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WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death

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Science Advances  21 Jun 2017:
Vol. 3, no. 6, e1700090
DOI: 10.1126/sciadv.1700090
  • Fig. 1 WNT antagonists synergize with taxanes.

    (A) Vantictumab (VAN) has greater tumor growth inhibition when combined with nab-paclitaxel than gemcitabine. OMP-PN13 was treated with vantictumab (25 mg/kg) every 2 weeks and with gemcitabine (gem; 25 mg/kg) or nab-paclitaxel (nab-pac; 30 mg/kg) every week (n = 7 to 9 per group). OMP-PN25 was treated with vantictumab (25 mg/kg) every 2 weeks and with gemcitabine (20 mg/kg) or nab-paclitaxel (15 mg/kg) every week (n = 5 to 10 per group). mAb, monoclonal antibody. (B) Ipafricept (IPA) promotes tumor growth inhibition when combined with weekly gemcitabine and nab-paclitaxel. Left: OMP-PN9 was treated with ipafricept (10 mg/kg) and gemcitabine (50 mg/kg) every week (n = 9 to 10 per group). Right: OMP-PN9 treated with ipafricept (25 mg/kg) every 2 weeks and gemcitabine (5 mg/kg) combined with nab-paclitaxel (10 mg/kg) every week (n = 7 to 8 per group). (C) Ipafricept results in greater tumor growth inhibition in combination with nab-paclitaxel than carboplatin in ovarian cancer. OMP-OV19 was treated with ipafricept (45 mg/kg) every 2 weeks and carboplatin (30 mg/kg) or nab-paclitaxel (7.5 mg/kg) every week (n = 8 per group). *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM.

  • Fig. 2 Nab-paclitaxel arrests cells at mitosis and promotes mitotic cell accumulation of β-catenin.

    Pancreatic adenocarcinoma OMP-PN13 was treated with gemcitabine and/or nab-paclitaxel every week with and without vantictumab (25 mg/kg) every 2 weeks for 6 weeks. (A) Tumors on day 41 were preserved as formalin-fixed, paraffin-embedded (FFPE), IHC was performed for pHH3 or β-catenin, and bound primary antibody was detected with horseradish peroxidase (HRP)–labeled secondary antibody with hematoxylin counterstain. Positive regions are labeled in brown, and nuclei are labeled in blue. Slides were imaged on an Aperio AT scanner with ×20 magnification. Representative images for control, gemcitabine, and nab-paclitaxel single agents are shown. (B) Nab-paclitaxel enriches for nuclear β-catenin. FFPE sections were costained for pHH3 and β-catenin and then with fluorescently labeled secondary antibodies, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were taken on an Olympus confocal microscope (magnification, ×100). (C) FFPE sections costained for pHH3 and β-catenin. Quantifications of cells expressing total β-catenin and pHH3-positive mitotic cells with β-catenin expression were performed. Total β-catenin was detected with HRP–3,3′-diaminobenzidine (DAB), and pHH3 was detected with alkaline phosphatase (AP)–Warp Red, with hematoxylin counterstain. Digital scans were performed on an Aperio AT scanner with ×20 magnification and were analyzed with Definiens Tissue Studio. *P < 0.05 combination versus chemotherapy. Data are means with four replicates.

  • Fig. 3 Ipafricept induces mitotic catastrophe when dosed sequentially before paclitaxel.

    Administration of vantictumab or ipafricept 2 days before paclitaxel is essential for blocking tumor growth. (A) UM-PE13 treated with vantictumab (25 mg/kg) on day 1 or 4 with paclitaxel (pac; 20 mg/kg) given on day 1 or 4 in 3-week cycles (n = 6 to 8 per group). (B) OMP-OV38 treated with ipafricept (25 mg/kg) on day 1 or 3 with paclitaxel (20 mg/kg) on day 1 or 3 in 2-week cycles (n = 9 per group). (C) OMP-OV19 treated with ipafricept (25 mg/kg) on day 1 and paclitaxel (20 mg/kg) on day 1 or 3 in 2-week cycles (n = 8 to 10 per group). *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM. (D) Tumors from OMP-OV38 as shown in (B) from study day 51 were processed as FFPE with IHC for pHH3 and β-catenin. β-Catenin was detected with HRP-DAB, and pHH3 was detected with AP–Warp Red, with hematoxylin counterstain (magnification, ×20). (E) Tumors from OMP-OV38 as shown in (B) from study day 51 were processed to RNA, and quantitative real-time polymerase chain reaction (PCR) was performed. Gene expression was normalized to control group using the relative quantification method. Data are means + SEM, four biological replicates per group; *P < 0.05, significant compared to paclitaxel.

  • Fig. 4 Ipafricept blocks selection of WNT active cell types by paclitaxel in ovarian cancer.

