Research ArticleCancer

Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells

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Science Translational Medicine  07 Sep 2016:
Vol. 8, Issue 355, pp. 355ra117
DOI: 10.1126/scitranslmed.aag1180
  • Fig. 1. Expression of BCL-2 proteins in Tet-off/Tet-on CML mice.

    (A) BM cells were collected from Tet-off/Tet-on mice, and the mRNA expression of Bcr-Abl, Bcl-XL, Mcl-1, and Bcl-2 was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Horizontal bars indicate the mean values. (B) SPADE tree analysis of mouse BM cell populations determined by CyTOF. (C) BCL-2, BCL-XL, MCL-1, BIM, BID, and BAX protein expression in BM cells from Tet-off and Tet-on mice determined and quantified by CyTOF. IgM, immunoglobulin M.

  • Fig. 2. Targeting leukemic cells and leukemic LSK cells in BCR-ABL–expressing mice by inhibiting BCL-2 and BCR-ABL.

    (A) BM cells obtained from Tet-off Scl-tTa–BCR-ABL/GFP CML mice were transplanted into wild-type (WT) recipient mice (0.6 × 106 cells per mouse) irradiated at 900 cGy. Mice were randomized after flow cytometric confirmation of the development of myeloproliferative disease and given a vehicle control (CON; n = 14), ABT-199 (100 mg/kg) (n = 15), nilotinib (50 mg/kg) (n = 16), or both agents (n = 16) daily by oral gavage. At the end of the 23-day treatment, three mice per group were sacrificed. BM and spleen cells were collected (arm I). The rest of the mice were followed for leukemia burden and survival (arm II). A separate set of mice was treated, and their BM cells were used for a second transplant (arm III). (B and C) BM (B) and spleen (C) (n = 3): numbers of leukemia LSK cells (GFP+LinSca-1+cKit+) in each treatment group are shown on the left, and numbers of leukemia cells (GFP+ WBCs), mature myeloid leukemia cells (GFP+Gr-1+Mac-1+), and leukemia progenitor cells (CMPs: GFP+LinSca-1cKit+CD34+FcγRII/IIIlo; GMPs: GFP+LinSca-1cKit+CD34+FcγRII/IIIhi) in each treatment group are shown on the right.

  • Fig. 3. Effect of targeting BCL-2 and BCR-ABL on leukemia in transgenic BCR-ABL–expressing mice.

    A subset of mice from Fig. 2A (arm II) underwent follow-up for examination of leukemia burden and survival. (A) Leukemia burden was assessed according to the number of total and GFP+ WBCs and neutrophils in mouse PB samples by flow cytometry at 8 and 12 weeks after treatment. (B) Survival curves for mice in the vehicle control, ABT-199, nilotinib, and combined treatment groups. D, days. Note that at the end of the 12-week treatments, three mice per group were sacrificed for additional experiments, which were not conducted. These mice were not included in the survival curves.

  • Fig. 4. The effect of combined inhibition of BCL-2 and BCR-ABL on leukemia LT-HSC frequency.

    A separate experiment was performed as described in Fig. 2A. At the end of treatment, BM cells from each group (1 × 106, 0.5 × 106, and 0.25 × 106 cells per mouse) plus 0.2 × 106 wild-type BM cells were transplanted into wild-type FBV/N recipient mice irradiated at 900 cGy (Fig. 2A; arm III). (A) PB leukemia burden at the end of treatments. (B) GFP+ PB cells obtained from mice 16 weeks after secondary transplant at 0.25 × 106 cells per mouse (left, plotted as mean ± SEM; right, individual animals). (C) PB engrafted/total transplanted mice and LT-HSC frequency in mice transplanted with BM cells from each treatment group. (D) BM GFP+ cells and Bcr-Abl mRNA expression in BM cells in mice 16 weeks after secondary transplant at 0.25 × 106 cells per mouse.

