Inhibition of JAK/STAT signaling has strong antileukemic efficacy in AML in vitro. (A) Human AML cell lines were either mono- or cocultured with MS-5 stromal cells and treated with 2 µM ruxolitinib or vehicle for 4 days. Absolute numbers of CD45⁺ cells were normalized to monocultured control (n = 6); for each pair, the mean change in cell count is given in percentage points. (B) In all, 5 × 10⁴ THP-1 or KG-1 cells were treated with 2 µM ruxolitinib or vehicle for 4 days, and then 200 cells were transferred to methylcellulose medium (n = 4). Number of colonies and cell numbers of single colonies were determined after 10 days. Representative colonies of THP-1 cells are shown below. (C) Primary colony formation of equal numbers of viable THP-1 cells after treatment with ruxolitinib or vehicle (n = 4). (D) Primary human AML cells or PBMCs as controls were either mono- or cocultured with HS-5 cells and treated with 2 µM ruxolitinib or vehicle for 4 days. Absolute numbers of CD45⁺ cells were normalized to monocultured control (n = 18-19 different AML or control donors; each primary sample was tested in triplicate); for each pair the change in cell count is given in percentage points. (E) Primary human AML cells were either mono- or cocultured with bone marrow–derived primary human mesenchymal stem and progenitor cells (HuMSPCs) and treated with 2 µM ruxolitinib or vehicle. Absolute numbers of CD45⁺ cells were normalized to monocultured control (n = 10 different AML and 2 different HuMSPC donors; each primary AML sample was tested in triplicate); for each pair, the change in cell count is given in percentage points. (F) CD33⁺ and CD33^– PBMCs from an individual AML patient were separated by FACS and treated with 2 µM ruxolitinib or vehicle in coculture with HS-5 stromal cells for 4 days. Left, representative flow cytometry plots gated on 4′,6-diamidino-2-phenylindole (DAPI)^– single cells after treatment with 2 µM ruxolitinib or vehicle. Right, absolute cell numbers of isolated CD33⁺ and CD33^– cells after ruxolitinib or vehicle treatment normalized to control (n = 6 different AML donors; each primary sample was tested in triplicate); for each pair, the change in cell count is given in percentage points. Data are shown as mean ± SEM. See also supplemental Figure 2. *P < .05; **P < .01; ***P < .001; ****P < .0001 determined by Student t test or Wilcoxon signed-rank test. SSC, side scatter.