The Imipridone ONC213 Targets α-Ketoglutarate Dehydrogenase to Induce Mitochondrial Stress and Suppress Oxidative Phosphorylation in Acute Myeloid Leukemia.

Eradication of acute myeloid leukemia (AML) is therapeutically challenging; many patients succumb to AML despite initially responding to conventional treatments. Here, we showed that the imipridone ONC213 elicits potent antileukemia activity in a subset of AML cell lines and primary patient samples, particularly in leukemia stem cells, while producing negligible toxicity in normal hematopoietic cells. ONC213 suppressed mitochondrial respiration and elevated α-ketoglutarate by suppressing α-ketoglutarate dehydrogenase (αKGDH) activity. Deletion of OGDH, which encodes αKGDH, suppressed AML fitness and impaired oxidative phosphorylation, highlighting the key role for αKGDH inhibition in ONC213-induced death. ONC213 treatment induced a unique mitochondrial stress response and suppressed de novo protein synthesis in AML cells. Additionally, ONC213 reduced the translation of MCL1, which contributed to ONC213-induced apoptosis. Importantly, a patient-derived xenograft from a relapsed AML patient was sensitive to ONC213 in vivo. Collectively, these findings support further development of ONC213 for treating AML. SIGNIFICANCE: In AML cells, ONC213 suppresses αKGDH, which induces a unique mitochondrial stress response, and reduces MCL1 to decrease oxidative phosphorylation and elicit potent antileukemia activity. See related commentary by Boët and Sarry, p. 950.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer.

Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for cancer patients with homologous recombination (HR) deficient tumors. The imipridone ONC206 is an orally bioavailable dopamine receptor D2 antagonist and mitochondrial protease ClpP agonist that has anti-tumorigenic effects in endometrial cancer via induction of apoptosis, activation of the integrated stress response and modulation of PI3K/AKT signaling. Both PARP inhibitors and imipridones are being evaluated in endometrial cancer clinical trials but have yet to be explored in combination. In this manuscript, we evaluated the effects of the PARP inhibitor olaparib in combination with ONC206 in human endometrioid endometrial cancer cell lines and in a genetically engineered mouse model of endometrial cancer. Our results showed that simultaneous exposure of endometrial cancer cells to olaparib and ONC206 resulted in synergistic anti-proliferative effects and increased cellular stress and apoptosis in both cell lines, compared to either drug alone. The combination treatment also decreased expression of the anti-apoptotic protein Bcl-2 and reduced phosphorylation of AKT and S6, with greater effects compared to either drug alone. In the transgenic model of endometrial cancer, the combination of olaparib and ONC206 resulted in a more significant reduction in tumor weight in obese and lean mice compared to ONC206 alone or olaparib alone, together with a considerably decreased Ki-67 and enhanced H2AX expression in obese and lean mice. These results suggest that this novel dual therapy may be worthy of further exploration in clinical trials.

Novel combination of imipridones and histone deacetylase inhibitors demonstrate cytotoxic effect through integrated stress response in pediatric solid tumors.

