Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL

Abstract

Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (T-ALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis.

Figure 1.

Fbxw7 is essential to maintain the adult hematopoietic pool. (A) Peripheral blood cell counts of Fbxw7-deficient (red closed circles, n = 15) and control (black closed circles, n = 15) mice after pIpC treatment. Results are shown as means ± SD. Red open circles indicate white blood cell counts of mice developing leukemia. (B) Absolute numbers of MNCs, Lin⁻ cells, and LSK CD34⁻ HSCs in Fbxw7-deficient and control BM at 4, 8, and 12 wk after pIpC treatment. Results are shown as means ± SD from six to eight independent experiments. (*) P < 0.05; (**) P < 0.01.

Figure 2.

Reconstitution capacity and quiescence are impaired in Fbxw7-deficient HSCs. (A) Competitive repopulation analysis. (Left) Recipient Ly5.1 mice (n = 6) were transplanted with 1500 Fbxw7-deficient or control LSK cells together with 4 × 10⁵ Ly5.1 × Ly5.2 competitor BM MNCs. Donor-derived chimerism of peripheral white blood cells was analyzed monthly after transplantation. (Right) Recipient Ly5.1 mice (n = 10) were transplanted with 4 × 10⁵ BM MNCs from Mx-1-Cre(+);Fbxw7^fl/− mice before pIpC treatment or with controls together with the same number of Ly5.1 × Ly5.2 competitor BM MNCs. pIpC treatment of recipient mice was performed 2 mo after transplantation. Donor-derived chimerism of peripheral white blood cells was then analyzed monthly after pIpC treatment. Results are shown as means ± SD. (B) Cell cycle status of LSK CD34⁻ cells of Fbxw7-deficient or control mice. BrdU was administered for 3 d to mark cells that entered S phase, and 7-amino-actinomycin D (7-AAD) was administered to detect DNA content. Data shown are representative FACS patterns derived from three independent experiments (left) and graphs showing the mean percentage of cells in G₀ (right). (C) LSK cells isolated from Fbxw7-deficient or control mice were stained with DAPI (blue) and anti-c-Myc antibody or anti-Notch1 antibody (green).

Figure 3.

The fate of Fbxw7-deficient hematopoietic cells is determined by p53 expression. (A) Peripheral blood cell counts of Fbxw7-deficient (WBC < 5000, red circle, n = 24; WBC > 5000, blue circle, n = 10) and control (gray circle, n = 13) mice 12 wk after pIpC treatment. Horizontal lines indicate mean values. Closed circles indicate the data for mice that developed leukemia ultimately. (B) Percentage of annexin V-positive apoptotic cells in Fbxw7-deficient (WBC < 5000, red bar; WBC > 5000, blue bar) and control (gray bar) BM MNCs (left) or LSK CD34⁻ HSCs (right) 12 wk after pIpC treatment. Results are shown as means ± SD from four independent experiments. (*) P < 0.05. (C) Western blot analysis of p53 in BM MNCs from control and Fbxw7-deficient mice 4 wk (left) and 12 wk (right) after pIpC treatment. Hsp70 was used as a loading control.

Figure 4.

Fbxw7 deficiency in adult hematopoietic cells leads to T-ALL. (A) Representative autopsy of leukemic Fbxw7-deficient mice. Thymus (T), liver (L), spleen (S), and lymph nodes (arrowheads) were massively swollen. (B) Representative histology of peripheral blood (PB), liver, kidney, and spleen from leukemic Fbxw7-deficient mice. (C) FACS analysis of BM MNCs and thymocytes from leukemic Fbxw7-deficient and control mice. Data shown are representative FACS patterns derived from five independent experiments. (D) Survival curves for mice injected with graded doses (1 × 10⁵, blue; 1 × 10⁶, red; or 1 × 10⁷ B, green) of leukemic Fbxw7-deficient BM MNCs. The average portion of CD4⁺CD8⁺ leukemic blasts in leukemic Fbxw7-deficient BM MNCs was 92.4 ± 3.1%. (E) Western blot analysis of Notch, c-Myc, and p53 in thymocytes from control, Fbxw7-deficient, and leukemic Fbxw7-deficient mice. (F) Survival curves for Fbxw7-deficient (red, n = 23), p53^−/−;Fbxw7-deficient (blue, n = 9), and control (black, n = 25) mice after pIpC treatment.