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ONC201 Selectively Induces Apoptosis in Cutaneous T-cell Lymphoma Cells via Activating Pro-Apoptotic Integrated Stress Response and Inactivating JAK/STAT and NF-κB Pathways

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ONC201 Selectively Induces Apoptosis in Cutaneous T-cell Lymphoma Cells via Activating Pro-Apoptotic Integrated Stress Response and Inactivating JAK/STAT and NF-κB Pathways

Xiao Ni et al. Oncotarget.

Abstract

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4+ malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V+ cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4+ malignant T cells, not in normal CD4+ T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Keywords: NHL; ONC201; TIC10; TRAIL; cancer.

Conflict of interest statement

CONFLICTS OF INTEREST R.S.T., J.E.A. and W.O. are employees and shareholders of Oncoceutics.

Figures

Figure 1
Figure 1. ONC201 inhibits cell growth in CTCL cell lines
H9, HH, Hut78, Mac2A, MJ, MyLa, PB2S, and SeAx cells were cultured in 96-well culture plates (5×104/well) with or without 1.25, 2.5, 5.0, and 10.0 μM of ONC201 for 48, 72, and 96 hrs, respectively. Cell viability was determined using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS). Data for 8 cell lines were presented with different doses at 3 time points (mean ± SD of triplicate determinations). *significant difference, p < 0.05.
Figure 2
Figure 2. ONC201 induces apoptosis in CTCL cell lines
H9, HH, Hut78, Mac2A, MJ, MyLa, PB2S, and SeAx cells (5×105/ml) were treated with or without 1.25, 2.5, 5.0, and 10.0 μM ofONC201 for 48, 72, and 96 hrs. Apoptotic cells were assessed by flow cytometry using the Annexin V-FITC Detection Kit. Data were presented as the percentage of Annexin V+ cells for all 8 cell lines with different doses at 3 time points (mean ± SD of triplicate determinations). *significant difference, p < 0.05.
Figure 3
Figure 3. ONC201 induces accumulation of sub-G1 portions in CTCL cell lines
HH, Hut78 and MJ cells (5×105) were treated with or without 1.25, 2.5, 5.0, and 10.0 μM of ONC201 for 48, 72, and 96 hrs. Cells were stained with PI, and sub-G1 distributions were determined by flow cytometry. (A) The percentages of sub-G1 portions for HH, Hut78, and MJ cells were presented with different doses at 3 time points (mean ± SD of triplicate determinations). *significant difference, p < 0.05. (B) Plots for representative paired cells with or without 5.0 μM of ONC201 for 96 hrs were presented. The % of sub-G1portion in each plot was indicated.
Figure 4
Figure 4. ONC201 selectively induces apoptosis in CD4+ malignant T cells
CD4+ T cells (5×105) from MF/SS patients and healthy donors were treated with or without 1.25, 2.5, 5.0, and 10.0 μM of ONC201 for 48 and 72 hrs. Apoptotic cells were assessed by flow cytometry using the Annexin V-FITC Detection Kit. (A) The percentages of Annexin V+ cells were presented for normal CD4+ T cells (mean, n=6) and malignant CD4+ T cells (mean, n=5; Patient #1 - #5) with different doses at two time points. (B) Dot plots for representative CD4+ T cells from a healthy donor (left) and Patient#5 with or without 5.0 μM of ONC201 for 72 hrs were presented.
Figure 5
Figure 5. ONC201 upregulates ATF4, downregulates Akt, and induces TRAIL in CTCL cell lines and primary Sézary cells
HH, Hut78, and MJ cells (A), and PBMCs from 3 MF/SS patients (Patient #6 - #8) (B) were treated with (1.25 or 5.0 μM) or without ONC201 for 72 hrs. The protein expression of eIF2α, p-eIF2α, ATF4, Akt, TRAIL, BAX, and C-PARP were assessed by western blot. The protein level of β-actin served as housekeeping gene control. All protein levels were semi-quantified using ImageJ system (NIH), and the levels in treated cells were compared with untreated control cells which were considered as 100%.
Figure 6
Figure 6. ONC201 downregulates JAK/STAT in CTCL cell lines and primary Sézary cells
HH, Hut78, and MJ cells (A), and PBMCs from 3 MF/SS patients (Patient #6 - #8) (B) were treated with (1.25 or 5.0 μM) or without ONC201 for 72 hrs. The protein expression of JAK3, pJAK3, STAT3, pSTAT3, pSTAT1, IRF7, and pIRF7 were assessed by western blot. Protein levels were semi-quantified as above, and the protein levels in treated cells were compared with untreated control cells which were considered as 100%.
Figure 7
Figure 7. ONC201 downregulates NF-κB and IL-32 expression in CTCL cell lines and primary Sézary cells
HH, Hut78, and MJ cells (A), and PBMCs from 3 MF/SS patients (Patient #6 - #8) (B) were treated with (1.25 or 5.0 μM) or without ONC201 for 72 hrs. The protein expression of NF-κB members and IL-32β were assessed by western blot. The protein level of β-actin served as housekeeping gene control. The protein levels were semi-quantified and the levels in treated cells were compared with untreated control cells which were considered as 100%.

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