c-Kit mutations functionally cooperate with loss of Dnmt3a in vivo. (A) Growth rate of mouse 32D cells transduced with WT c-Kit, c-Kit^V750M, or c-Kit^D814V. Data represent mean ± SEM of 3 independent experiments. (B) Growth rate of mouse 32D cells transduced with WT c-Kit, c-Kit^V750M, or c-Kit^D814V in the presence of the c-Kit ligand SCF (10 ng/mL). Data represent mean ± SEM of 3 independent experiments. (C) Western blot to assess c-Kit signaling transducers Jnk1 and Jnk2 in 32D cells in the absence of IL-3 and SCF. Constitutive phosphorylation of Jnk1 and Jnk2 in c-Kit^V750M and c-Kit^D814V cells indicates ligand-independent signaling indicative of gain-of-function mutations. (D) Methocult serial replating of control and Dnmt3a-KO bone marrow progenitors transduced with c-Kit variants. In a Dnmt3a-KO background, c-Kit^D814V produced significantly more colonies in the second plate. Data represent mean ± SEM of 3 independent experiments. (E) Flow cytometry profiles of secondary Methocult colonies showing Gr-1⁻ Mac-1⁻ gated cells. c-Kit^D814V drives mast cell (Gr-1⁻ Mac-1⁻ c-Kit⁺ FcεR1⁺) proliferation, whereas c-Kit^V750M maintains an immature progenitor phenotype (Gr-1⁻ Mac-1⁻ c-Kit⁺ FcεR1⁻ Sca-1⁺ CD150⁺) in a Dnmt3a-KO background. (F) Survival curve of mice transplanted with control or Dnmt3a-KO bone marrow progenitors transduced with c-Kit^D814V. (G) Bone marrow flow cytometry plots of mice transplanted with Dnmt3a-KO c-Kit^D814V reveal 3 distinct pathologies: B-ALL, T-ALL, and mastocytosis with myeloid blasts. (H) Peripheral blood lineage distribution of surviving mice (6 months posttransplant) transplanted with control or Dnmt3a-KO c-Kit^V750M showing expansion of B cells only in mice receiving Dnmt3a-KO c-Kit^V750M cells. Transplant data are compiled from 2 independent cohorts. * P < .05, ** P < .01, *** P < .001.