Figure 1.
Prolonged XPO1 inhibition is necessary to elicit significant antileukemic activity in NPM1-mutated AML in vitro. (A) Fluorescence microscopy of NPM1c-GFP OCI-AML3 treated for 18 hours with selinexor 100 nM. After 18 hours, selinexor was removed from the medium, and cells were left in culture for the following 24 hours taking pictures at 5, 7, 9, 22, and 24 hours after drug washout. Hoechst 33342 was used to stain the nuclei. Original magnification ×100; scale bar, 5 μm. (B) Principal component analysis plot derived from the means (N = 2) of the fragments per kilobase of transcript per million mapped reads (FPKM) values of parental OCI-AML3 cells collected at 24 hours treated with either DMSO or selinexor 50 nM and OCI-AML3 cells collected at 72 hours treated with either DMSO, selinexor 50 nM ST (24 hours selinexor + 48 hours fresh medium) or selinexor 50 nM CT (72 hours selinexor). (C) Volcano plots depicting differentially expressed genes in parental OCI-AML3 cells treated for 72 hours with selinexor 50 nM CT (72 hours selinexor) and 72 hours selinexor 50 nM ST (24 hours selinexor + 48 hours fresh medium), compared with DMSO. Log2FC and log10p andjusted values are shown on the x- and y-axis, respectively. Genes belonging to the HOXA (red), HOXB (blue), and MEIS/PBX (green) families are highlighted. (D) Gene set enrichment analysis of RNA-sequencing data from parental OCI-AML3 cells treated for 72 hours with selinexor CT or DMSO. Enrichment plots for “Hay_bone_marrow_neutrophils” and “Hallmark_P53_pathway” gene sets are shown. Complete Gene Ontology and Molecular Signature Database enrichment data are provided in supplemental Table 2. (E) HOXA9, HOXA10, and MEIS1 expression by quantitative polymerase chain reaction (qPCR) in dTAG OCI-AML3 cells after 24 hours treatment with either DMSO or dTAG 500 nM, and 72 hours treatment with either DMSO, dTAG-13 500 nM ST (24 hours dTAG-13 + 48 hours fresh medium) or dTAG-13 500 nM CT (72 hours dTAG-13). N = 3, mean ± standard error of the mean (SEM), Tukey multiple comparison test. (F) Flow cytometry quantification of CD11b, expressed as MFI FC relative to DMSO in dTAG OCI-AML3 cells on day 11 following treatment with DMSO, dTAG-13 500 nM 2 days per week or dTAG-13 500 nM 5 days per week. N = 4, mean ± SEM, Tukey multiple comparison test. (G) Histogram plot representing NPM1c-GFP levels demonstrate efficient degradation of NPM1c upon dTAG-13 treatment. (H) Histogram plot representing CD11b expression analyzed by flow cytometry on day 11 in dTAG OCI-AML3 cells treated with either DMSO, dTAG-13 500 nM 2 days per week or dTAG-13 500 nM 5 days per week. d/w, days per week; FC, fold change; H33342, Hoechst 33342; MFI, median fluorescence intensity; ns, not significant; padjust, adjusted P value; PC, principal component; Seli, selinexor.

Prolonged XPO1 inhibition is necessary to elicit significant antileukemic activity in NPM1-mutated AML in vitro. (A) Fluorescence microscopy of NPM1c-GFP OCI-AML3 treated for 18 hours with selinexor 100 nM. After 18 hours, selinexor was removed from the medium, and cells were left in culture for the following 24 hours taking pictures at 5, 7, 9, 22, and 24 hours after drug washout. Hoechst 33342 was used to stain the nuclei. Original magnification ×100; scale bar, 5 μm. (B) Principal component analysis plot derived from the means (N = 2) of the fragments per kilobase of transcript per million mapped reads (FPKM) values of parental OCI-AML3 cells collected at 24 hours treated with either DMSO or selinexor 50 nM and OCI-AML3 cells collected at 72 hours treated with either DMSO, selinexor 50 nM ST (24 hours selinexor + 48 hours fresh medium) or selinexor 50 nM CT (72 hours selinexor). (C) Volcano plots depicting differentially expressed genes in parental OCI-AML3 cells treated for 72 hours with selinexor 50 nM CT (72 hours selinexor) and 72 hours selinexor 50 nM ST (24 hours selinexor + 48 hours fresh medium), compared with DMSO. Log2FC and log10p andjusted values are shown on the x- and y-axis, respectively. Genes belonging to the HOXA (red), HOXB (blue), and MEIS/PBX (green) families are highlighted. (D) Gene set enrichment analysis of RNA-sequencing data from parental OCI-AML3 cells treated for 72 hours with selinexor CT or DMSO. Enrichment plots for “Hay_bone_marrow_neutrophils” and “Hallmark_P53_pathway” gene sets are shown. Complete Gene Ontology and Molecular Signature Database enrichment data are provided in supplemental Table 2. (E) HOXA9, HOXA10, and MEIS1 expression by quantitative polymerase chain reaction (qPCR) in dTAG OCI-AML3 cells after 24 hours treatment with either DMSO or dTAG 500 nM, and 72 hours treatment with either DMSO, dTAG-13 500 nM ST (24 hours dTAG-13 + 48 hours fresh medium) or dTAG-13 500 nM CT (72 hours dTAG-13). N = 3, mean ± standard error of the mean (SEM), Tukey multiple comparison test. (F) Flow cytometry quantification of CD11b, expressed as MFI FC relative to DMSO in dTAG OCI-AML3 cells on day 11 following treatment with DMSO, dTAG-13 500 nM 2 days per week or dTAG-13 500 nM 5 days per week. N = 4, mean ± SEM, Tukey multiple comparison test. (G) Histogram plot representing NPM1c-GFP levels demonstrate efficient degradation of NPM1c upon dTAG-13 treatment. (H) Histogram plot representing CD11b expression analyzed by flow cytometry on day 11 in dTAG OCI-AML3 cells treated with either DMSO, dTAG-13 500 nM 2 days per week or dTAG-13 500 nM 5 days per week. d/w, days per week; FC, fold change; H33342, Hoechst 33342; MFI, median fluorescence intensity; ns, not significant; padjust, adjusted P value; PC, principal component; Seli, selinexor.

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