Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
275–280
Year published:
DOI:
doi:10.1038/nbt.1607
Received
Accepted
Published online

Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML)1, 2, 3. We have previously reported4 that CD34+CD38 human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rγnull mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

At a glance

Figures

  1. Quiescent LSCs enter cell cycle after in vivo G-CSF treatment.
    Figure 1: Quiescent LSCs enter cell cycle after in vivo G-CSF treatment.

    (a,b) Representative contour plots of hCD34+CD38 LSCs in bone marrow of recipients engrafted with LSCs isolated from case 3 and 5. In a and b, histograms demonstrate nearly complete replacement of the recipient bone marrow with human AML cells (left panels). The phenotypic isolation of hCD34+CD38 LSCs is shown in the middle panels. At baseline, the majority of bone marrow LSCs are quiescent G0 cells, as demonstrated by Hoechst 33342-low PyroninY-negative phenotype (a). After in vivo G-CSF treatment, LSCs enter cell cycle as indicated by increase in PyroninY-positive cells (indicating RNA synthesis), Hoechst 33342-high cells (indicating DNA synthesis), resulting in decreased frequency of cells in G0 phase (b). (c) Recipient bone marrow LSCs in G0 phase decreased with G-CSF treatment (open circles) compared with control recipients (filled circles). Horizontal bars indicate means + s.e.m. Number of control and experimental (G-CSF) recipients, respectively, from AML case 1 (n = 7, n = 6, P = 0.0010); case 2 (n = 9, n = 6, P < 0.0001); case 3 (n = 10, n = 5, P < 0.0001); case 4 (n = 6, n = 4, P = 0.0016); case 5 (n = 5, n = 6, P = 0.0043); case 6 (n = 5, n = 5, P = 0.0050); case 7 (n = 3, n = 5, P = 0.0004); each by two-tailed t-test.

  2. Quiescent human AML cells within the bone marrow endosteal region enter the cell cycle after in vivo G-CSF treatment.
    Figure 2: Quiescent human AML cells within the bone marrow endosteal region enter the cell cycle after in vivo G-CSF treatment.

    (af) Immunofluorescence labeling for human CD45 and Ki67 in the peripheral (ac) and the central zones (df) of bone marrow from a primary humal AML cell recipient with bone marrow human AML chimerism of 98.5%. The peripheral zone contains both the endosteal and perivascular regions and the central zone contains the perivascular region, framed by white rectangles on the images. (ac) CD45+ human AML cells in the endosteal region are largely Ki67 (cell cycle quiescent), whereas in the perivascular region, CD45+ human AML cells are Ki67+ (cycling). Serial vertical sectional images through the bone section confirm that the endosteal region is nearly devoid of Ki67+ nuclei. (df) In contrast, the central zone of the bone marrow, including the perivascular region, is enriched for Ki67+ cycling human CD45+ AML cells, again confirmed by serial vertical sectional images. (gi) Immunofluorescence labeling for human CD45 and Ki67 in the peripheral zone of bone marrow from a recipient with bone marrow human AML chimerism of 97.8%. The peripheral zone contains both the endosteal and perivascular regions, framed by white rectangles on the images. CD45+ human AML cells in the endosteal region enter cell cycle after in vivo cytokine treatment, as demonstrated by their Ki67 expression. Serial vertical sectional images through the bone section confirm that nuclei of human CD45+ AML cells are adjacent to the endosteum express Ki67. (j,k) Three-dimensional reconstruction of recipient bone sections was performed using serial sectional images. At steady state, the AML cells adjacent to the bone marrow endosteum are cell cycle quiescent (Ki67) (j). After in vivo G-CSF treatment, AML cells within the bone marrow endosteal region became Ki67+, indicating that they had entered the cell cycle. CD45 (red), Ki67 (green), DAPI (blue) and merged images are shown (k). (a,d,g) 20× magnification, scale bars, 50 μm. (d,e,h) 40× magnification, scale bars, 20 μm.

  3. Cell cycle entry potentiates chemotherapy-induced apoptosis of LSCs in vivo, reduces LSC frequency and leads to superior survival of secondary recipients.
    Figure 3: Cell cycle entry potentiates chemotherapy-induced apoptosis of LSCs in vivo, reduces LSC frequency and leads to superior survival of secondary recipients.

