Research ArticleCancer

PP2A inhibition sensitizes cancer stem cells to ABL tyrosine kinase inhibitors in BCR-ABL+ human leukemia

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Science Translational Medicine  07 Feb 2018:
Vol. 10, Issue 427, eaan8735
DOI: 10.1126/scitranslmed.aan8735
  • Fig. 1 LB100 and LB102 specifically inhibit PP2A phosphatase activity and the growth of BCR-ABL+ cells.

    (A) The inhibitory effects of LB100 and LB102 on phosphatase activity of protein phosphatase 2A (PP2A) were measured in the presence of various concentrations of either LB100 or LB102 for 15 min in a phosphatase assay. (B) K562 cells were treated with 5 μM LB100/LB102, and PP2A phosphatase activity was measured at the time points indicated using a PP2A immunoprecipitation (IP) phosphatase assay kit (left). K562 and K562 imatinib mesylate–resistant (K562-IMR) cells were treated with 5 μM LB100/LB102, and BV173 cells were treated with 2.5 μM LB100/LB102 and then assayed for phosphatase activity after 12 hours (right). (C) K562 and K562-IMR cells were treated with IM (0.5 μM) with or without LB100/LB102 (2.5 or 5 μM) for 48 hours and assayed for cell viability. The cell lines were treated with 0.5 μM IM (K562) or 5 μM IM (K562-IMR) with or without 5 μM LB100/LB102 and assayed for cell proliferation (D) and apoptosis (E). The fraction of apoptotic cells was determined by annexin V+ staining. (F) Representative Western blot analysis of total and cleaved caspase 8 and caspase 3 in K562 cells after 24 and 48 hours of treatment. Data are mean and SEM from three independent experiments. P values were calculated using paired two-tailed Student’s t test. Ctrl, untreated cells.

  • Fig. 2 Inhibition of PP2A disrupts cell cycle control and induces mitotic arrest in CML cells.

    (A) Representative fluorescence-activated cell sorting (FACS) plots showing cell cycle distribution in K562 and K562-IMR cells after treatment with 5 μM LB100 alone or in combination with IM (0.5 and 5 μM, respectively). Arrows indicate enhancement in the G2-M population after LB100 treatment. (B) Representative confocal microscopy images of K562 cells stained with an anti–α-tubulin antibody (green) and DNA binding dye 4′,6-diamidino-2-phenylindole (DAPI) (blue) in the presence or absence of 5 μM LB100. Scale bars, 10 μm. (C) Percentage of noncatastrophic cells after treatment with 5 μM LB100 or 5 μM LB102. Values were determined by counting the number of catastrophic cells, which displayed disrupted spindles with multipolar cell division, in PP2A inhibitor–treated cells compared to control cells. At least 100 cells per treatment group were counted in the field of view, and the percentages of noncatastrophic cells were calculated and expressed as percentages of untreated control cells. Data are mean and SEM from three independent experiments. P values were calculated using paired two-tailed Student’s t test.

  • Fig. 3 Combination treatment with PP2A inhibitors and IM disrupts phosphorylation and expression of key signaling proteins.

    K562-IMR cells were treated with 5 μM IM and 5 μM LB100/LB102, either alone or in combination for 24 or 48 hours, and cell lysates were harvested for Western blotting of (A) key signaling proteins after 48 hours and (B) phosphorylation of specific β-catenin residues after 24 and 48 hours. Representative Western blots from three repeats are shown. (C) RNA from the treated cells was used for quantitative reverse transcription polymerase chain reaction analysis of β-catenin downstream target genes cyclin D1 (CCND1), MYC, TCF1, and LEF1 in K562-IMR cells after 48 hours of treatment. (D) Schematic diagram of how BCR-ABL–mediated tyrosine phosphorylation (Y86) and PP2A-mediated threonine and serine dephosphorylation (T41, S45) change in response to tyrosine kinase inhibitors (TKIs) and PP2A inhibitors in chronic myeloid leukemia (CML) cells. Data are mean and SEM from three independent experiments. P values were calculated using paired two-tailed Student’s t test. ERK, extracellular signal–regulated kinase; JNK, c-Jun N-terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 4 Knockdown of PP2A-C sensitizes K562-IMR cells to IM treatment.

