Efficacy of ONC201 in Desmoplastic Small Round Cell Tumor

Abstract

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare sarcoma tumor of adolescence and young adulthood, which harbors a recurrent chromosomal translocation between the Ewing's sarcoma gene (EWSR1) and the Wilms' tumor suppressor gene (WT1). Patients usually develop multiple abdominal tumors with liver and lymph node metastasis developing later. Survival is poor using a multimodal therapy that includes chemotherapy, radiation and surgical resection, new therapies are needed for better management of DSRCT. Triggering cell apoptosis is the scientific rationale of many cancer therapies. Here, we characterized for the first time the expression of pro-apoptotic receptors, tumor necrosis-related apoptosis-inducing ligand receptors (TRAILR1-4) within an established human DSRCT cell line and clinical samples. The molecular induction of TRAIL-mediated apoptosis using agonistic small molecule, ONC201 in vitro cell-based proliferation assay and in vivo novel orthotopic xenograft animal models of DSRCT, was able to inhibit cell proliferation that was associated with caspase activation, and tumor growth, indicating that a cell-based delivery of an apoptosis-inducing factor could be relevant therapeutic agent to control DSRCT.

Copyright © 2018. Published by Elsevier Inc.

Figures

Figure 1

TRAIL death receptor expression in DSRCT cell line in untreated cells and after treatment with ONC201. (A) Flow cytometric analysis of surface expression of decoy receptors (DcR1 and DcR2), death receptor proteins DR4 and DR5 in JN-DSRCT cells show expression of DR5 in untreated control cells. There is marked increase in DR5 expression after 72 hours of treatment with 1.66 μM of ONC201. (B) Immunofluorescence stains of DSRCT cells (left) at 40× and normal colon 20× (right) for nuclei (blue) and for DR5 (red).

Figure 2

Effects of ONC201 on DSRCT cells. (A) DSRCT Cell proliferation was assessed using MTT in vitro cell-based proliferation assay after 72-hour exposure to ONC201. Histograms represent mean values of three independent experiments. (B) Curve representative of DSRCT cell survival after 72-hour exposure to ONC201 using MTT cell-based assay. The IC50 value of DSRCT cells to ONC201 is reported in the legend. (C) Colony formation assay 10 days after exposure to variable concentrations of ONC201. (D) Columns represent mean values of three independent experiments and bars represent standard deviation for the relative colony formation of DSRCT cells after 10 days of exposure to ONC201.

Figure 3

Activation of extrinsic pathway of apoptosis by ONC201 in JN-DSRCT-1. (A) ONC-201 induces cell death in JN-DSRCT cells. Flow cytometric analysis of cell viability by propodium iodide (PI) shows marked apoptosis after 72 hours of treatment with 1.66 μM of ONC201 (B) Western blotting analysis for caspase 8, cleaved caspase 8, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP and GAPDH on lysates from DSRCT cells cultures with ONC201 (1.66 μM) for 72 hours. Western blot shows activation of the extrinsic pathway of apoptosis with increased protein expression of cleaved caspase 8, 3, and PARP after treatment with ONC201.

Figure 4

Necropsy of metastatic organs in DSRCT xenograft mouse model and histology observance. (A-E) Metastasis of the brain (A-B), liver (C-D), pancreas (E) and spleen (F) in two different mice. (G) Representative H&E analysis of pancreatic metastasis. (H) Representative H&E analysis of intraperitoneal metastasis show typical infiltrative nests of small round blue cells. Scale bars represent 200 μm in G and H.

Figure 5

In vivo imaging of DSRCT xenograft mouse model before and after ONC201 treatment. (A) In vivo bioluminescence imaging of immunocompromised NSG mice IP-implanted, with luciferase transfected JN-DSRCT cells. From left to right, one control mouse (no IP implantation, two mice IP-injected with 106 luciferase-JN-DSRCT cells, and two mice injected with 2 × 106 luciferase-JN-DSRCT cells, are visualized for tumor size via bioluminescence measurement 21 days post injection. (B) Monitoring DSRCT tumor growth and ONC201 treatment response after intraperitoneal implantation of DSRCT-luciferase cell line in immunodeficient mice. Luminescent intensity of photons emitted from each tumor from control and ONC201 treated mice after injections with 106 luciferase-JN-DSRCT cells.

Figure 6

Treatment of a new orthotopic model of DSRCT with ONC201. A-H Each panel represents the same 4 of 5 mice over time treated with high dose (100 mg/kg) of ONC 201. Panels A-E represents bioluminescent signal 2, 3, 4, 5 and 6 weeks after implantation. Panels F-H represent mice after withdrawal of treatment at weeks 7-9. H. Two mice demonstrated a complete response without rebound growth after 6 weeks of treatment and 3 weeks of drug withdrawal, one mouse had a partial response with rebound growth and one mouse had no response.

Supplementary Figure 1

Immunohistochemical analysis for DR4 (A) and DR5 (B) in two different human primary DSRCT tumors