ONC201 is the founding member of a novel class of anti-cancer compounds called imipridones that is currently in Phase II clinical trials in multiple advanced cancers. Since the discovery of ONC201 as a p53-independent inducer of TRAIL gene transcription, preclinical studies have determined that ONC201 has anti-proliferative and pro-apoptotic effects against a broad range of tumor cells but not normal cells. The mechanism of action of ONC201 involves engagement of PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt/ERK inactivation, and consequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animals models, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors confirmed that ONC201 is exceptionally well-tolerated and established the recommended phase II dose of 625 mg administered orally every three weeks defined by drug exposure comparable to efficacious levels in preclinical models. Clinical trials are evaluating the single agent efficacy of ONC201 in multiple solid tumors and hematological malignancies and exploring alternative dosing regimens. In addition, chemical analogs that have shown promise in other oncology indications are in pre-clinical development. In summary, the imipridone family that comprises ONC201 and its chemical analogs represent a new class of anti-cancer therapy with a unique mechanism of action being translated in ongoing clinical trials.

BRCA1 and BRCA2 are involved in control of DNA repair by homologous recombination (HR). Germline mutations in these genes substantially increase lifetime risk of developing breast, ovarian and other cancers. BRCA-deficient tumors show increased sensitivity to therapies that target defective HR. Sporadic tumors lacking germline BRCA mutations but sharing the molecular features of BRCA-mutant tumors may respond to these types of therapies as well. PARP inhibitors represent a form of targeted therapy for HR-deficient tumors that have been approved in ovarian cancer and are being tested in clinical trials in breast cancer, metastatic castration-resistant prostate cancer, and glioblastoma. Our lab has previously identified a small molecule called ONC201 in a screen for compounds capable of inducing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Our lab has previously reported that ONC201 induces an integrated stress response that involves ATF4/CHOP and inactivates pro-survival kinases Akt and ERK. The latter leads to decreased phosphorylation of transcription factor FOXO3a, its nuclear translocation, and induction of transcription of the target gene TRAIL. A first-in-human clinical trial showed that the compound is well tolerated, achieves a therapeutic pharmacokinetic profile, exhibits biological activity. It has since entered multiple phase I/II trials. ONC201 has anti-proliferative and pro-apoptotic effects in a wide range of tumor types including BRCA-deficient breast and ovarian cancers (n=10), with GI50 values in the low micromolar range. Treatment with ONC201 induces surface TRAIL and inhibits Akt activity in BRCA deficient breast and ovarian cancers. PARP inhibitors have been previously shown to upregulate DR5 through transcription factor CHOP, sensitizing solid tumors to TRAIL. Resistance to PARP inhibitors can occur through PI3K/Akt pathway activation, and PI3K/MEK blockade improves their anti-tumor effects. We observed synergy between ONC201 and PARP inhibitors olaparib and rucaparib in BRCA-deficient breast and ovarian cancer cell lines in cell viability assays with combination indices (CI) ranging from 0.4 - 0.8. Robust synergy was also observed in prostate cancer and glioblastoma cells. The mechanisms of the observed synergy are currently under investigation. These results indicate that ONC201 possesses single agent activity in BRCA-deficient cancer cells and that the combination of ONC201 with PARP inhibitors represents a promising synergistic therapeutic approach that could be exploited in multiple solid tumors. Citation Format: Marie D. Baumeister, Ozan C. Küçükkase, Varun V. Prabhu, David T. Dicker, Josh E. Allen, Wafik S. El-Deiry. ONC201 shows efficacy in BRCA-deficient cancer cells and synergy with PARP inhibitors in glioblastoma, breast, prostate, and ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3212. doi:10.1158/1538-7445.AM2017-3212

