C-KIT with D816Y/V Mutations Cooperate with CBFB-MYH11 to Accelerate Leukemogenesis in Mice.

Abstract 274

CBF leukemia is a group of acute myeloid leukemia (AML) caused by the fusion genes involving core binding factors (CBFs), which include RUNX1 and CBFB. C-KIT mutations are common in CBF leukemias. For example, 10–45% of AML cases with inv(16), which generates a fusion gene between CBFB and MYH11, carry activating mutations in c-KIT. In general, the presence of c-KIT mutations in inv(16) AML suggests poor clinical prognosis. Therefore, it is important to verify that c-KIT mutations cooperate with CBFB-MYH11, and to investigate the underlying mechanism during leukemogenesis. Here, we transduced bone marrow (BM) cells from conditional Cbfb-MYH11 knockin mice with retroviral vectors carrying c-KIT mutations,D816V and D816Y (located in the kinase domain of c-KIT and can cause constitutive activation of c-KIT), which are common in inv(16) AML. Wild type c-KIT and empty retroviral vectors were used as controls. The transduced BM cells were then transplanted to sub-lethally irradiated recipient mice. In 2–5 months, 60% of mice carrying D816Y and 50% of mice carrying D816V c-KIT mutations developed leukemia, while none of the mice transduced with the control vectors developed leukemia up to one year after transplantation. In addition, BM cells from wildtype mice transduced with the c-KIT mutant vectors did not induce leukemia >5 months after transplantation. Analysis of signaling pathways revealed that Stat3-serine phosphorylation and P44/42 MAPK pathways, but not AKT and Stat5 pathways, were activated (phosphorylated) in the leukemia cells from the Cbfb-MYH11 knockin mice expressing mutated c-KIT. In vitro colony forming assays indicated that Cbfb-MYH11 BM cells expressing c-KIT D816V/Y mutants produced similar number of colonies as Cbfb-MYH11 BM cells transduced with wild type c-KIT control vector. However, they had increased CFU-G and CFU-GEMM and decreased CFU-E colonies, suggesting that the c-KIT mutants drove Cbfb-MYH11 BM cells towards an earlier progenitor state. Secondary transplantation with 1×106 leukemia cells from donors with c-KIT mutations showed an average life span of three weeks compared to six weeks from donors without c-KIT mutation. Further limiting dilution transplantation with 100 leukemia cells from one donor with c-KIT mutation led to lethal leukemia in 4 out of 5 mice 2.5 months after transplantation, indicating a very high frequency of leukemia initiating cells in this sample. These data suggest a strong cooperation between the mutant c-KIT and Cbfb-MYH11 for leukemogenesis in mice. This mouse model can also serve as a clinically relevant model for small chemical screening and the development of novel therapeutic approaches.