KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice

Abstract

KIT mutations are the most common secondary mutations in inv(16) acute myeloid leukemia (AML) patients and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis. Here, we transduced wild-type and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations. KIT transduction caused massive BM Lin(-) cell death and fewer colonies in culture that were less severe in the KI cells. D816Y KIT but not wild-type KIT enhanced proliferation in Lin(-) cells and led to more mixed lineage colonies from transduced KI BM cells. Importantly, 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, whereas no control mice died. Results from limiting dilution transplantations indicate higher frequencies of leukemia-initiating cells in the leukemia expressing mutated KIT. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis.

Figure 1

Differentiation potential of BM cells transduced with D816Y/V KIT and control vectors. (A) Total numbers of colonies from 10⁵ cultured WT (wt) or Cbfb^+/56m; Tg(Mx1-Cre) BM cells transduced with retroviral vectors containing WT KIT, KIT^D816V or GFP and sorted for GFP expression. Wt + GFP indicates WT BM cells transduced with GFP vector alone; Wt + KIT wt, WT BM cells transduced with WT KIT vector; and MxCre-CM + GFP, KIT wt, or D816Y, Cbfb^+/56m; Tg(Mx1-Cre) BM cells transduced with GFP, Wt KIT, or KIT^D816V vector, respectively. The numbers above the bars are average colony numbers for the group. (B) Ratio of total colony numbers from 10⁵ cultured WT or Cbfb^+/56m; Tg(Mx1-Cre) (MxCre-CM) BM cells transduced with GFP versus those transduced with WT KIT. (C) Types of colonies formed from transduced Cbfb^+/56m; Tg(Mx1-Cre) BM cells. CFU indicates colony-forming unit; G, granulocyte; E, erythrocyte; MM, monocyte and megakaryocyte; and GM, granulocyte and monocyte. All BM cells were sorted for GFP⁺ cells before plating. (D) Comparison of annexin V⁺ cell numbers (percentage) between the KIT (both WT and 816 mutants)–transduced (GFP⁺) and untransduced (GFP⁻) BM lin⁻ cell populations, for both WT and Cbfb^+/56m; Tg(Mx1-Cre). (E) Comparison of annexin V⁺ cell numbers (percentage) between the WT and Cbfb^+/56m; Tg(Mx1-Cre) BM lin⁻ cell populations that were transduced with WT KIT or KIT^816Y/V. (F) BrdU⁺ cells in KIT (WT and 816 mutants)–transduced BM cells.

Figure 2

Lineage contribution of KIT-transduced bone marrow cells in transplanted mice. PB cells were collected at the indicated times after transplantation from the recipient mice, stained for the indicated markers, and analyzed by FACS. (A) WT BM cells transduced with GFP. (B) WT BM cells transduced with KIT^D816. (C) Cbfb^+/56m; Tg(Mx1-Cre) BM cells transduced with GFP. (D) Cbfb^+/56m; Tg(Mx1-Cre) BM cells transduced with KIT^D816Y. (E) PB from a mouse that was starting to develop leukemia that was transplanted with Cbfb^+/56m; Tg(Mx1-Cre) BM cells expressing KIT^D816Y. CD3/4/8 indicates combination staining of CD3, CD4, and CD8.

Figure 3

Leukemia development in mice transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y/V BM cells. (A) Survival curves of transplanted mice. Black dashed line with squares indicate mice transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y BM cells (N = 13); black dotted line with triangles, mice transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816V BM cells (N = 10); gray line with diamonds, mice transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT or Cbfb^+/56m; Tg(Mx1-Cre)/GFP BM cells (N = 18); and gray dotted line, mice transplanted with KIT (both WT and D816 mutants)–transduced WT BM cells (N = 15). (B) H&E stained lung tissue (left) and Wright Giemsa–stained leukemia cells in PB (middle and right, respectively) from a Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y leukemic mouse. White arrow in the left panel indicates a cell in mitosis. Black arrow in the middle panel indicates a cell that has been enlarged in the right panel. (Imager D2, Zeiss; 20×, 40×, and 63× plan-Apochromat objective lenses; AxioVision 4.8 acquision software; AxioCam HRC, Zeiss). (C) Mitotic cell count from leukemic spleen sections of Cbfb^+/56m; Tg(Mx1-Cre) mice (MxCre-CM; N = 3) and Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y (MxCre-CM + KIT mu; N = 3) mice. (D) FACS analysis of PB cells from a leukemic mouse transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y BM cells.

Figure 4

Expression of human KIT protein in the transplanted mice. (A) Leukemic cells from spleens of mice transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816V or Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y BM cells. (B) Splenocytes from a mouse (nonleukemic) transplanted with Cbfb^+/56m; Tg(Mx1-Cre)/KIT wt BM cells. (C) Leukemia cell line ME-1. The cells in panels A-C were stained with an anti–human KIT antibody and analyzed by FACS. In panels A through C, the cells in the left-hand panels were unstained and those in the right-hand panels were stained with the anti–human KIT. Cells in the boxes are GFP⁺ and human KIT⁺. (D) Western blot of leukemic spleen cells from Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y (MxCre-CM + KIT mu) and Cbfb^+/56m; Tg(Mx1-Cre) (MxCre-CM) mice using the anti–human KIT antibody. Actin was probed as the loading control.

Figure 5

Accelerated leukemia development and increased LICs by KIT^D816V/Y. (A) Disease latency in secondarily transplanted mice with leukemic cells of Cbfb^+/56m; Tg(Mx1-Cre) (black diamonds) and Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816V/Y (gray squares). Each recipient mouse was given 10⁶ leukemia cells. (B) Leukemia incidences from limiting dilution transplantation with Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y leukemia cells (N = 4; black lines) and Cbfb^+/56m; Tg(Mx1-Cre) leukemia cells (N = 2; gray lines). Numbers of cells injected per mouse are shown on x-axis. N = 5 in each dose group. (C) Genomic Southern blot hybridization for clonality analysis. Each lane is from one secondarily transplanted mouse. Panels A, B, and C are 3 different donors (lanes not labeled had degraded DNA). (D) Leukemia cell viability in culture after PKC412 treatment. Gray line indicates Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y mice (N = 4); and black line, Cbfb^+/56m; Tg(Mx1-Cre) mice (N = 5; *P < .01).

Figure 6

Phosphorylation status of MAPK/Stat3/Stat5/Akt in leukemia cells. Western blot analysis of leukemia cells from Cbfb^+/56m; Tg(Mx1-Cre)/KIT^D816Y (MxCre-CM + KIT mut) mice and Cbfb^+/56m; Tg(Mx1-Cre) mice (MxCre-CM) and human ME-1 cells for the indicated proteins and their phosphorylated products.