Lgb321

The MOLM-14, MV4-11, OCI-AML2, OCI-AML3, THP-1, and HL-60 human AML cell lines were cultured in α-modified Eagle’s medium (αMEM) supplemented with 10% fetal calf serum (FCS) (47 (link)). We evaluated these cell lines for FLT3-ITD, FLT3-TKD, NPM1, n-ras, IDH1, IDH2, DNMT3A, and TP53 gene mutations (table S2). Ba/F3 cells transfected or not with ITD-mutated FLT3 with or without a TKD mutation (F691L, D835Y, S935A, or S935D) were grown in RPMI with 10% FCS, supplemented with 10% WEHI-3–conditioned medium as a source of interleukin-3 (IL-3) in the case of parental Ba/F3 cells (48 (link)). Dox (Sigma-Aldrich) was used in vitro at a concentration of 200 ng/ml in cell culture experiments (49 (link)–51 (link)) and at 200 μg/ml in mouse experiments. The endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Sigma-Aldrich) was used at 10 μM as a control for calcium mobilization. We also used AC220, PKC412, sorafenib, and crenolanib (all from Selleck Chemical) at various concentrations as indicated. AC220 was also used at 1 mg/kg in mouse experiments. Murine IL-3 and FLT3-L were purchased from PeproTech. The pan-Pim kinase inhibitor LGB321 was from Novartis Pharmaceutical (25 (link)). The construction of mammalian expression plasmids for PIM1, PIM2, Pim2, Pim2 kinase domain K61A mutant (kinase-dead or KD), and various FLT3-ITD mutants is described in the Supplementary Materials.