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. 2025 Jan 15;17(1):104-115.
doi: 10.62347/IBUS3598. eCollection 2025.

Small cell lung cancer and prostate cancer cells with varying neuroendocrine differentiation markers show sensitivity to imipridone ONC201/TIC10

Elizabeth Ding  1   2 , Maximillian Pinho-Schwermann  1   2   3 , Shengliang Zhang  1   2   3 , Connor Purcell  1   2 , Wafik S El-Deiry  1   2   3   4   5
Affiliations

Small cell lung cancer and prostate cancer cells with varying neuroendocrine differentiation markers show sensitivity to imipridone ONC201/TIC10

Elizabeth Ding et al. Am J Transl Res. .

Abstract

Objectives: To investigate whether neuroendocrine differentiation (NED) markers, activation of the integrated stress response (ISR), and TRAIL pathway alter neuroendocrine tumor (NET) cell death and ONC201 sensitivity.

Methods: We conducted cell viability assays to determine ONC201 sensitivity. Western blot analysis was performed to evaluate NED, ISR, and TRAIL pathway markers. Expression levels of NED markers were compared between cell lines with and without BRN2 overexpression.

Results: Prostate cancer (PCa) and small cell lung cancer (SCLC) cell lines (N = 6) were sensitive to ONC201. Endogenous NET marker levels varied across PCa and SCLC cells. Transient BRN2 overexpression slightly reduced some NET markers while maintaining the sensitivity of PCa cells to ONC201.

Conclusions: PCa cell lines exhibit sensitivity to ONC201, with variability of NED features. These findings are relevant to the design of future studies evaluating imipridone efficacy in PCa and suggest that non-NET patients could be included in such studies.

Keywords: BRN2; ONC201; Prostate cancer; SOX2; neuroendocrine differentiation; small cell lung cancer.

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Conflict of interest statement

W.S.E-D. is a co-founder of Oncoceutics, Inc., a subsidiary of Chimerix. Dr. El-Deiry has disclosed his relationship with Oncoceutics/Chimerix and potential conflict of interest to his academic institution/employer. He is fully compliant with NIH and institutional policy managing this potential conflict of interest.

Figures

Figure 1
Figure 1
IC50 curves for PCa and SCLC cell lines with ONC201 treatment. A. PCa and SCLC cell viability after 72 h of ONC201 treatment measured with CellTiterGlo (CTG) reagent. IC50 curves of cell lines (right). Representative images of cell viability (left). B. Reported IC50 values of cell lines treated with ONC201. PCa: Prostate cancer; SCLC: small cell lung cancer.
Figure 2
Figure 2
Basal expression levels of NED and TRAIL markers in PCa and SCLC. A. Western blot of endogenous TRAIL and NED marker expression in PCa and SCLC. B. Western blot of cPARP expression in PCa and SCLC. C. Western blot of SOX2 marker expression in SCLC. For all cell lines, membranes were collected from the same set of lysates and probed with a loading control. Ran and actin were used as loading controls. NED: neuroendocrine differentiation; PCa: Prostate cancer; SCLC: small cell lung cancer.
Figure 3
Figure 3
Characterization of NED spectrum model in PCa, SCLC, and NET. Characterization was based on basal neuroendocrine differentiation marker expression levels and scientific literature. NED: neuroendocrine differentiation; PCa: Prostate cancer; SCLC: small cell lung cancer.
Figure 4
Figure 4
Early expression of TRAIL pathway proteins in response to ONC201. DU145 was treated with the IC50 value of 3.10 μM ONC201, and 22RV1 was treated with the IC50 value of 1.16 μM ONC201. DU145 and 22RV1 were treated with ONC201 for 12, 24, and 48 h. All membranes were collected from the same set of lysates and probed with a loading control.
Figure 5
Figure 5
Transient overexpression of BRN2 alters PCa sensitivity to ONC201. A-D. Dose-response curves for 72 h ONC201 treatment after BRN2 plasmid overexpression compared to the control plasmid in PC3 (top left), DU145 (top right), LNCaP (bottom left), and 22RV1 (bottom right) cell lines. BRN2 was transiently overexpressed in cells using a 0.8 ug/uL BRN2 plasmid and 0.8 ug/uL pcDNA3.1 eGFP control plasmid for 48 h before ONC201 treatment for 72 h. Cell viability was measured through the addition of CellTiterGlo (CTG) reagent. PCa: Prostate cancer.
Figure 6
Figure 6
Overexpression of BRN2 alters NED marker expression. PCa cell lines were transiently overexpressed with 0.8 μg/μL BRN2 plasmid and 0.8 μg/μL pcDNA3.1 eGFP control plasmid for 48 h. Membranes were probed with a loading control. All membranes were analyzed from one set of lysates. NED: neuroendocrine differentiation; PCa: Prostate cancer.
Figure 7
Figure 7
BRN2 overexpression in combination with ONC201 treatment alters NED marker and TRAIL pathway expression in DU145 and LNCaP. Cells were transfected with 0.8 μg/μL BRN2 plasmid and 0.8 μg/μL pcDNA3.1 eGFP control plasmid for 48 h before treating with IC50 doses of ONC201 for 48 h. The membrane was probed with a loading control and analyzed from one set of lysates. NED: neuroendocrine differentiation.
Figure 8
Figure 8
Colony formation assays of PCa cell lines following ONC201 treatment. A. Representative images of biological replicates after 7 days (top to bottom: PC3, DU145, 22RV1). B. Colony formation assay quantification for PC3, DU145, and 22RV1 cell lines (left to right) as mean ± SD. Treatment groups were compared using one-way ANOVA tests. PCa: Prostate cancer.
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