  • Fig. 5. Targeting of BCL-2 and BCR-ABL in bulk, CD34+CD38, and CD34+CD38+ leukemia cells from BC CML patients.

    (A) Cells from TKI-resistant BC CML patients (Table 1; n = 6) were treated with ABT-199, nilotinib (Nil), or both. Apoptosis in bulk, CD34+CD38, and CD34+CD38+ cells was assessed after 48 hours. (B) Normal BM cells were treated with ABT-199, nilotinib, or both. Apoptosis in CD34+ cells was assessed after 48 hours. (C) Cells from TKI-resistant BC CML patients were treated with ABT-199, nilotinib, or both for 24 hours. Protein expression in the cells was examined using immunoblot, and mRNA expression was determined using real-time RT-PCR analysis. (D) Cells from TKI-resistant BC CML patients were treated with siRNAs against Bcl-XL, Mcl-1, or both for 24 hours and then with ABT-199 for 48 hours. Cell death was then assessed. AnnV, annexin V.

  • Fig. 6. Targeting of BCL-2 and BCR-ABL in proliferating and quiescent CD34+ cells from TKI-resistant BC CML patients.

    (A) CFSE-stained cells were treated with ABT-199, nilotinib, or both. Apoptosis in proliferating (P) and quiescent (Q) CD34+ cells was assessed after 48 hours. Upper panel shows flow cytometric profiles of cells from patient 8 (Pt 8) before and after treatment. Lower panel shows the results of six treated patient samples, where each dot represents the results for one patient sample. (B) Expression of BCL-2, BCL-XL, and MCL-1 in cells from CML patient samples cocultured with MSCs (cocx) for 24 hours or 5 days was examined using immunoblot. (C) CFSE-labeled cells (Table 1; n = 6) were treated with ABT-199, nilotinib, or both with or without MSC coculture. Apoptosis was assessed in proliferating and quiescent CD34+ cells after 48 hours.

  • Table 1. Patient characteristics and in vitro treatments.

    Pt, patients; No, number; MSC, mesenchymal stromal cell; siRNA, small interfering RNA; CECA, cyclophosphamide, etoposide, carboplatin, and cytosine arabinoside; P/Q, apoptosis in proliferating and quiescent cells; WB, Western blot; % Q cells, percentage of quiescent cells over total CD34+ cells; KD, knockdown.

    Pt.
    no.
    SourceBlast
    (%)
    BCR-ABL
    status
    Treatments and
    responses
    In vitro treatment
    and assays
    % Q
    cells
    Days of
    coculture
    with MSC
    Cells in which
    apoptosis was
    determined
    WB/
    PCR
    Bcl-XL/
    Mcl-1 KD
    1PB91T315I, E255KResistant to imatinib; treated with
    chemotherapy and dasatinib
    P/QBulk/CD34+38/
    CD34+38+
    9.46
    2PB89T315I, E255VResistant to imatinib, dasatinib,
    nilotinib, and ponatinib
    P/QBulk/CD34+38
    CD34+38+
    Protein/
    RNA
    siRNAs15.97
    3PB83H396RFailed imatinib, dasatinib,
    and nilotinib
    P/QBulk/CD34+38
    CD34+38+
    siRNAs22.110
    4aPB89T315I, E255BResistant to imatinib, nilotinib,
    dasatinib, and ponatinib; treated
    with CECA, ponatinib, and dasatinib
    Bulk/CD34+38
    CD34+38+
    Protein/
    RNA
    siRNAs
    5PB80No mutationResistant to imatinib and dasatinibBulkProtein/
    RNA
    6BM93No mutationFailed imatinib; treated with nilotinibBulk/CD34+38
    CD34+38+
    4bPB62T315I, E255VResistant to imatinib, dasatinib,
    and ponatinib; treated with
    nilotinib, decitabine, and dasatinib
    P/Q7.76
    7PB24No mutationResistant to imatinib; treated
    with nilotinib
    P/Q6.88
    8PB11No mutationResistant to imatinib, dasatinib,
    nilotinib, and ponatinib; treated
    with decitabine, dasatinib, and
    bosutinib
    P/Q14.47

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/355/355ra117/DC1

    Fig. S1. Characteristics of Scl-tTa–BCR-ABL transgenic mice.