There is a demonstrated need for new chemotherapy options in pediatric oncology, as pediatric solid tumors continue to plateau at 60% with event-free survival. Imipridones, a novel class of small molecules, represent a potential new therapeutic option, with promising pre-clinical data and emerging clinical trial data in adult malignancies. ONC201, ONC206, and ONC212 are imipridones showing pro-apoptotic anti-cancer response. Using cell viability assays, and protein immunoblotting, we were able to demonstrate single-agent efficacy of all 3 imipridones inducing cell death in pediatric solid tumor cell lines, including osteosarcoma, malignant peripheral nerve sheath tumors, Ewing sarcoma (EWS), and neuroblastoma. ONC201 displayed IC50 values for non-H3K27M-mutated EWS cell lines ranging from 0.86 µM (SK-N-MC) to 2.76 µM (RD-ES), which were comparable to the range of IC50 values for H3K27M-mutated DIPG cells lines (range 1.06 to 1.56 µM). ONC212 demonstrated the highest potency in single-agent cell killing, followed by ONC206, and ONC201. Additionally, pediatric solid tumor cells were treated with single-agent therapy with histone deacetylase inhibitors (HDACi) vorinostat, entinostat, and panobinostat, showing cell killing with all 3 HDACi drugs, with panobinostat showing the greatest potency. We demonstrate that dual-agent therapy with combinations of imipridones and HDACi lead to synergistic cell killing and apoptosis in all pediatric solid tumor cell lines tested, with ONC212 and panobinostat combinations demonstrating maximal potency. The imipridones induced the integrated stress response with ATF4 and TRAIL receptor upregulation, as well as reduced expression of ClpX. Hyperacetylation of H3K27 was associated with synergistic killing of tumor cells following exposure to imipridone plus HDAC inhibitor therapies. Our results introduce a novel class of small molecules to treat pediatric solid tumors in a precision medicine framework. Use of impridones in pediatric oncology is novel and shows promising pre-clinical efficacy in pediatric solid tumors, including in combination with HDAC inhibitors.

ONC201/TIC10 plus TLY012 anti-cancer effects via apoptosis inhibitor downregulation, stimulation of integrated stress response and death receptor DR5 in gastric adenocarcinoma.

Gastric adenocarcinoma typically presents with advanced stage when inoperable. Chemotherapy options include non-targeted and toxic agents, leading to poor 5-year patient survival outcomes. Small molecule ONC201/TIC10 (TRAIL-Inducing Compound #10) induces cancer cell death via ClpP-dependent activation of the integrated stress response (ISR) and up-regulation of the TRAIL pathway. We previously found in breast cancer, pancreatic cancer and endometrial cancer that ONC201 primes tumor cells for TRAIL-mediated cell death through ISR-dependent upregulation of ATF4, CHOP and TRAIL death receptor DR5. We investigated the ability of ONC201 to induce apoptosis in gastric adenocarcinoma cells in combination with recombinant human TRAIL (rhTRAIL) or PEGylated trimeric TRAIL (TLY012). AGS (caspase 8-, KRAS-, PIK3CA-mutant, HER2-amplified), SNU-1 (KRAS-, MLH1-mutant, microsatellite unstable), SNU-5 (p53-mutant) and SNU-16 (p53-mutant) gastric adenocarcinoma cells were treated with ONC201 and TRAIL both in cell culture and in vivo. Gastric cancer cells showed synergy following dual therapy with ONC201 and rhTRAIL/TLY012 (combination indices < 0.6 at doses that were non-toxic towards normal fibroblasts). Synergy was observed with increased cells in the sub-G1 phase of the cell cycle with dual ONC201 plus TRAIL therapy. Increased PARP, caspase 8 and caspase 3 cleavage after ONC201 plus TRAIL further documented apoptosis. Increased cell surface expression of DR5 with ONC201 therapy was observed by flow cytometry, and immunoblotting revealed ONC201 upregulation of the ISR, ATF4, and CHOP. We observed downregulation of anti-apoptotic cIAP-1 and XIAP in all cells except AGS, and cFLIP in all cells except SNU-16. We tested the regimen in an organoid model of human gastric cancer, and in murine sub-cutaneous xenografts using AGS and SNU-1 cells. Our results suggest that ONC201 in combination with TRAIL may be an effective and non-toxic option for the treatment of gastric adenocarcinoma by inducing apoptosis via activation of the ISR, increased cell surface expression of DR5 and down-regulation of inhibitors of apoptosis. Our results demonstrate in vivo anti-tumor effects of ONC201 plus TLY012 against gastric cancer that could be further investigated in clinical trials.

Anti-Tumor and Anti-Invasive Effects of ONC201 on Ovarian Cancer Cells and a Transgenic Mouse Model of Serous Ovarian Cancer.