ONC201, a novel first-in-class, orally active anti-tumor agent that upregulates the cytotoxic ligand TRAIL (Allen et al., Sci. Trans. Med., 2013; Wagner et al., Oncotarget, 2014), activates the integrated stress response leading to tumor cell upregulation of TRAIL death receptor 5 (Kline et al., Sci. Sig., in press). ONC201 is active against bulk tumor and cancer stem cells (Prabhu et al., Can. Res., 2015). ONC201 is under clinical development by Oncoceutics, and is being evaluated in multiple phase I/II clinical trials. Results of the first-in-human ONC201 study presented at the 2015 AACR-NCI-EORTC meeting (Stein et al., Abstract C138) demonstrated ONC201 to be safe in humans, to exhibit predicted PK and sustained PD characteristics, and revealed a preliminary efficacy signal. As patients were dosed on an every 3-week schedule, based on supportive preclinical data, we investigated dose-intensification of ONC201 to determine whether a higher dose/frequency schedule might impact efficacy with limited toxicity. We hypothesized that ONC201 may be effective in dose-intensified schedule and may inhibit metastases. We tested a range of ONC201 doses including 25, 50, and 100 mg/kg and frequencies including every 4, 3, 2, 1 week as well as twice a week dosing. In colon and triple-negative breast cancer we observed that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo. Frequency effects were more pronounced at lower doses and dose-dependency was more impactful with less frequent schedules. We noted a potent anti-metastasis effect of ONC201 in vivo, not previously reported, as a function of both increased ONC201 dose and frequency of administration. ONC201 inhibits cancer cell migration and invasion in vitro in a TRAIL-dependent manner. We found ONC201 more potently suppresses Akt and ERK in tumors in vivo in a dose- and frequency-dependent manner, whereas its effect on TRAIL serum levels appeared to be impacted by frequency. We observed accumulation of CD3+/NK1.1+ cells within ONC201-treated tumors in athymic nude mice that lack T-cells. Accumulation of CD3+/NK1.1 cells within ONC201-treated tumors was more pronounced with dose intensification that correlated with superior efficacy. In summary, we have uncovered a potent anti-metastasis effect of ONC201 coupled with the appearance of CD3+/NK1.1+ cells within ONC201-treated tumors. We are further evaluating the biomarker characteristics and immune function of the CD3+/NK1.1+ cells and the relationship of their intra-tumoral accumulation to observed anti-tumor effects of ONC201, including in fully immunocompetent mice. Our results suggest that clinical investigation of both dose and frequency of ONC201 administration is warranted and is being evaluated in an ongoing clinical trial (NCT02609230). Citation Format: Jessica Wagner, Christina L.B. Kline, Marie Baumeister, Wafik S. El-Deiry. Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3000.

Breast cancer is a major cause of cancer related death in United States women, and there is an urgent need for novel targeted therapies with low toxicity and increased efficacy. The TRAIL pathway has been of interest in the field of oncology, but resistance has been identified in cancer cell lines and primary tumors including those of the breast. Only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL, with other breast cancer molecular subtypes being resistant (Rahman et al., Breast Cancer Res. Treat., 2008). Clinical translation of these findings has been limited, as TRAIL receptor agonistic antibodies have shown minimal effects. We identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10 (Allen et al., Sci. Trans. Med., 2013) that functions through dual inhibition of AKT/ERK, induction of the ATF4/CHOP pathway (Kline et al., Submitted, 2015) and cytotoxic effects dependent on TRAIL/DR5 upregulation. Recently, ONC201 completed its first-in-man phase 1 clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-EORTC meeting). The objective of this work was to investigate ONC201 efficacy in TNBC (TRAIL-sensitive) and non-TNBC (TRAIL-resistant) cells. Using cell viability assays, we demonstrate IC50 values for ONC201 in the low micromolar range for both TNBC (n = 6) and non-TNBC cells (n = 5). These doses are achievable based on human PK. The evaluated non-TNBC cell lines, including those with estrogen receptor positivity and HER2 amplifications, have been shown to be highly resistant to TRAIL therapy. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in both TNBC and non-TNBC cells. We also show that a single dose of ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 triple negative human breast cancer xenograft model. The effects of ONC201 on well-known mechanisms of TRAIL-resistance, such as increases in inhibitor of apoptosis (IAP) family proteins, were investigated to explain sensitivity of TRAIL-resistant breast cancer cells to ONC201. We observed that ONC201 mediates a decrease in expression of IAP family proteins XIAP, c-IAP1, and c-IAP2 across TNBC and non-TNBC cell lines. We have shown that XIAP levels correlate with sensitivity to ONC201-induced apoptosis (Kline et al., Submitted, 2015). We also examined levels of death receptors DR4 and DR5 using flow cytometry, as a known mechanism of TRAIL resistance involves constitutive death receptor endocytosis from the cell surface (Zhang et al., Mol. Cancer Res., 2008). Interestingly, increases in cell surface DR4 and DR5 levels are greater in TNBC cell lines when compared with non-TNBC cell lines. Overall, our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC and non-TNBC subtypes. These effects are observed regardless of the TRAIL sensitivity of the cells, and may be mediated through mechanisms involving downregulation of anti-apoptotic proteins and upregulation of cell surface death receptors. Our work will further understanding of these mechanisms and contribute to development of a preclinical rationale for the use of ONC201 as a treatment for breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Christina L.B. Kline, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A16.