    Fig. S2. Expression of proapoptotic Bcl-2 mRNAs in BM cells of Tet-off/on CML mice and in vitro treatment of mouse BM cells.

    Fig. S3. Enumeration of BM and spleen GFPLSK cells in each group after treatment.

    Fig. S4. Targeting of BCL-2 and BCR-ABL in bulk, CD34+38, and CD34+38+ leukemia cells from TKI-resistant BC CML patients.

    Fig. S5. Targeting of BCL-2 and BCR-ABL with TKIs in bulk, CD34+38, and CD34+38+ leukemia cells from TKI-resistant BC CML patients.

    Fig. S6. Targeting of BCL-2 and BCR-ABL in proliferating and quiescent CD34+ leukemia cells from TKI-resistant BC CML patients.

    Table S1. Primers used for real-time PCR.

    Table S2. Antibody panel for CyTOF analysis.

    Table S3. Data values for Fig. 1C.

    Table S4. Data values for Fig. 2B.

    Table S5. Data values for Fig. 2C.

    Table S6. Data values for Fig. 3A.

    Table S7. Data values for Fig. 3B.

    Table S8. Data values for Fig. 4A.

    Table S9. Data values for Fig. 5A.

    Table S10. Data values for Fig. 5B.

    Table S11. Data values for Fig. 5D.

    Table S12. Data values for Fig. 6B.

    Table S13. Data values for Fig. 6C.

  • Supplementary Material for:

    Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells

    Bing Z. Carter,* Po Yee Mak, Hong Mu, Hongsheng Zhou, Duncan H. Mak, Wendy Schober, Joel D. Leverson, Bin Zhang, Ravi Bhatia, Xuelin Huang, Jorge Cortes, Hagop Kantarjian, Marina Konopleva, Michael Andreeff*

    *Corresponding author. Email: bicarter{at}mdanderson.org (B.Z.C.); mandreef{at}mdanderson.org (M.A.)

    Published 7 September 2016, Sci. Transl. Med. 8, 355ra117 (2016)
    DOI: 10.1126/scitranslmed.aag1180

    This PDF file includes:

    • Fig. S1. Characteristics of Scl-tTa–BCR-ABL transgenic mice.
    • Fig. S2. Expression of proapoptotic Bcl-2 mRNAs in BM cells of Tet-off/on CML mice and in vitro treatment of mouse BM cells.
    • Fig. S3. Enumeration of BM and spleen GFPLSK cells in each group after treatment.
    • Fig. S4. Targeting of BCL-2 and BCR-ABL in bulk, CD34+38, and CD34+38+ leukemia cells from TKI-resistant BC CML patients.
    • Fig. S5. Targeting of BCL-2 and BCR-ABL with TKIs in bulk, CD34+38, and CD34+38+ leukemia cells from TKI-resistant BC CML patients.
    • Fig. S6. Targeting of BCL-2 and BCR-ABL in proliferating and quiescent CD34+ leukemia cells from TKI-resistant BC CML patients.
    • Table S1. Primers used for real-time PCR.
    • Table S2. Antibody panel for CyTOF analysis.
    • Table S3. Data values for Fig. 1C.
    • Table S4. Data values for Fig. 2B.
    • Table S5. Data values for Fig. 2C.
    • Table S6. Data values for Fig. 3A.
    • Table S7. Data values for Fig. 3B.
    • Table S8. Data values for Fig. 4A.
    • Table S9. Data values for Fig. 5A.
    • Table S10. Data values for Fig. 5B.
    • Table S11. Data values for Fig. 5D.
    • Table S12. Data values for Fig. 6B.
    • Table S13. Data values for Fig. 6C.

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