    K562-IMR cells were transduced with short hairpin RNA (shRNA) targeting the PP2A catalytic C subunit (shPP2A-C) or scrambled shRNA (Scr) and treated with 5 μM IM and 5 μM LB100, either alone or in combination, for 48 hours. After 48 hours, some of the cells were used to make cell lysates for Western blotting [representative blot is shown in (A), whereas the rest of the cells were used for cell viability and apoptosis assays (B)]. Data are mean and SEM from three independent experiments. P values were calculated using paired two-tailed Student’s t test. (C) Distinct Abelson helper integration site–1 (AHI-1) prey proteins, including PP2A subunit B (PR55α, P63151), identified by IP coupled to mass spectrometry (IP-MS) in 293T cells transfected with two different human AHI-1 constructs: N-terminal FLAG-tagged AHI-1 (N-AHI-1) and C-terminal FLAG-tagged AHI-1 (C-AHI-1). The percent sequence coverage, statistical confidence [log(e)], and the number of unique peptides observed are presented; the results are from three replicate IPs.

  • Fig. 5 PR55α, a B subunit of PP2A, and β-catenin interact with AHI-1.

    (A) PR55α was immunoprecipitated from cell lysates of 293T cells transfected with a human influenza hemagglutinin (HA)–tagged full-length AHI-1 construct (left) and BaF3 cells stably transduced with full-length AHI-1 (right). The immunoprecipitates were probed with anti–AHI-1 antibodies and an anti-PR55α antibody. Mouse serum was used as an immunoglobulin G (IgG) control to replace a specific IP antibody as a negative control. Western blots (WB) shown are representative of three independent experiments. (B) HA was immunoprecipitated from 293T cells overexpressing HA-tagged full-length AHI-1. The immunoprecipitates were probed with either anti–β-catenin or anti–AHI-1 antibody. (C) AHI-1 was immunoprecipitated from cell lysates of BCR-ABL–transduced BaF3 cells or BCR-ABL–transduced BaF3 cells overexpressing full-length AHI-1. The immunoprecipitates were then probed with either anti–β-catenin or anti–AHI-1 antibody. (D) Schematic diagram of how targeting the AHI-1–mediated complex by dual inhibition of PP2A and BCR-ABL disrupts several key signaling molecules and activities of PP2A/β-catenin and downstream target genes, leading to reduced survival and increased TKI response of drug nonresponder cells.

  • Fig. 6 Combination treatment with PP2A inhibitors and TKIs effectively targets CD34+ CML cells.

    CD34+ normal bone marrow (NBM, n = 3) and CML cells from IM nonresponders (n = 5) were cultured for 72 hours with single and combination treatments of 5 μM IM, 150 nM dasatinib (DA), and 2.5 μM LB100/LB102. After 72 hours, (A) viability and (B) apoptotic cells were measured. (C) CD34+ NBM and CML cells from IM nonresponders were plated in colony-forming cell (CFC) assays with single and combination treatments of TKIs and PP2A inhibitors. CFCs were expressed as a percentage of colonies from untreated control cells. (D) Long-term culture-initiating cell (LTC-IC) assays were performed from CD34+ CML cells obtained from the same CML patient samples that were used to perform CFC assays with or without indicated inhibitors, alone or in combination. DMSO, dimethyl sulfoxide.

  • Fig. 7 Treatment with LB100 and DA reduces engraftment of leukemic blast cells and prolongs survival of leukemic mice.

    (A) BV173-YFP (yellow fluorescent protein) cells were intravenously injected into sublethally irradiated interleukin-2 receptor γ-chain–deficient (NSG) mice. Treatment with DA or LB100, alone or in combination, was initiated 2 weeks after transplantation for 2 weeks. A noninvasive bioluminescent imaging assay was performed 1 week after the end of treatment, and representative images from each treatment group (out of 6 to 8 mice) are presented. (B) One mouse from each group was sacrificed, and hematopoietic tissues were collected for analysis 7 weeks after transplantation. Images and weights of spleen and liver from each mouse are shown. (C) Hematoxylin and eosin staining of spleen and liver of mice with or without treatments. Arrows indicate areas where infiltrated leukemic cells are found. Scale bar, 0.1 mm. (D) Fold difference in BCR-ABL transcripts in BM, spleen, and liver of treated groups compared to vehicle-treated control mice. (E) Representative bioluminescent images from a negative control mouse (ctrl), a DA-treated mouse, and a mouse that received the combination (out of 4 to 8 mice per group) 10 weeks after transplantation. Mice from vehicle and LB100 treatment groups had already died. (F) FACS plots of engraftment of human leukemic cells (YFP+CD19+) in BM and spleen from mice in each treatment group and percentages of these double-positive cells. (G) Images and weights of spleen and liver from each mouse. (H) Fold difference in BCR-ABL transcripts from BM and spleen of treated mice compared to vehicle-treated controls. (I) Western blots of whole protein extracts from the BM of mice treated with vehicle, DA, or DA plus LB100. (J) Overall survival of leukemic mice treated with vehicle, LB100 (1.5 mg/kg), and/or DA (15 mg/kg), alone or in combination once a day for 2 weeks. Median survival with the combination of LB100 and DA versus LB100 alone was 83 days versus 52 days, ratio = 0.65, 95% confidence interval (CI) = 0.226 to 1.879, P = 0.0011; versus DA alone, 71 days, ratio = 0.924, 95% CI = 0.298 to 2.865, P = 0.0184. Ctrl, mice without injection of BV173-YFP cells, as a negative control. Vehicle, mice injected with BV173-YFP cells treated with propylene glycol, as a positive control. ND, not detectable.

