Figure 1.
GSK621 induces preapoptotic surface CALR exposure in murine and human cells. (A) C1498 cells were treated at the indicated concentrations with GSK621 for 48 hours, and the percentage apoptotic cells was measured by flow cytometry using annexin V/propidium iodide (PI) staining (early apoptotic cells: annexin V–positive/PI−; late apoptotic: annexin V–positive/PI+). Ordinary two-way analysis of variance (ANOVA) with Dunnett multiple comparisons test. (B) Flow cytometry of C1498 cells used as controls for surface CALR staining. CALR KO or constitutive cell surface CALR were stained with a CALR–AF647 antibody or isotype control. Graphs represent mean fluorescence intensity (MFI) ± standard deviation (SD). (C) Fold change in MFI relative to vehicle of surface CALR staining on live, unpermeabilized cells by flow cytometry (MFI ± standard error of the mean) after 6 hours of treatment with the indicated drugs (GSK621, MTO, or AraC); n = 3 biological replicates. Ordinary two-way ANOVA with Dunnett multiple comparisons test. (D) Schematic of the procedure to generate MLL-AF9 transformed AML cells from leukemia-bearing mice. After multiple replatings in methylcellulose supplemented with IL-6 (10 ng/mL), stem cell factor (SCF, 10 ng/mL), and IL-3 (6 ng/mL), colonies were enriched in granulocyte colony-forming units (CFU-G; picture). Colonies were then transferred in liquid media and grown in suspension. (E) MLL-AF9 cells were treated at various concentrations with GSK621 for 48 hours and percentages of apoptotic cells were measured as described earlier. Ordinary two-way ANOVA with Šidák multiple comparisons test. (F) Representative dot plots of surface CALR staining in unpermeabilized MLL-AF9 cells treated for 24 hours with GSK621 30 μM or dimethyl sulfoxide (DMSO). (G) MFI of surface CALR staining measured on live, unpermeabilized MLL-AF9 cells; n = 3 replicates. Paired t test. (H) Percent of CALR+ PI− blasts of 3 samples from patients with AML after treatment with GSK621 (20 μM), MTO (0.5 nM), or DMSO. Human AML samples 1 and 2 were cryopreserved, thawed, and treated with GSK621. Sample 3 was received fresh and treated immediately. Ordinary one-way ANOVA with Dunnett multiple comparisons test. (I) Representative dot plots for sample 3.

GSK621 induces preapoptotic surface CALR exposure in murine and human cells. (A) C1498 cells were treated at the indicated concentrations with GSK621 for 48 hours, and the percentage apoptotic cells was measured by flow cytometry using annexin V/propidium iodide (PI) staining (early apoptotic cells: annexin V–positive/PI−; late apoptotic: annexin V–positive/PI+). Ordinary two-way analysis of variance (ANOVA) with Dunnett multiple comparisons test. (B) Flow cytometry of C1498 cells used as controls for surface CALR staining. CALR KO or constitutive cell surface CALR were stained with a CALR–AF647 antibody or isotype control. Graphs represent mean fluorescence intensity (MFI) ± standard deviation (SD). (C) Fold change in MFI relative to vehicle of surface CALR staining on live, unpermeabilized cells by flow cytometry (MFI ± standard error of the mean) after 6 hours of treatment with the indicated drugs (GSK621, MTO, or AraC); n = 3 biological replicates. Ordinary two-way ANOVA with Dunnett multiple comparisons test. (D) Schematic of the procedure to generate MLL-AF9 transformed AML cells from leukemia-bearing mice. After multiple replatings in methylcellulose supplemented with IL-6 (10 ng/mL), stem cell factor (SCF, 10 ng/mL), and IL-3 (6 ng/mL), colonies were enriched in granulocyte colony-forming units (CFU-G; picture). Colonies were then transferred in liquid media and grown in suspension. (E) MLL-AF9 cells were treated at various concentrations with GSK621 for 48 hours and percentages of apoptotic cells were measured as described earlier. Ordinary two-way ANOVA with Šidák multiple comparisons test. (F) Representative dot plots of surface CALR staining in unpermeabilized MLL-AF9 cells treated for 24 hours with GSK621 30 μM or dimethyl sulfoxide (DMSO). (G) MFI of surface CALR staining measured on live, unpermeabilized MLL-AF9 cells; n = 3 replicates. Paired t test. (H) Percent of CALR+ PI− blasts of 3 samples from patients with AML after treatment with GSK621 (20 μM), MTO (0.5 nM), or DMSO. Human AML samples 1 and 2 were cryopreserved, thawed, and treated with GSK621. Sample 3 was received fresh and treated immediately. Ordinary one-way ANOVA with Dunnett multiple comparisons test. (I) Representative dot plots for sample 3.

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