    (a) Reduced LSC survival as measured by percent active caspase-3 cells in bone marrow hCD34+CD38 LSCs from recipients after chemotherapy with cell cycle induction (open circles) compared with chemotherapy alone (filled circles). Horizontal bars indicate means + s.e.m. Number of control and experimental (G-CSF) recipients, respectively, from AML case 1. Number of recipients receiving chemotherapy alone or chemotherapy with cell cycle induction, respectively, from AML case 1 (n = 3 each, P = 0.0127); case 2 (n = 4 each, P = 0.0006); case 3 (n = 7, n = 4, P = 0.0001); case 4 (n = 4 each, P < 0.0001); case 5 (n = 4 each, P = 0.0005); case 6 (n = 5 each, P < 0.0001); case 7 (n = 5 each, P = 0.0003); each by two-tailed t-test. (b) HE and TUNEL staining of bone sections from recipients after chemotherapy alone reveal apoptosis in the central region of the bone marrow whereas cells abutting the endosteum remain viable (*). In contrast, bone marrow of recipient following chemotherapy with cell cycle induction shows apoptosis in the endosteal region (+) in addition to the central zone. Scale bars, 10 μm. (c) Peripheral blood hCD45+ AML cell engraftment time course of serial transplant recipients at graft doses of 2 × 102, 2 × 103, 2 × 104 and 2 × 105 hCD34+ AML cells, from 6–24 weeks post-transplantation. The findings are summarized in Supplementary Table 4. Blue and red symbols and lines, respectively, indicate recipients of bone marrow hCD34+ cells from Ara-C alone- and Ara-C post-cytokine–treated AML-engrafted mice. Case 1, []; case 2, []; case 3, []; case 4, []; case 5, []; case 6, [●]; case 7, []. (d) From AML-engrafted mice receiving either chemotherapy alone or chemotherapy with cell cycle induction, bone marrow hCD34+ cells were harvested and retransplanted at doses of 2 × 102, 2 × 103, 2 × 104 and 2 × 105 into new recipients that were followed for 24 weeks. Recipients of bone marrow hCD34+ cells from AML-engrafted mice treated with chemotherapy with cell cycle induction (red) demonstrated higher overall survival compared with recipients of mouse bone marrow hCD34+ cells (blue) treated with chemotherapy alone, as estimated by the Kaplan-Meier method (P < 0.0001 for comparison of recipients within a given graft dose and for all recipients combined). (e,f) Central role of cell cycle-quiescent human primary AML LSCs in AML relapse and induction of cell cycle entry as a therapeutic strategy targeting LSCs. (e) In the past, disease relapse was attributed to the inability to completely eliminate AML cells by chemotherapy. (f) The current model maintains that chemotherapy-resistant LSCs that survive and repopulate the bone marrow prompt AML relapse. One mechanism through which LSCs successfully evade chemotherapy is their cell cycle quiescence within the bone marrow endosteal niche. Induction of cell cycle entry sensitizes these LSCs to chemotherapy, leading to reduced potential for relapse.

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Author information

Affiliations

  1. Research Unit for Human Disease Models, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.

    • Yoriko Saito,
    • Nahoko Suzuki,
    • Mariko Tomizawa-Murasawa,
    • Akiko Sone,
    • Yuho Najima,
    • Shinsuke Takagi,
    • Yuki Aoki &
    • Fumihiko Ishikawa
  2. Department of Hematology, Toranomon Hospital, Tokyo, Japan.

    • Naoyuki Uchida,
    • Shinsuke Takagi,
    • Atsushi Wake &
    • Shuichi Taniguchi
  3. Nippon Becton Dickinson Company, Tokyo, Japan.

    • Satoshi Tanaka
  4. The Jackson Laboratory, Bar Harbor, Maine, USA.

    • Leonard D Shultz

Contributions

Y.S., L.D.S. and F.I. designed the study, analyzed the data and wrote the manuscript. N.U., A.W. and S. Taniguchi provided clinical samples, information and discussion. Y.S., S. Tanaka, M.T.-M., N.S., A.S. and F.I. performed the experiments. S. Takagi and Y.A. analyzed the data. Y.N. analyzed the data and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (9M)

    Supplementary Figs. 1–5 and Supplementary Tables 1–5

Movies

  1. Supplementary Movie 1 (9M)

    This movie shows a cross-sectional view through a three-dimensional reconstruction of the recipient femur shown in Fig. 2j. The bone section was labeled for human CD45 (red), Ki67 (green) and DAPI (blue). The serial cross-sectional images along the long axis of the bone demonstrate that there is little to no Ki67 expression by hCD45+DAPI+ AML cells in the BM endosteal region adjacent to the bone at baseline.

  2. Supplementary Movie 2 (8M)

    This movie shows a cross-sectional view through a three-dimensional reconstruction of the recipient femur shown in Fig. 2k. The bone section was labeled for human CD45 (red), Ki67 (green) and DAPI (blue). The serial cross-sectional images along the long axis of the bone demonstrate the appearance of Ki67+hCD45+DAPI+ cells within the BM endosteal region following G-CSF treatment.

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