DTPs survive lethal doses of FLT3 inhibitors by upregulating inflammatory pathways. (A) Representative images (n = 3) of FACS analysis of MV4-11 cells following treatment with 100 nM quizartinib, gilteritinib, midostaurin, and sorafenib for 48 hours. Cells are labeled with APC-Annexin-V and 7-AAD. (B) Cell-cycle analysis was performed after MV4-11 cells were treated with DMSO or 100 nM quizartinib (Quiz) for 48 hours immediately after treatment (0 days) or after recovery in drug-free media for 28 days. Surviving cells were isolated by Ficoll centrifugation, stained with propidium iodide, and analyzed by FACS. (C) DTPs were isolated by Ficoll centrifugation after 48 hours of quizartinib treatment. The surviving DTPs as well as untreated parental cells were used for quantification of dead cells (Annexin-V positive) by APC-Annexin-V and 7-AAD staining and FACS analysis following further treatment with DMSO or 100 nM quizartinib for 48 hours (data normalized to DMSO control). (D) A representative immunoblot (n = 3) showing that phosphorylation of FLT3 and STAT5, but not p42/44 MAPK, is abolished in DTPs (MV4-11 cells that survived 100 nM quizartinib treatment of the indicated times). (E) A heat map of differentially expressed genes relative to untreated parental MV4-11 cells at the indicated time points (green, downregulated; red, upregulated). Pathway analysis of downregulated (i) and upregulated (ii) genes at 48 hours after treatment with 100 nM quizartinib. RNA-sequencing was conducted in 3 independent experiments with similar results. GO, Gene Ontology.