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Targeting BCL-2 and ABL/LYN in Philadelphia chromosome–positive acute lymphoblastic leukemia

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Science Translational Medicine  31 Aug 2016:
Vol. 8, Issue 354, pp. 354ra114
DOI: 10.1126/scitranslmed.aaf5309
  • Fig. 1.

    TKI-sensitive leukemia cells with higher BCL-2/MCL-1 ratio respond to venetoclax. (A) Dose-response curves of three leukemic cell lines known to be kinase-driven. Cells were treated with increasing concentrations of venetoclax for 72 hours, then assayed with MTS as an indirect measure of viability. Absorbance at 490 nm was measured and normalized to untreated cells, defined as 100%. RCH, black pyramids; K562, blue triangles; SUPB15, red diamonds. Each point represents the mean ± SEM (n = 3). (B) Drug sensitivity of Ph+ALL samples 12-149 and 12-225 to increasing concentrations of dasatinib (black to gray bars) and venetoclax (shades of red bars). Cells were treated with increasing concentrations of each drug over 48 to 72 hours, then assayed for viability using MTS and normalized to no-drug control. Each bar represents the mean ± SD [n = 3, with the exception of 12-225 (n = 1)]. (C) Immunoblot analysis of leukemic cells. Samples were blotted for MCL-1, BCL-2, BCL-xL, and tubulin. Samples 07-112 and 11-064 are primary TCF3-PBX1 samples. The immunoblot represents three independent experiments that were subsequently quantified for the BCL-2/MCL-1 ratio normalized to SUPB15. Each bar represents the mean ± SD (n = 3). The graph on the right shows the average ratio for Ph+ cells compared to the average ratio for TCF3-PBX1 (*P = 0.0193, unpaired two-tailed t test). (D) Dose response of RCH cells to venetoclax with MCL-1 siRNA treatment. RCH cells were treated with MCL-1 (red triangles) or nonspecific (NS) (black diamonds) siRNA, then treated with increasing concentrations of venetoclax for 72 hours and assayed by MTS. Results were normalized to no drug in the NS siRNA control. Each point represents the mean ± SEM (n = 3). A portion of the no-drug–treated cells were harvested 24 hours after siRNA treatment, separated by SDS–polyacrylamide gel electrophoresis (PAGE), and blotted for MCL-1 and tubulin (inset).

  • Fig. 2.

    Combination of venetoclax and dasatinib is synergistic. (A) Response of SUPB15 cells to increasing concentrations of dasatinib and venetoclax. Cells were treated with dasatinib and venetoclax for 72 hours, then assessed with MTS. Results were then normalized to no-drug control. The graph shows cells with no venetoclax (black circles), with 1 nM venetoclax (red pyramids), with 5 nM venetoclax (blue diamonds), with 10 nM venetoclax (green pyramids), and with 50 nM venetoclax (gray triangles). Each point represents the mean ± SEM (n = 3). (B) Isobologram analysis showing the CI values for dasatinib and venetoclax treatment of SUPB15 cells. Cells were incubated with various concentrations of dasatinib and venetoclax at fixed ratios and assessed at 72 hours via MTS. The CI values comparing the IC50 in relation to the drug, alone and in combination, were calculated and plotted by CalcuSyn (Biosoft). Points falling below the diagonal line indicate synergy. Each point represents a different concentration of the combination of dasatinib and venetoclax. (C) Isobologram analysis for the treatment of 12-149 cells with the combination of dasatinib and venetoclax. Fresh leukemic cells were treated as above and assessed with MTS, and then CI values were calculated and plotted with CalcuSyn software. Each point represents a different dose combination of dasatinib and venetoclax. (D) Time course of exposure to drug over 48 hours, assessed for apoptosis by annexin V staining. SUPB15 cells were treated with 50 nM dasatinib (blue bars), 50 nM venetoclax (red bars), or their combination (green bars) for 1, 4, 24, and 48 hours. At each time point, the sample was stained with annexin V and counted using Guava ViaCount. The dotted line indicates baseline annexin V staining in untreated cells. Each bar represents the mean ± SD (n = 3). (E) Immunoblot of SUPB15 cells treated with drugs for 4 and 24 hours. Cells were incubated with 50 nM dasatinib, 50 nM venetoclax, or their combination, and then harvested and immunoblotted for phosphorylated (p-) CRKL, cleaved (cl-) PARP, caspase 9, and tubulin.

