Asxl1^−/− promotes ERK hyperactivation and CMML-like phenotypes in Nras^G12D/+ mice. Control (C; Vav-Cre), Asxl1^−/− (Asxl1^−/−), Nras^G12D/+ (Nras), and Nras^G12D/+;Asxl1^−/− (NA) mice were euthanized at age 6 weeks. Quantification of (A) spleen weight, (B) white blood cells (WBCs) in peripheral blood, and (C) numbers of monocytes and neutrophils in peripheral blood. (D-E) Frequencies and absolute numbers of (D) LSK cells and (E) MP cells in bone marrow (BM; including femurs and tibias) and spleen (SP). (F-G) Cell cycle analysis of (F) LSK cells and (G) MP cells from bone marrow using Ki67 and 4′,6-diamidino-2-phenylindole (DAPI). (H) A total of 5 × 10⁴ bone marrow cells were plated in duplicate and cultured in semisolid medium with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). Colony numbers were counted after 7 days in culture. (I) Bone marrow cells were serum- and cytokine-starved for 2 hours at 37°C. Cells were then stimulated with or without 2 ng/mL of murine GM-CSF for 10 minutes at 37°C. Levels of pERK1/2 were measured using phospho-flow cytometry in Lin^–/lowc-Kit⁺ cells. Data are presented as mean + standard deviation (SD). *P < .05; **P < .01; ***P < .001.