  • Fig. 8 LB100 enhances the efficacy of DA against engrafted BCR-ABL+ stem cells in a BCR-ABL transgenic mouse model.

    (A) Experimental design using the SCLtTA/TRE-BCR-ABL transgenic mouse model. (B) A group of mice was sacrificed and analyzed 6.5 weeks after transplantation. Images and weights of spleen and liver from each mouse are shown. (C) FACS plots from harvested BM showing gating strategies for detection of stem cells [long-term (LT)–HSCs; CD45.2+linSca1+cKit+Flt3CD48CD150+]. The absolute cell numbers calculated for LT-HSCs (red) and short-term (ST)–HSCs (black) per sample are indicated. (D) FACS plots from peripheral blood collected at the end point, when mice displayed >20% weight loss, increased white blood cell counts (3- to 5-fold), breathing difficulties, reduced alertness/responsiveness, lethargy, etc., showing BCR-ABL+ granulocytes (CD45.2+Gr1+Mac1+) with percentage of total CD45.2+ cells (black) and percentage of Gr1+Mac1+ cells (red) outlined. (E) Overall survival of mice treated with vehicle, LB100 (1.5 mg/kg), and/or DA (15 mg/kg), alone or in combination. Median survival with the combination versus LB100 alone was >82 days versus 60 days, and that with the combination versus DA alone was >82 days versus 75 days, as indicated in the figure.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/427/eaan8735/DC1

    Materials and Methods

    Fig. S1. Identification of a growth inhibitory compound CAN in AHI-1–transduced CML cells.

    Fig. S2. Synergistic cytotoxicity in CML cells by combination of PP2A inhibitors with IM.

    Fig. S3. Disruption of cell cycle control and abnormal formation of mitotic spindles in CML cells by PP2A inhibitors.

    Fig. S4. Reduced phosphorylation and expression of signaling proteins in K562 cells after treatment with PP2A inhibitors and IM.

    Fig. S5. Effects of PP2A inhibitors and IM on CD34+ NBM cells.

    Fig. S6. Analysis of mice treated with LB100, DA, or a combination, 7 weeks after transplantation.

    Fig. S7. Analysis of mice treated with LB100, DA, or a combination, 10 weeks after transplantation.

    Fig. S8. Analysis of progenitor populations in the BM of BCR-ABL transgenic mice.

  • Supplementary Material for:

    PP2A inhibition sensitizes cancer stem cells to ABL tyrosine kinase inhibitors in BCR-ABL+ human leukemia

    Damian Lai, Min Chen, Jiechuang Su, Xiaohu Liu, Katharina Rothe, Kaiji Hu, Donna L. Forrest, Connie J. Eaves, Gregg B. Morin, Xiaoyan Jiang*

    *Corresponding author. Email: xjiang{at}bccrc.ca

    Published 7 February 2018, Sci. Transl. Med. 10, eaan8735 (2018)
    DOI: 10.1126/scitranslmed.aan8735

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification of a growth inhibitory compound CAN in AHI-1–transduced CML cells.
    • Fig. S2. Synergistic cytotoxicity in CML cells by combination of PP2A inhibitors with IM.
    • Fig. S3. Disruption of cell cycle control and abnormal formation of mitotic spindles in CML cells by PP2A inhibitors.
    • Fig. S4. Reduced phosphorylation and expression of signaling proteins in K562 cells after treatment with PP2A inhibitors and IM.
    • Fig. S5. Effects of PP2A inhibitors and IM on CD34+ NBM cells.
    • Fig. S6. Analysis of mice treated with LB100, DA, or a combination, 7 weeks after transplantation.
    • Fig. S7. Analysis of mice treated with LB100, DA, or a combination, 10 weeks after transplantation.
    • Fig. S8. Analysis of progenitor populations in the BM of BCR-ABL transgenic mice.

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