ONC201 Exerts p53-Independent Cytotoxicity Through TRAIL and DR5 Induction In Mantle Cell Lymphomas
Authors: Ishizawa Jo , Kojima Kensuke , Dilip Archana , Ruvolo Vivian R , Carter Bing Z , Allen Joshua E , Neelapu Sattva S. , McDonnell Timothy J. , Talekar Mala Kiran , El-Deiry Wafik S. , Kwak Larry W. , Andreeff Michael 
Published: October 2019

Abstract
ONC201 (TIC10) is a novel small molecule that induces TRAIL-dependent apoptosis in various cancer cell types and is under development to enter a first-in-man study in advanced cancer patients .It was identified in a screen for small molecules capable of up-regulating endogenous TRAIL gene transcription in a p53-independent manner (Allen JE et al, Sci Transl Med., 2013). ONC201 triggers FOXO3a activation through dual inhibition of ERK and AKT, which transcriptionally upregulates TRAIL and TNFRSF10B (TRAIL-R2/DR5) in solid tumors. Because PI3K/AKT and MEK/ERK activation have been shown to be major contributors to drug resistance, ONC201 is potentially promising since it not only promotes TRAIL activation, but also upregulates its pro-apoptotic receptor DR5.
Here we report the anti-lymphoma effects of ONC201 in MCL, a presently incurable disease. We treated three human MCL cell lines with wild-type p53 (Z-138, JVM-2, and Granta-519) and two similar lines with mutant p53 (MINO and Jeko-1) with ONC201. A 72-hour ONC201 treatment induced apoptosis in all MCL cell lines. Surprisingly, the p53 mutant MINO and Jeko-1 cells were more susceptible in apoptosis assays to ONC201 than cells with wild-type p53 (Fig.1) The effective concentrations inducing cell killing (as measured by annexin V positivity) in 50%/75% of the cells in the Z-138, JVM-2, MINO, Jeko-1, and Granta-519 cells were 9.9/>10, >10/>10, 2.6/5.2, 2.7/4.6 and >10/ >10 micromolar, respectively. We also treated five primary human MCL samples (three with wild-type p53 and two with mutant p53), and found that one of the two mutant p53 samples was highly sensitive to ONC201 as were the three samples with wild-type p53. One mutant p53 sample that was less sensitive to ONC201, was also resistant to Nutlin-3a and Ibrutinib suggesting an extremely drug-resistant phenotype. Real-time PCR analysis revealed that both DR5 and TRAIL mRNAs were transcriptionally upregulated in the primary MCL samples (a relative ratio of 7.25 compared to 3.13 in controls) after 72-hour treatment with ONC201.
To determine the significance of p53 functional status in ONC201-induced apoptosis, p53 wild-type Z-138 and JVM-2 cells were stably transduced with lentivirus encoding either negative control shRNA or p53-specific shRNA and were exposed to ONC201 and results demonstrated complete p53-independence. Normal human bone marrow cells and mesenchymal stem cells were completely resistant to the cytotoxic effects of ONC201, which illustrated this agent's low toxicity against normal tissues.
In order to examine the role of p53 activation in ONC201-induced apoptosis in MCL cells, we combined ONC201 with the MDM2 inhibitor Nutlin-3a. The combination cytotoxic effects of this combination were synergistic in p53 wild-type Z-138 and JVM-2 cells (combination index 0.87 and 0.63, respectively). Similar synergistic effects of ONC201 combined with the BTK inhibitor Ibrutinib were observed in Z-138 and MINO cells (combination index 0.63 and 0.61, respectively). This combination also triggered synergistic apoptotic effects in two primary MCL samples with combination indexes of 0.0011 and 0.073, respectively.

Conclusion
ONC201 induces p53-independent apoptosis in MCL cells, and may have significant clinical impact by targeting both p53 wild type and p53 mutant drug-resistant MCL cells. ONC201 exerts synergistic effects with MDM2 and BTK inhibitors that may be explored clinically.

Disclosures:
Allen: Drug Company: Employment. Andreeff:Oncoceutics: SAB Other.