Figure 1.
Gilteritinib induces S100A8/A9 expression and subsequently confers gilteritinib resistance. (A) Expression of S100 proteins in MOLM13 and MOLM13-RES xenograft models treated with vehicle or gilteritinib, 30 mg/kg once daily for 5 days per week (n = 8-10 mice per treatment cohort). AML cells were isolated from bone marrow at the study end point and analyzed by RNA-seq. (B) Expression of S100A8 and S100A9 by RT-PCR in cells obtained from the MOLM13 and MOLM13-RES xenograft models to validate the RNA-seq data. (C) Immunoblot of S100A8 and S100A9 expression in the MOLM13-RES xenograft model treated with vehicle or gilteritinib. Representative image of 3 to 4 samples per cohort. An immunoblot analysis was performed on Li-Cor Odyssey Fc, quantitated by Image Studio Software, and normalized to the actin control, as indicated above each blot. (D) Expression of S100A8 and S100A9 by RT-PCR in FLT3-ITD+ cells treated with 10 nM gilteritinib for 24 hours (n = 3). In all graphs, error bars represent the standard deviation. (E) Immunoblot of S100A8 and S100A9 expression in MOLM13 and MOLM13-RES cells treated with dimethyl sulfoxide (DMSO) or 10 nM gilteritinib for up to 24 hours. Representative blot of 3 separate experiments. An immunoblot was captured on Li-Cor Odyssey Fc, quantified by Image Studio Software, and normalized to actin control and then DMSO for 1 hour, as indicated above each blot. (F) Intracellular free-calcium assay of FLT3-ITD+ AML cell lines treated with 10 nM gilteritinib for 24 hours (n = 9). Cell growth assay of S100A9 overexpressed (G) or knocked down (H) in MOLM13 cells. One representative growth curve from 3 separate experiments (n = 3). Data were normalized to vector DMSO (G) or EV DMSO (H) at 24 hours. Statistical analysis was performed between the overexpression construct and vector or NT and shA9. (I) Cell growth assay of MOLM13 cells treated with 100 nM PLX51107 and 10 nM gilteritinib, separately or in combination. One representative growth curve from 3 separate experiments (n = 3). Data were normalized to DMSO at 24 hours. Statistical analysis was performed between the combination and gilteritinib-alone treatment groups. (J) Human primary AML cells (patient sample 370) with the indicated mutations were treated with DMSO, 150 nM gilteritinib, 500 nM PLX51107, and 2.5 µM tasquinimod, separately or in combination for 96 hours. Data were normalized to DMSO (CellTiter-Glo assay; n = 3 replicates). Statistical analysis was performed between the combination and gilteritinib-alone treatment groups. *P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant, by 2-tailed, unpaired Student t test. EV, empty vector; Gilt, gilteritinib; NT, non-targeting short hairpin RNA control; PLX, PLX51107; shA9, S100A9 short hairpin RNA; TasQ, tasquinimod; RFU, relative fluorescence units.

Gilteritinib induces S100A8/A9 expression and subsequently confers gilteritinib resistance. (A) Expression of S100 proteins in MOLM13 and MOLM13-RES xenograft models treated with vehicle or gilteritinib, 30 mg/kg once daily for 5 days per week (n = 8-10 mice per treatment cohort). AML cells were isolated from bone marrow at the study end point and analyzed by RNA-seq. (B) Expression of S100A8 and S100A9 by RT-PCR in cells obtained from the MOLM13 and MOLM13-RES xenograft models to validate the RNA-seq data. (C) Immunoblot of S100A8 and S100A9 expression in the MOLM13-RES xenograft model treated with vehicle or gilteritinib. Representative image of 3 to 4 samples per cohort. An immunoblot analysis was performed on Li-Cor Odyssey Fc, quantitated by Image Studio Software, and normalized to the actin control, as indicated above each blot. (D) Expression of S100A8 and S100A9 by RT-PCR in FLT3-ITD+ cells treated with 10 nM gilteritinib for 24 hours (n = 3). In all graphs, error bars represent the standard deviation. (E) Immunoblot of S100A8 and S100A9 expression in MOLM13 and MOLM13-RES cells treated with dimethyl sulfoxide (DMSO) or 10 nM gilteritinib for up to 24 hours. Representative blot of 3 separate experiments. An immunoblot was captured on Li-Cor Odyssey Fc, quantified by Image Studio Software, and normalized to actin control and then DMSO for 1 hour, as indicated above each blot. (F) Intracellular free-calcium assay of FLT3-ITD+ AML cell lines treated with 10 nM gilteritinib for 24 hours (n = 9). Cell growth assay of S100A9 overexpressed (G) or knocked down (H) in MOLM13 cells. One representative growth curve from 3 separate experiments (n = 3). Data were normalized to vector DMSO (G) or EV DMSO (H) at 24 hours. Statistical analysis was performed between the overexpression construct and vector or NT and shA9. (I) Cell growth assay of MOLM13 cells treated with 100 nM PLX51107 and 10 nM gilteritinib, separately or in combination. One representative growth curve from 3 separate experiments (n = 3). Data were normalized to DMSO at 24 hours. Statistical analysis was performed between the combination and gilteritinib-alone treatment groups. (J) Human primary AML cells (patient sample 370) with the indicated mutations were treated with DMSO, 150 nM gilteritinib, 500 nM PLX51107, and 2.5 µM tasquinimod, separately or in combination for 96 hours. Data were normalized to DMSO (CellTiter-Glo assay; n = 3 replicates). Statistical analysis was performed between the combination and gilteritinib-alone treatment groups. *P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant, by 2-tailed, unpaired Student t test. EV, empty vector; Gilt, gilteritinib; NT, non-targeting short hairpin RNA control; PLX, PLX51107; shA9, S100A9 short hairpin RNA; TasQ, tasquinimod; RFU, relative fluorescence units.

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