C. Leah B. Kline

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases.

ONC201 triggers an apoptotic cellular stress response in both solid and blood tumors. Stressing cancer cells to death The anticancer drug ONC201 triggers cell death in various tumor types. A pair of papers (see also the Focus by Greer and Lipkowitz) show that ONC201 activated cell stress pathways that depended on the activation of the transcription factor ATF4. Kline et al. showed this stress response to ONC201 occurred in cells derived from various types of solid tumors, in which ATF4 activation led to an increase in the abundance of the proapoptotic protein TRAIL and its receptor DR5. Ishizawa et al. demonstrated that in acute myeloid leukemia and mantle cell lymphoma, ONC201 triggered apoptosis and inhibited mTORC1 signaling, a pathway that promotes cell growth and proliferation. The findings reveal more details about ONC201’s mechanism of action, potentially enabling patient stratification and future development to improve its efficacy. ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation– and cell survival–promoting kinases Akt and ERK and induces cell death through the proapoptotic protein TRAIL. ONC201 is currently in early-phase clinical testing for various malignancies. We found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the transactivator CHOP, and the TRAIL receptor DR5. ATF4 was not activated in ONC201-resistant cancer cells, and in ONC201-sensitive cells, knockdown of ATF4 or CHOP partially abrogated ONC201-induced cytotoxicity and diminished the ONC201-stimulated increase in DR5 abundance. The activation of ATF4 in response to ONC201 required the kinases HRI and PKR, which phosphorylate and activate the translation initiation factor eIF2α. ONC201 rapidly triggered cell cycle arrest, which was associated with decreased abundance of cyclin D1, decreased activity of the kinase complex mTORC1, and dephosphorylation of the retinoblastoma (Rb) protein. The abundance of X-linked inhibitor of apoptosis protein (XIAP) negatively correlated with the extent of apoptosis in response to ONC201. These effects of ONC201 were independent of whether cancer cells had normal or mutant p53. Thus, ONC201 induces cell death through the coordinated induction of TRAIL by an ISR pathway.

Discovery and clinical introduction of first-in-class imipridone ONC201

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.

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies

ABSTRACT ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1–8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitts lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkins lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitts lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitts lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkins lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

Anti-tumor effects of ONC201 in combination with VEGF-inhibitors significantly impacts colorectal cancer growth and survival in vivo through complementary non-overlapping mechanisms

BackgroundSmall molecule ONC201 is an investigational anti-tumor agent that upregulates intra-tumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 therapy in advanced cancer patients has been completed and the drug has progressed into Phase II trials in several cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease has a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents.MethodsWe investigated the potential combination of VEGF inhibitors such as bevacizumab and its murine-counterpart; along with other anti-angiogenic agents and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) models. We utilized non-invasive imaging and immunohistochemistry to determine potential mechanisms of action.ResultsOur results demonstrate significant tumor regression or complete tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. Our results indicate that ONC201 and anti-angiogenic agents act through distinct mechanisms while increasing tumor cell death and inhibiting proliferation.ConclusionWith the use of both a murine VEGF inhibitor in syngeneic models, and bevacizumab in human cell line-derived xenografts, we demonstrate that ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising approach for further testing in the clinic for the treatment of CRC.

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.

Abstract A060: Targeting DRD2 dysregulation in recurrent glioblastoma with imipridone ONC201: predictive and pharmacodynamic clinical biomarker analyses

Background: ONC201, an imipridone that is a selective antagonist of the G protein-coupled receptors dopamine receptor D2 (DRD2) and D3 (DRD3), has exhibited biologic activity and an exceptional safety profile in a phase II study in bevacizumab-naive recurrent glioblastoma (Arrillaga et a.l, 2017). Single-agent ONC201 efficacy has been observed in preclinical glioblastoma models in addition to robust penetrance of the blood-brain barrier (Allen et al., 2013). DRD2 antagonism induces tumor cell apoptosis via the same signaling pathways affected by ONC201. In addition, DRD2 is expressed on NK and other immune cells and DRD2 antagonism can induce their activation. Methods: Cell viability assays were performed with ONC201 in >1000 Genomic of Drug Sensitivity in Cancer (GDSC) cell lines and NCI60. Immunohistochemistry staining of DRD2/DRD5 was performed in glioblastoma tissue microarrays and archival tumor tissues. Whole exome sequencing was performed in RKO cells with acquired resistance to ONC201. DRD5 wild-type and mutant constructs were generated for overexpression studies. ELISA was used to quantitate serum prolactin and immune effector (perforin) levels. Intratumoral drug concentrations were evaluated by LC-MS assays conducted on glioblastoma tissue resected from patients following the second dose of 625mg ONC201. Results: Evaluation of ONC201 in GDSC cell lines confirmed broad anticancer efficacy with high sensitivity (~1-3 µM) in human brain cancer. The Cancer Genome Atlas (TCGA) revealed that DRD2 is highly expressed in glioblastoma relative to other dopamine receptors and that genetic aberrations are rare. High expression of DRD2 occurred in primary, rather than secondary, glioblastoma and was associated with a poor prognosis. Immunohistochemistry of tissue microarrays revealed DRD2 overexpression in glioblastoma relative to normal brain. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among glioblastoma cell lines in the NCI60 panel. Interestingly, expression of DRD5, a D1-like dopamine receptor that counteracts DRD2 signaling, was significantly inversely correlated with ONC201 potency in the NCI60 and GDSC datasets (P 5 month had no detectable expression of DRD5, unlike those with PFS Citation Format: Varun Vijay Prabhu, Neel Madhukar, C. Leah B. Kline, Rohinton Tarapore, Wafik El-Deiry, Olivier Elemento, Faye Doherty, Alexander VanEngelenburg, Jessica Durrant, Andrew Zloza, Cyril Benes, Isabel Arrillaga, Wolfgang Oster, Joshua E. Allen. Targeting DRD2 dysregulation in recurrent glioblastoma with imipridone ONC201: predictive and pharmacodynamic clinical biomarker analyses [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A060.