ONC201 is a promising first-in-class small molecule that has been reported to have anti-neoplastic activity in various types of cancer through activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as well as activation of mitochondrial caseinolytic protease P (ClpP). The present study was to explore the anti-tumor potential effect of ONC201 in ovarian cancer cell lines and in a transgenic mouse model of high grade serous ovarian cancer under obese (high fat diet) and lean (low fat diet) conditions. ONC201 significantly suppressed cell proliferation, induced arrest in G1 phase, and increased cellular stress and apoptosis, accompanied by dual inhibition of the AKT/mTOR/S6 and MAPK pathways in OC cells. ONC201 also resulted in inhibition of adhesion and invasion via epithelial-mesenchymal transition and reduction of VEGF expression. Pre-treatment with the anti-oxidant, N-acetylcysteine (NAC), reversed the ONC201-induced oxidative stress response, and prevented ONC201-reduced VEGF and cell invasion by regulating epithelial-mesenchymal transition protein expression. Knockdown of ClpP in ovarian cancer cells reduced ONC201 mediated the anti-tumor activity and cellular stress. Diet-induced obesity accelerated ovarian tumor growth in the KpB mouse model. ONC201 significantly suppressed tumor growth, and decreased serum VEGF production in obese and lean mice, leading to a decrease in tumoral expression of Ki-67, VEGF and phosphorylation of p42/44 and S6 and an increase in ClpP and DRD5, as assessed by immunohistochemistry. These results suggest that ONC201 may be a promising therapeutic agent to be explored in future clinical trials in high-grade serous ovarian cancer.

Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma.

PURPOSE: Novel therapeutic targets are critical to unravel for the most common primary brain tumor in adults, glioblastoma (GBM). We have identified a novel synthetic lethal interaction between ClpP activation and HDAC1/2 inhibition that converges on GBM energy metabolism. EXPERIMENTAL DESIGN: Transcriptome, metabolite, and U-13C-glucose tracing analyses were utilized in patient-derived xenograft (PDX) models of GBM. Orthotopic GBM models were used for in vivo studies. RESULTS: We showed that activation of the mitochondrial ClpP protease by mutant ClpP (Y118A) or through utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (panobinostat) or selective (romidepsin) HDAC inhibitors caused synergistic reduction of viability in GBM model systems, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. This effect was partially mediated by activation of apoptosis along with activation of caspases regulated chiefly by Bcl-xL and Mcl-1. Knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant substantially rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, respectively. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC inhibitors and imipridones prolonged host survival more potently than single treatments or vehicle in vivo. CONCLUSIONS: Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

ONC206 has anti-tumorigenic effects in human ovarian cancer cells and in a transgenic mouse model of high-grade serous ovarian cancer.

ONC206, a dopamine receptor D2 (DRD2) antagonist and imipridone, is a chemically modified derivative of ONC201. Recently, ONC206 and other imipridones were identified as activators of the mitochondrial protease ClpP, inducing downstream pathways that allow them to selectively target cancer cells. Clinical trials showed that ONC201, the first in class imipridone, was well tolerated and exhibited tumor regression in some solid tumors. Our goal was to evaluate the effect of ONC206 on cell proliferation and tumor growth in ovarian cancer cell lines and in a transgenic mouse model of high grade serous ovarian cancer (KpB model). ONC206 was more potent than ONC201 in inhibiting cell proliferation, as evidenced by a 10-fold decrease in IC50 for the SKOV3 and OVCAR5 cell lines. This was accompanied by the results that ONC206 significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, and inhibited adhesion and invasion in vitro. Treatment of obese and non-obese KpB mice with ONC206 elevated Bip and ClpP expression and reduced KI67, BCL-XL and DRD2 expression in the ovarian tumors. Our findings demonstrate that ONC206 has anti-tumorigenic effects in ovarian cancer as previously demonstrated by ONC201 but appears to be as well tolerated and more potent. Thus, ONC206 deserves further evaluation in clinical trials.

Quantitation of the next-generation imipridone ONC206 in human plasma by a simple and sensitive UPLC-MS/MS assay for clinical pharmacokinetic application.

