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. 2018 May 1;9(5):445.
doi: 10.1038/s41419-018-0478-0.

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells

Karen Legler  1 , Charlotte Hauser  2 , Jan-Hendrik Egberts  2 , Anna Willms  1 , Carola Heneweer  3   4 , Susann Boretius  3   5 , Christoph Röcken  6 , Claus-Christian Glüer  7 , Thomas Becker  2 , Michael Kluge  8   9 , Oliver Hill  8 , Christian Gieffers  8 , Harald Fricke  8 , Holger Kalthoff  1 , Johannes Lemke  10 , Anna Trauzold  11   12
Affiliations

The novel TRAIL-receptor agonist APG350 exerts superior therapeutic activity in pancreatic cancer cells

Karen Legler et al. Cell Death Dis. .

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.

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Conflict of interest statement

M.K., O.H., C.G. and H.F. are full term employees at APOGENIX AG. The remaining authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. APG350 induces cell death in PDAC cells.
PDAC cells were treated for 24 h with indicated concentrations of soluble TRAIL or APG350. Viability of PancTuI a or Colo357 b was determined using crystal violet staining. Values are means ± SD (n = 6). PancTuI c and Colo357 d were stimulated with TRAIL or APG350 (both in conc. 1.7 nM) for 24 h and the expression and cleavage of caspase-8, Bid and PARP were analyzed in whole-cell lysates by western blot. β-Actin was used as a gel-loading control. PancTuI e and Colo357 f cells were seeded into six-well plates with a density of 500 (upper part) or 5000 (lower part) cells/well, treated with 1.7 nM TRAIL or APG350 for 24 h and clonogenic survival was determined by crystal violet staining. Shown are representative results out of three independent experiments performed
Fig. 2
Fig. 2. APG350 induces pro-inflammatory signaling in PDAC cells.
PancTuI a and Colo357 c cells were treated with TRAIL or APG350 (both in conc. 1.7 nM) for 3 h. The phosphorylation of MAP kinases p38, p-JNK and ERK1/ERK2 as well as of IκBα was analyzed by western blotting in whole-cell lysates by usage of phospho-specific antibodies. As control, the total expression levels of corresponding proteins and the cellular levels of β-actin were determined in parallel. PancTuI b and Colo357 d cells were treated with indicated concentrations of TRAIL or APG350 for 24 h. IL-8 concentration in cell culture supernatants was determined by ELISA. Shown are means ± SD (n = 6)
Fig. 3
Fig. 3. Overexpression of Bcl-xL influences apoptotic and pro-inflammatory APG350-induced signaling.
a Whole-cell lysates of different PDAC cell lines were tested for the expression of Bcl-xL via western blot. *Colo357 wild type cells used for the generation of stable cell line-overexpressing Bcl-xL. **Another charge of Colo357 cells line. b–d Colo357 cells stably overexpressing Bcl-xL (Colo357/Bcl-xL) and the corresponding mock-transfected control cells (Colo357/vector) were treated with TRAIL (1.7 nM) or APG350 (1.7 nM) and the apoptotic and pro-inflammatory responses were analyzed. b Cells were treated for 24 h. Whole-cell lysates were analyzed for the expression and cleavage of caspase-8, Bid and PARP by western blot. The level of β-actin was determined in parallel and served as a gel-loading control. c Viability of the cells was determined by crystal violet staining. Values are means ± SD (n = 6). *p < 0,05. d Cells were treated for 3 h. Phosphorylation of MAP kinases and IκBα was analyzed by western blot using phosphorylation-status detecting antibodies. As an equal gel-loading control, the total levels of corresponding proteins as well as the level of β-actin were determined in parallel. e IL-8 concentration was determined by ELISA in cell culture supernatants of cells treated with TRAIL (1 nM) or APG350 (1 nM) for 24 h. Shown are means ± SD (n = 6)
Fig. 4
Fig. 4. Effects of APG350 on primary tumor growth.
PancTuI cells were orthotopically inoculated into SCID/beige mice. Four days later, mice were randomized and treated i.p. with TRAIL (3 mg/kg body weight), APG350 (3 mg/kg body weight) or PBS for 5 following days. The animals were sacrificed 33 days post op. a Tumor growth was monitored on day 12, 19, 25 and 33 post op by ultrasound imaging with mice in supine position and sagittal transducer orientation. Shown are representative time-course ultrasound images of one animal per group. b Tumor volume measured in 3D motion are shown as median with standard deviation. Significance was tested using the Mann–Whitney U-test, *p < 0.05. c Tumor weight is shown as box plots with median; top of each box, 75th percentile; bottom, 25th percentile. Significance was tested using the Mann–Whitney U-test, *p < 0.05, **p < 0.01
Fig. 5
Fig. 5. Effects of APG350 on primary tumor growth in Colo357 tumors with or without Bcl-xL overexpression.
Colo357/vector or Colo357/Bcl-xL cells were orthotopically inoculated into SCID/beige mice. Four days later, mice were randomized and treated i.p. with TRAIL (3 mg/kg body weight), APG350 (3 mg/kg body weight) or with PBS for 5 following days. The animals were sacrificed 33 days post op (Colo357/Bcl-xL-bearing mice) or 50 days post op (Colo357/vector-bearing mice). a Representative images on day 33 after tumor cell inoculation are shown. b, c Tumor volumes measured in 3D motion are shown as median with standard deviation. Significance was tested using the Mann–Whitney U-test, *p < 0.05. d, e Tumor weight are shown as box plots with median; top of each box, 75th percentile; bottom, 25th percentile. Significance was tested using the Mann–Whitney U-test, *p < 0.05
Fig. 6
Fig. 6. Effects of APG350 treatment on recurrent tumor growth and metastasis.
PancTuI cells were injected orthotopically into pancreas of SCID bg mice. Fourteen days later primary tumor resection was performed via subtotal pancreatectomy. Four days later mice were randomized and treated i.p. with TRAIL (3 mg/kg body weight), APG350 (3 mg/kg body weight) or PBS for 5 consecutive days. On day of sacrifice (day 36), tumors were removed and weighed. Metastases were macroscopically identified and counted. Representative MRI scan pictures showing recurrent tumor growth a and liver metastases b in PBS-, TRAIL- or APG350-treated animals on day 22 after primary tumor resection. c Effect of TRAIL and APG350 on PancTuI-derived tumors. Recurrent tumor weight is shown as box plots with median; top of each box, 75th percentile; bottom of box, 25th percentile. Significance was tested by Mann–Whitney U-test, *p < 0.05. d Number of mice that developed recurrent tumor only or recurrent tumor and liver metastases. e Paraffin-embedded tumors were sectioned and stained with antibodies against Ki67. Each tumor was scanned and Ki67 positive cells were evaluated per 100 tumor cells. f Correlation between recurrent tumor weight and degree of proliferation as detected by counting Ki67-positive cells. g Images showed CD31 stained tumor cells in TRAIL, APG350- or PBS-treated tissue
Fig. 7
Fig. 7. Effects of a combination of BH3-mimetics with TRAIL or APG350 on PDAC cell viability.
PancTuI a, Colo357/vector b and Colo357/Bcl-xL c were pretreated with Navitoclax or Venetoclax (both in conc. 5 µM) for 2 h and stimulated for additional 24 h with TRAIL or APG350 (both in conc. 1.7 nM). Cell viability was determined using crystal violet staining. Values are means ± SD of four independent experiments n = 6. ***p < 0.001
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