistical analyses are highlighted. Mean plus or minus standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001. Fruc, fructose; Glc, glucose; Man, Mannose; mRNA, messenger RNA; P, phosphate.

N-glycosylation inhibitors preferentially reduce growth of mutant CALR-transformed cells and reduce MPL surface expression. (A) Overview of N-glycosylation biosynthesis and endoplasmic reticulum (ER)-resident trimming proteins (labeled in black) differentially required for growth of CALRΔ52-transformed cells (compared with EV or CALRΔ52 cells grown in the presence of IL3) as determined by scoring as a “hit” in either of our 2 CRISPR screens (negative log2 fold change > 1). After biosynthesis of the N-glycan tree (dolichol-P-P-GlcNAc2Man9Glc3), it is transferred to a newly synthesized glycoprotein such as MPL (red line) by the OST complex. OST complex members that scored in our screens include OST4, OSTC, STT3A, RPN1, RPN2, and DAD1. CALR then binds and folds the glycoprotein. The graph also depicts the mode of action of N-glycosylation inhibitors (labeled in red) used in B-I. (B) Growth curves of BA/F3-MPL-EV or -CALRΔ52–expressing cells in the presence or absence of IL3, respectively. Cells were treated with DMSO, 10 μM or 50 μM 2-deoxyglucose (2-DG). Data were normalized to DMSO control for each cell line. (C) Normalized geometric mean MPL (CD110) cell surface expression of BA/F3-MPL-EV, -CALRΔ52, or –Jak2V617F–expressing cells following 2-DG treatment for 24 hours. EV control cells were grown in the presence of IL3, CALRΔ52, and Jak2V617F cells in the absence of IL3. (D-I) Growth curves and MPL surface expression as described in panels B-C. DMSO, 1 μM tunicamycin (D-E), DMSO, 1 μM or 10 μM ML414, 5-[(Dimethylamino)sulfonyl]-N-(5-methyl-2-thiazolyl)-2-(1-pyrrolidinyl)-benzamide (NGI1) (F-G), and DMSO, 20, or 50 μM castanospermine (H-I). Statistical significance in panels B, D, F, and H was calculated using 2-sided Student t tests at the 72-hour time point. Growth curves were performed in duplicate 2 to 4 times. Statistical significance in panels C, E, G, and I was determined using 1-way analysis of variance. MPL measurements were performed in duplicate 2 to 3 times. The most important statistical analyses are highlighted. Mean plus or minus standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001. Fruc, fructose; Glc, glucose; Man, Mannose; mRNA, messenger RNA; P, phosphate.

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