  • Fig. 3.

    Dual inhibition of ABL and LYN in Ph+ALL enhances the effectiveness of treatment with TKI and venetoclax. (A) TKIs’ effects on apoptosis when combined with venetoclax. SUPB15 cells were treated with 5000 nM imatinib, 500 nM nilotinib, 50 nM dasatinib, or 50 nM ponatinib, alone (black bars) or in combination with 50 nM venetoclax (gray bars), for 4 hours. The samples were then stained with annexin V and analyzed. Baseline untreated annexin V staining was subtracted from each bar. Each bar represents the mean ± SD (n = 3). (B) Effect of TKIs on the expression of the proapoptotic BCL-2 family member BIM and the antiapoptotic members BCL-1 and MCL-1. Cells were treated with TKIs as in (A) for 24 hours, then immunoblotted for BIM, BCL-2, MCL-1, and tubulin. (C) Effect of the TKIs and venetoclax on other proapoptotic molecules. Cells were treated with drugs for 24 hours, then immunoblotted for BAX, BID, PUMA, NOXA, cleaved PARP, and tubulin. (D) Effect of the TKIs and venetoclax on kinase activity. SUPB15 cells were treated with the indicated drugs for 24 hours, then immunoblotted for phosphorylated and total CRKL, BTK, and LYN; tubulin was probed as a loading control. (E) Viability of SUPB15 cells after knockdown with ABL1, LYN, or BTK siRNA. Cells were treated with NS (black bar), BTK (red bar), LYN (green bar), or ABL (blue bar) siRNA for 72 hours and assessed with MTS. Results were normalized to cells treated with NS siRNA. Each bar represents the mean ± SD (n = 3). (F) Apoptosis was assessed in SUPB15 cells treated with siRNA for NS, LYN, ABL1, or ABL1 + LYN, alone or in combination with 50 nM venetoclax. Twenty-four hours after electroporation, treated cells were split to receive no treatment (black bars) or 50 nM venetoclax (gray bars) for 4 hours and assessed for annexin V staining. Each bar represents the mean ± SD (n = 3). A portion of the sample was also harvested, and the cell lysate was immunoblotted for ABL1 or LYN (inset).

  • Fig. 4.

    Combination of venetoclax and dasatinib is tolerable and efficacious in mice xenografted with Ph+ALL. (A) NSG mice engrafted with Ph+ALL cells were treated with a combination of venetoclax and dasatinib. NSG mice were injected with primary patient sample 12-225. Upon peripheral engraftment of 5 to 10% human CD45, animals were treated with the combination of dasatinib (40 mg/kg) and venetoclax (25 mg/kg) (red squares) or monitored without treatment (black circles) for up to 4 weeks. Each point represents the mean ± SEM from each animal (n = 3). Control animals quickly became moribund upon reaching about 10% chimerism and were euthanized within 1 week. At the time of euthanasia, the spleen weights were measured. Each bar in the graph on the right represents the mean ± SD weight of the spleens (n = 3; *P = 0.0158, unpaired two-way t test). (B to D) Long-term treatment with low-dose dasatinib and venetoclax. A separate group of mice were injected with patient sample 12-149. After 2 weeks, mice were treated with dasatinib (5 mg/kg daily), venetoclax (5 mg/kg daily), the combination (each drug at 5 mg/kg daily), or vehicles for 5 days each week. For the combination cohort, there was separation (for a minimum of 2 hours) between dasatinib and venetoclax treatment because of the different solubilities of the vehicle solutions. Mice were assessed for engraftment every 1 to 2 weeks and euthanized when they achieved >10% hCD45 cells in the peripheral blood or displayed any symptoms of systemic illness. (B) Kaplan-Meier curves of engraftment-free survival over 90 days for cohorts treated with control (black line, n = 4), venetoclax (blue line, n = 5), dasatinib (red line, n = 5), or the combination (5/5) (green line, n = 5). (C) Assessment of spleen weight in each cohort after harvest. Each point represents the mean ± SD weight of individual spleens (**P = 0.0018, *P = 0087, unpaired two-way t test). (D) Hematoxylin and eosin staining of bone marrow from the femurs of the treated mice. Scale bars, 100 μm.

