Gilteritinib-induced upregulation of S100A9 is mediated through loss ofBCL6 enrichment at the S100A9 promoter. (A) Putative hits with a greater than twofold change in the S100A8 promoter (left) and S100A9 promoter (middle) from the transcription factor screen. The overlapping hits with a greater than fivefold change resulted in RUNX2 and BCL6 (right). (B) Relative luciferase activity in a promoter assay with luciferase-expressing vectors containing S100A8/A9 promoters or empty vector and transfected with BCL6 (promoter assay; n = 4). In all graphs, error bars represent the standard deviation. (C) BCL6 enrichment at the S100A8 or S100A9 promoter in MOLM13 cells treated with 10 nM gilteritinib or dimethyl sulfoxide (DMSO) for 24 hours. Enrichment was normalized to input (chromatin immunoprecipitation assay; n = 4). (D) Relative luminescence in MOLM13 cells treated with 1 µM BI-3802 for 24 and 48 hours. Data were normalized to DMSO at 24 hours (CellTiter-Glo Assay; n = 3). (E) Expression of S100A8 and S100A9 by RT-PCR in MOLM13 cells treated with 10 nM gilteritinib or 1 µM BI-3802 for 24 hours (n = 3). (F) Immunoblot of BCL6 and S100A9 expression in MOLM13 cells treated with 10 nM gilteritinib ± 1 µM BI-3802 for 24 hours. Representative image of 2 separate experiments. The immunoblot was captured on film and normalized to the glyceraldehyde phosphate dehydrogenase (GAPDH) control. (G) Cell growth assay of MOLM13 cells pretreated with BI-3802 (1 µM; 24 hours) followed by cotreatment with gilteritinib (10 nM) for 48 hours. Data were normalized to DMSO at 24 hours (n = 9). *P < 0.05; **P < 0.01; ***P < 0.001, by 2-tailed, unpaired Student t test.