KB003 impacts in vitro decreases in proliferation and viability in monocytic precursors. (A) The flow cytometry gating strategy used to determine the viability of specific subpopulations in each CMML sample is shown. Immature myeloids were defined as CD33-positive and CD14-negative. Immature monocytes were defined as CD33-positive and CD14-positive. Myeloid progenitor cells were further analyzed using CD34 and CD38 as shown. (B-D) Ten different CMML patient samples were tested (each in duplicate) to determine the viability of CMML subpopulations in the presence of increasing doses of KB003 and GM-CSF. Only viable cells were considered in the analysis, and all data were normalized to the no drug–treated group. Myeloid subpopulations were grouped as shown, and a one-way analysis of variance was done to compare the percentage of cells within the viable gate at each dose of KB003. Significant P values represent a comparison within each subpopulation. (E) A heat map was generated using DMSO (0) and increasing doses (as shown) to each individual JAK2 inhibitor (ruxolitinib, SD-1029, CYT-387, TG-101348). The percentage of GM-CSF (10 ng/mL)–dependent pSTAT5-responsive cells as measured by flow cytometry is shown relative to the maximal GM-CSF response in the presence of DMSO (drug vehicle control). Six individual CMML patient samples were analyzed (P1-P6). (F) Dose-response curves for 5 CMML BM-MNC subsets CD33+/CD14+ (Fi) and CD33+/CD38+ (Fii) treated for 48 hours with a representative JAK2 inhibitor (SD-1029). Viability is relative to the DMSO drug vehicle control and was measured using a viability stain (DAPI) by flow cytometry.