    (A) Ipafricept is active when dosed either 1 or 2 days before paclitaxel. Ipafricept dosed 2 days before paclitaxel promotes mitotic catastrophe. Ipafricept (25 mg/kg) was dosed in 2-week cycles on the first day of each cycle. Paclitaxel (20 mg/kg) was dosed every 2 weeks, either on day 2, 3, or 5 of the cycle. Tumors at the end of study were paraffin-embedded and imaged for pHH3 and β-catenin (magnification, ×20). *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM; n = 8 to 10 per group. Q2W, once every 2 weeks. (B) Ipafricept dosed 2 days before paclitaxel, in 2-week cycles, prevents the accumulation of drug-resistant, WNT pathway–active ovarian tumor cells. Ipafricept (25 mg/kg) was dosed on days 1, 15, and 29 (blue arrows), and paclitaxel (20 mg/kg) was dosed on days 3, 17, and 31 (red arrows). Time course study: four to five tumors per treatment group per time point; time points were from days 2 to 7, 14, 21, 28, and 35, relative to first ipafricept dose on day 1. IHC performed from FFPE whole-tumor sections for pHH3, Aurora B, and LEF1. Digital scans were performed on an Aperio AT scanner and analyzed with Definiens Tissue Studio. Positive tumor nuclei from the control group of each time point were normalized to “1,” where a fold change of 1 implies no change, and the treatment groups are displayed as fold change as compared to the control from each corresponding time point and are mean + SEM; *P < 0.05 versus control. Representative images are from study day 35 (magnification, ×20).

  • Fig. 5 Sequential dosing of vantictumab and paclitaxel potentiates mitotic cell death in breast cancer.

    (A) OMP-B90 and UM-PE13 breast tumors respond to sequential vantictumab followed by paclitaxel. For OMP-B90, vantictumab (25 mg/kg) was administered on day 1 in 2-week cycles, for a total of six cycles. Paclitaxel (10 mg/kg) was given on day 1 or 3 in weekly cycles. For UM-PE13, vantictumab (25 mg/kg) was given on day 1 or 3, every other week, whereas paclitaxel (10 mg/kg) was given on day 3 every week. *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM; n = 6 to 9 per group. (B) Vantictumab promotes P21 induction and blockade of cyclin expression in OMP-B90. Four tumors from each treatment group were collected on day 5 of the sixth cycle and processed as FFPE. Dual IHC was performed for β-catenin (HRP-DAB; brown) and pHH3 (AP–Warp Red), with hematoxylin counterstain. IHC was performed for P21, cyclin E2, and cyclin B1 (HRP-DAB; brown) with hematoxylin counterstain. Digital scans were performed on an Aperio AT scanner and analyzed with Definiens Tissue Studio. *P < 0.05 combination versus chemotherapy. Data are means with four replicates.

  • Fig. 6 Sequential ipafricept followed by paclitaxel reduces the paclitaxel resistant CSC population.

    (A) OMP-OV40 was treated with ipafricept (45 mg/kg on days 1, 15, and 29) for three cycles total and/or with paclitaxel (15 mg/kg on days 5, 12, 19, 32, and 39). Thirteen days after the last ipafricept dose and 3 days after the last paclitaxel dose, tumors were processed into single cells for tumorigenicity study. *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM; n = 9 per group. (B) For the tumorigenicity study, tumors were collected from four mice per treatment group, as shown in (A), and these four tumors were mixed together to prepare the cells for the limiting dose serial transplantation assay in (B). Tumor cells were injected into recipient mice at two cell doses, 100 and 1000 cells (n = 9 to 10 mice per cell dose). The number of tumors that grew to more than 100 mm3 by day 78 after injection was used to calculate the CSC frequency. Data are means + SEM; P < 0.01, significant to control.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1700090/DC1

    fig. S1. Nab-paclitaxel arrests cells at mitosis and synergizes with WNT antagonists.

    fig. S2. Higher doses of WNT antagonists administered infrequently are more active than corresponding dosages administered weekly.

    fig. S3. Ipafricept induces mitotic catastrophe when dosed sequentially before paclitaxel.

    fig. S4. Ipafricept blocks selection of WNT-active cell types by paclitaxel in ovarian cancer.

    fig. S5. Paclitaxel up-regulates Aurora B and pHH3 by 24 hours up to 72 hours.

    table S1. PDX tumors.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Nab-paclitaxel arrests cells at mitosis and synergizes with WNT antagonists.
    • fig. S2. Higher doses of WNT antagonists administered infrequently are more active than corresponding dosages administered weekly.
    • fig. S3. Ipafricept induces mitotic catastrophe when dosed sequentially before paclitaxel.
    • fig. S4. Ipafricept blocks selection of WNT-active cell types by paclitaxel in ovarian cancer.
    • fig. S5. Paclitaxel up-regulates Aurora B and pHH3 by 24 hours up to 72 hours.
    • table S1. PDX tumors.

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