ERK-D domain targeting inhibited cellular proliferation of JAK2V617F-dependent cells. (A) Space filling model of ERK2 and the D docking domain (orange pocket region) complexed with a MAPK docking peptide (MKNK1, blue colored structure). (B) Selective inhibition of ERK2-mediated phosphorylation of RSK by D-domain inhibitor (#76). SCID.adh cells were pretreated with U0126 or #76 inhibitor and then stimulated with PMA. Immunoblot analysis of protein extracts was performed, using the following antibodies: anti-EGR1, anti–phospho-RSK, anti-RSK, anti–phospho-ERK, anti- ERK, and anti–β-actin, which served as a loading control. (C-D) Colony forming analysis was performed on JAK2V617F-transduced HSPCs treated with vehicle control, U0126 or #76. Cells (3 × 10³) were plated on M3434 medium and cultured for 7 days. The number of colonies was scored and compared. Data are expressed as the mean ± standard deviation (SD). **P < .01. (E) Human SET-2 cells expressing JAK2V617F were treated with vehicle control, U0126 (10 µM), or #76 at the indicated doses. Cell viability and proliferation were measured each day for 3 days and normalized to untreated cells at day 0 and are expressed as the mean ± SD. **P < .01; ***P < .001. (F) Human SET-2 cells were implanted subcutaneously into Il2rg^−/−Rag2^−/− mice and left to reach a size of 40 mm³ before treatment every other day by intraperitoneal injection with vehicle control (dimethyl sulfoxide) or #76 at 10 mg/kg. Tumor size and cell recovery upon tumor disaggregation are expressed as the mean ± SD (n = 6 per condition). Results in panels B-E were derived from at least 3 independent experiments.