Abstract 3245: Preclinical evaluation of the imipridone family of small molecules, including analogues of clinical-stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212

We previously identified a novel, potent anti-cancer small molecule ONC201, which upregulates the integrated stress response (ISR) through ATF4/CHOP/DR5 and acts as a dual inactivator of Akt and ERK, leading to TRAIL gene activation. After completing a first-in-human phase I clinical trial that revealed exceptional safety, therapeutic pharmacokinetic (PK) profile and tumor engagement, ONC201 is under investigation in several advanced cancer Phase I/II trials. Given the unique imipridone core chemical structure of ONC201, we synthesized a family of analogues in an effort to identify additional chemical family members with distinct therapeutic properties. Based on in vitro potency improvements in human cancer cell lines and therapeutic window approximations with normal human fibroblasts, select analogues were investigated in animals for toxicity, maximum tolerated dose (MTD), and antitumor efficacy. ONC212 is one of the most promising new imipridones that was further evaluated to establish the PK profile, oral bioavailability, and efficacy in tumor types that are less sensitive to ONC201. Compared to ONC201, we noted distinct and more rapid kinetics of activity as well as improved potency in multiple human cancer cell lines in vitro. ONC212 has a broad therapeutic window, an acceptable PK profile, and is orally well-tolerated in mice. With no evidence of toxicity at efficacious doses in both colon and triple negative breast cancer, we have begun further evaluation of antitumor efficacy studies in ONC201-resistant tumor types. Efficacy studies with ONC212 are ongoing in melanoma models that are sensitive to ONC212 but less sensitive to ONC201 in vitro. Preliminary data indicates potent tumor growth reduction by ONC212 in vivo in ONC201-resistant melanoma xenografts. With a wide safety margin, potent antitumor activity in ONC201-insenstive tumors, and drug-like characteristics, ONC212 is being further developed as a drug candidate from the new imipridone class of compounds that complements the spectrum of activity of ONC201. Citation Format: Jessica Wagner, C. Leah Kline, Gary Olson, Bhaskara Nallaganchu, Richard Pottorf, Varun Prabhu, Martin Stogniew, Joshua Allen, Wafik El-Deiry. Preclinical evaluation of the imipridone family of small molecules, including analogues of clinical-stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212 [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 3245. doi:10.1158/1538-7445.AM2017-3245

Abstract 124: Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy

ONC201, a first-in-class oral anti-tumor agent, upregulates the pro-apoptotic immune cytokine TRAIL and activates the integrated stress response leading to upregulation of death receptor 5 in bulk tumor and cancer stem cells. We previously demonstrated that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo while suppressing Akt/ERK signaling in tumors in a dose/frequency-dependent manner (Wagner et al., AACR, 2016). We also provided evidence that ONC201 exhibits a potent anti-metastatic effect (Wagner et al., AACR, 2016). We observe accumulation and activation of TRAIL-secreting NK+ cells within ONC201-treated tumors in C57/BL6, Balb/c, and athymic nude tumor-bearing mice. Importantly, ONC201 exerts in vivo anti-tumor efficacy on tumor cell lines that are ONC201-resistant in vitro, including acquired stable resistance. Using the NK-depleting antibody GM1, we demonstrate that the activation and TRAIL secretion of NK cells by ONC201 significantly contributes to in vivo anti-tumor efficacy, including TRAIL/ONC201-resistant tumors. We are currently investigating how ONC201 recruits NK cells to the tumor by examining NK-recruiting chemokine factors within the tumor site. We have also demonstrated upregulation of CD3+ T cells by ONC201 in syngeneic mice. Finally, we observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients upon ONC201 administration in the clinic. Our results demonstrate novel and potentially significant increases in cytotoxic NK cell recruitment to tumors. The results offer a unique pathway of immune stimulation for cancer therapy that may be combined with immune checkpoint or targeted cancer therapy strategies. We are currently investigating the role of NK cells and CD3+ cells in ONC201’s ability to inhibit metastasis by using a metastatic model that involves surgically removing the primary tumor and allowing metastases to grow in vivo before treatment. These findings indicate that ONC201 possess immunomodulatory activity and provide a rationale for combining ONC201 with PD-1/PDL-1 inhibitors, a combination we are currently testing in syngeneic immunocompetent mouse models. Citation Format: Jessica Wagner, C. Leah Kline, Lanlan Zhou, Andrew Zloza, Charles Chesson, Jenna Newman, Howard Kaufman, Joseph Bertino, Mark Stein, Wafik El-Deiry. Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy [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 124. doi:10.1158/1538-7445.AM2017-124