  • Fig. 5.

    The amount of MCL-1 is regulated by LYN and STAT5 in Ph+ALL. (A) Amounts of MCL-1 and BCL-2 after drug treatment. SUPB15 and mononuclear cells from the spleen of animals engrafted with 12-149x were treated with 50 nM venetoclax, 50 nM dasatinib, or the combination for 24 hours, then harvested to quantify MCL-1, BCL-2, and tubulin. (B) The effects of combination treatment with TKIs and venetoclax on MCL-1 and BIM. SUPB15 cells were treated with 50 nM venetoclax alone or combined with 5000 nM imatinib, 500 nM nilotinib, 50 nM dasatinib, or 50 nM ponatinib for 16 hours, harvested, and immunoblotted for MCL-1, BIM, and tubulin. (C) The effects of LYN siRNA treatment on MCL-1. Twenty-four hours after electroporation of SUPB15 cells with NS or LYN siRNA, the cells were treated with 50 nM venetoclax or vehicle for an additional 24 hours, then immunoblotted for LYN, MCL-1, and tubulin. (D) STAT5 phosphorylation after treatment with TKIs. SUPB15 cells were treated with 5000 nM imatinib, 500 nM nilotinib, 50 nM dasatinib, 50 nM ponatinib, or 500 nM saracatinib for 16 hours, then immunoblotted for phospho-STAT5, total STAT5, and tubulin. (E) Immunoprecipitation with anti-LYN antibody. Cell lysate from SUPB15 cells was incubated with anti-LYN antibody or rabbit immunoglobulin G (IgG) overnight at 4°C. Antibody was recovered using Protein G Sepharose and immunoblotted for STAT5 and LYN.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/354/354ra114/DC1

    Fig. S1. Dose-response curves for SUPB15 cells treated with the combination of cytotoxic chemotherapy and venetoclax.

    Fig. S2. Dose-response curves for SUPB15 cells treated with the combination of TKI and venetoclax.

    Fig. S3. Phosphoproteome Profiler Array comparing imatinib versus dasatinib.

    Table S1. Combination indices of TKIs and cytotoxic chemotherapy with venetoclax.

  • Supplementary Material for:

    Targeting BCL-2 and ABL/LYN in Philadelphia chromosome–positive acute lymphoblastic leukemia

    Jessica T. Leonard, Joelle S. J. Rowley, Christopher A. Eide, Elie Traer, Brandon Hayes-Lattin, Marc Loriaux, Stephen E. Spurgeon, Brian J. Druker, Jeffrey W. Tyner, Bill H. Chang*

    *Corresponding author. Email: changb{at}ohsu.edu

    Published 31 August 2016, Sci. Transl. Med. 8, 354ra114 (2016)
    DOI: 10.1126/scitranslmed.aaf5309

    This PDF file includes:

    • Fig. S1. Dose-response curves for SUPB15 cells treated with the combination of cytotoxic chemotherapy and venetoclax.
    • Fig. S2. Dose-response curves for SUPB15 cells treated with the combination of TKI and venetoclax.
    • Fig. S3. Phosphoproteome Profiler Array comparing imatinib versus dasatinib.
    • Table S1. Combination indices of TKIs and cytotoxic chemotherapy with venetoclax.

    [Download PDF]

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