The HDACi panobinostat demonstrates therapeutic efficacy in a mouse model of A/E9a;Nras^G12D-driven but not M/E;Nras^G12D-driven AML. (A) Western blot analysis of whole-cell lysates prepared from cell lines K562 and Kasumi-1 and spleen cells (spl) isolated from a WT and an A/E9a;Nras^G12D leukemia recipient mouse using an antibody to AML1 (upper panel). Membrane was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (bottom panel). (B) Flow cytometry analysis of c-Kit and Mac-1 expression in GFP-positive spleen cells isolated from an A/E9a;Nras^G12D leukemia recipient mouse. A representative flow cytometry plot is shown. (C) Immunoprecipitation/western blot analysis of the interaction between A/E9a and HDAC1 in A/E9a;Nras^G12D leukemic cells treated with DMSO (D) or 16 nM panobinostat (P) for 6 hours. A control mouse immunoglobulin G (IgG) and antibody to AML1 (AML) were used for immunoprecipitation; antibodies to HDAC1 and AML1 were used for western blotting. The results shown are representative of 3 independent experiments. (D-F) Total white blood cells (WBC), flow cytometry analysis of leukemic cells in peripheral blood, and BLI of C57BL/6 mice bearing A/E9a;Nras^G12D tumors treated with panobinostat or vehicle using the standard treatment regimen. In panels D and E, each data point represents an individual mouse, and horizontal bars represent mean value. ***P < .0001. (G) Kaplan-Meier survival curves of treated A/E9a;Nras^G12D leukemia recipient mice following initiation of therapy (n = 6 for vehicle, n = 5 for panobinostat; median survival benefit 84 days, P = .0006). Dotted line indicates final day of treatment. (H) BLI of M/E;Nras^G12D leukemia recipient mice treated with panobinostat or vehicle using the standard treatment regimen. (I) Kaplan-Meier survival curves of treated M/E;Nras^G12D leukemia recipient mice following initiation of therapy (n = 6 for vehicle, n = 6 for panobinostat; median survival benefit 6 days, P = .0015).