Marie D. Ralff

ONC201 demonstrates antitumor effects in both triple-negative and non–triple-negative breast cancers through TRAIL-dependent and TRAIL-independent mechanisms

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G1 phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290–8. ©2017 AACR.

Preclinical evaluation of the imipridone family, analogues of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212

ABSTRACT Anti-cancer small molecule ONC201 upregulates the integrated stress response (ISR) and acts as a dual inactivator of Akt/ERK, leading to TRAIL gene activation. ONC201 is under investigation in multiple clinical trials to treat patients with cancer. Given the unique imipridone core chemical structure of ONC201, we synthesized a series of analogs to identify additional compounds with distinct therapeutic properties. Several imipridones with a broad range of in vitro potencies were identified in an exploration of chemical derivatives. Based on in vitro potency in human cancer cell lines and lack of toxicity to normal human fibroblasts, imipridones ONC206 and ONC212 were prioritized for further study. Both analogs inhibited colony formation, and induced apoptosis and downstream signaling that involves the integrated stress response and Akt/ERK, similar to ONC201. Compared to ONC201, ONC206 demonstrated improved inhibition of cell migration while ONC212 exhibited rapid kinetics of activity. ONC212 was further tested in >1000 human cancer cell lines in vitro and evaluated for safety and anti-tumor efficacy in vivo. ONC212 exhibited broad-spectrum efficacy at nanomolar concentrations across solid tumors and hematological malignancies. Skin cancer emerged as a tumor type with improved efficacy relative to ONC201. Orally administered ONC212 displayed potent anti-tumor effects in vivo, a broad therapeutic window and a favorable PK profile. ONC212 was efficacious in vivo in BRAF V600E melanoma models that are less sensitive to ONC201. Based on these findings, ONC212 warrants further development as a drug candidate. It is clear that therapeutic utility extends beyond ONC201 to include additional imipridones.

Role of Dopamine Receptors in the Anticancer Activity of ONC201

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201’s interaction with DRD2 plays a role in ONC201’s anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type–specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201’s anticancer effects. Although ONC201’s anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201’s anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201’s ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.

Cancer stem cell-related gene expression as a potential biomarker of response for first-in-class imipridone ONC201 in solid tumors

Cancer stem cells (CSCs) correlate with recurrence, metastasis and poor survival in clinical studies. Encouraging results from clinical trials of CSC inhibitors have further validated CSCs as therapeutic targets. ONC201 is a first-in-class small molecule imipridone in Phase I/II clinical trials for advanced cancer. We have previously shown that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs via Akt/ERK inhibition and DR5/TRAIL induction. In this study, we demonstrate that the anti-CSC effects of ONC201 involve early changes in stem cell-related gene expression prior to tumor cell death induction. A targeted network analysis of gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates stem cell pathways such as Wnt signaling and modulates genes (ID1, ID2, ID3 and ALDH7A1) known to regulate self-renewal in colorectal, prostate cancer and glioblastoma. ONC201-mediated changes in CSC-related gene expression were validated at the RNA and protein level for each tumor type. Accordingly, we observed inhibition of self-renewal and CSC markers in prostate cancer cell lines and patient-derived glioblastoma cells upon ONC201 treatment. Interestingly, ONC201-mediated CSC depletion does not occur in colorectal cancer cells with acquired resistance to ONC201. Finally, we observed that basal expression of CSC-related genes (ID1, CD44, HES7 and TCF3) significantly correlate with ONC201 efficacy in >1000 cancer cell lines and combining the expression of multiple genes leads to a stronger overall prediction. These proof-of-concept studies provide a rationale for testing CSC expression at the RNA and protein level as a predictive and pharmacodynamic biomarker of ONC201 response in ongoing clinical studies.

Anti-pancreatic cancer activity of ONC212 involves the unfolded protein response (UPR) and is reduced by IGF1-R and GRP78/BIP

Pancreatic cancer is chemo-resistant and metastasizes early with an overall five-year survival of ∼8.2%. First-in-class imipridone ONC201 is a small molecule in clinical trials with anti-cancer activity. ONC212, a fluorinated-ONC201 analogue, shows preclinical efficacy in melanoma and hepatocellular-cancer models. We investigated efficacy of ONC201 and ONC212 against pancreatic cancer cell lines (N=16 including 9 PDX-cell lines). We demonstrate ONC212 efficacy in 4 in-vivo models including ONC201-resistant tumors. ONC212 is active in pancreatic cancer as single agent or in combination with 5-fluorouracil, irinotecan, oxaliplatin or RTK inhibitor crizotinib. Based on upregulation of pro-survival IGF1-R in some tumors, we found an active combination of ONC212 with inhibitor AG1024, including in vivo. We show a rationale for targeting pancreatic cancer using ONC212 combined with targeting the unfolded-protein response and ER chaperones such as GRP78/BIP. Our results lay the foundation to test imipridones, anti-cancer agents, in pancreatic cancer, that is refractory to most drugs.

Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax–/– tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti–PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

ONC201 Targets AR and AR-V7 Signaling, Reduces PSA, and Synergizes with Everolimus in Prostate Cancer

Androgen receptor (AR) signaling plays a key role in prostate cancer progression, and androgen deprivation therapy (ADT) is a mainstay clinical treatment regimen for patients with advanced disease. Unfortunately, most prostate cancers eventually become androgen-independent and resistant to ADT with patients progressing to metastatic castration-resistant prostate cancer (mCRPC). Constitutively activated AR variants (AR-V) have emerged as mediators of resistance to AR-targeted therapy and the progression of mCRPC, and they represent an important therapeutic target. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant, and its expression correlates with ADT resistance. ONC201/TIC10 is the founding member of the imipridone class of small molecules and has shown anticancer activity in a broad range of tumor types. ONC201 is currently being tested in phase I/II clinical trials for advanced solid tumors, including mCRPC, and hematologic malignancies. There has been promising activity observed in patients in early clinical testing. This study demonstrates preclinical single-agent efficacy of ONC201 using in vitro and in vivo models of prostate cancer. ONC201 has potent antiproliferative and proapoptotic effects in both castration-resistant and -sensitive prostate cancer cells. Furthermore, the data demonstrate that ONC201 downregulates the expression of key drivers of prostate cancer such as AR-V7 and downstream target genes including the clinically used biomarker PSA (KLK3). Finally, the data also provide a preclinical rationale for combination of ONC201 with approved therapeutics for prostate cancer such as enzalutamide, everolimus (mTOR inhibitor), or docetaxel. Implications: The preclinical efficacy of ONC201 as a single agent or in combination, in hormone-sensitive or castration-resistant prostate cancer, suggests the potential for immediate clinical translation. Mol Cancer Res; 16(5); 754–66. ©2018 AACR.

TRAIL pathway targeting therapeutics

ABSTRACT Introduction: Despite decades of focused research efforts, cancer remains a significant cause of morbidity and mortality. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is capable of inducing cell death selectively in cancer cells while sparing normal cells. Areas covered: In this review, the authors cover TRA therapy and strategies that have been undertaken to improve their efficacy, as well as unconventional approaches to TRAIL pathway activation including TRAIL-inducing small molecules. They also discuss mechanisms of resistance to TRAIL and the use of combination strategies to overcome it. Expert commentary: Targeting the TRAIL pathway has been of interest in oncology, and although initial clinical trials of TRAIL receptor agonists showed limitations, novel approaches represent the future of TRAIL-based therapy.