ONC206 is an imipridone derivative that is being developed clinically as a single agent given orally in a first-in-human trial (NCT04541082). This ongoing clinical trial requires pharmacokinetic analysis of ONC206 to fully characterize its pharmacologic profile. There is currently no published bioanalytical method for ONC206 quantitation. To understand the clinical pharmacokinetics of ONC206, a sensitive yet simple uHPLC-MS/MS method for quantitation of ONC206 in human plasma was developed. Protein-precipitation allowed rapid and sensitive bioanalytical measurement of ONC206 in human plasma. A Phenomenex Kinetex C18 (50 ×2.1 mm, 1.3 µm, 100 Å) analytical column achieved symmetrical and sharp chromatography peaks of ONC206 and the internal standard, [2H]7-ONC206, which were detected using multiple reaction monitoring. The assay calibration range was 1-500 ng/mL and was best fit by a linear regression model (r2 > 0.99732 ± 0.0010). The method proved accurate (< ± 9% deviation), precise (<11%CV), selective and specific with no interference and low inter-lot matrix variability. ONC206 demonstrated excellent short-term, long-term, and multiple freeze-thaw cycle stability in solution and human plasma. This fully validated method was used to quantitate ONC206 plasma concentrations from patients enrolled in the aforementioned clinical trial at the NCI to demonstrate its clinical applicability.

Phase II Study of ONC201 in Neuroendocrine Tumors including Pheochromocytoma-Paraganglioma and Desmoplastic Small Round Cell Tumor.

PURPOSE: Tumor dopamine-like DRD2 receptor expression is higher in pheochromocytoma-paraganglioma (PC-PG) compared with other cancers. ONC201 is a bitopic DRD2 antagonist with preclinical ONC201 activity in desmoplastic small round cell tumor (DSRCT). PATIENTS AND METHODS: Patients (N = 30) with neuroendocrine tumors were treated on this investigator-initiated trial (NCT03034200). ONC201 dose and schedule were 625 mg orally weekly in cohorts A (PC-PG) + B (other neuroendocrine tumors) and 625 mg orally on 2 consecutive days each week in cohort C, which included 5 responding patients. The primary endpoint was radiographic response measured using RECIST. Secondary endpoints included progression-free survival, overall survival, and safety. RESULTS: In arm A (n = 10; all PC-PG), 50% (5/10) exhibited a partial response (PR) and 2 additional patients had stable disease (SD) >3 months. Median duration of therapy for arm A patients was 9 months (range: 1.5-33 months) with 5 patients treated >1 year. In arm B (n = 12), there were 1 PR (DSRCT) and 2 SD (DSRCT; neuroblastoma) >3 months. Median duration of therapy in arm A was 18 months (range: 1-33 months) and arm B was 3 months (range: 1.5-33 months). Arm C PC-PG (N = 8) showed 1 PR and 7 SD at 3 months, with median duration of therapy >10 months. There was no decline in Karnofsky performance status at week 12 for 28 of 30 patients and no dose modification due to treatment-related adverse events. CONCLUSIONS: Oral ONC201 was well tolerated in patients with metastatic neuroendocrine tumors and associated with clinical benefit, including tumor responses, particularly in some patients with DSRCT and the majority of patients with PC-PG. See related commentary by Owen and Trikalinos, p. 1748.

Extracellular Matrix in Synthetic Hydrogel-Based Prostate Cancer Organoids Regulate Therapeutic Response to EZH2 and DRD2 Inhibitors.

Following treatment with androgen receptor (AR) pathway inhibitors, ≈20% of prostate cancer patients progress by shedding their AR-dependence. These tumors undergo epigenetic reprogramming turning castration-resistant prostate cancer adenocarcinoma (CRPC-Adeno) into neuroendocrine prostate cancer (CRPC-NEPC). No targeted therapies are available for CRPC-NEPCs, and there are minimal organoid models to discover new therapeutic targets against these aggressive tumors. Here, using a combination of patient tumor proteomics, RNA sequencing, spatial-omics, and a synthetic hydrogel-based organoid, putative extracellular matrix (ECM) cues that regulate the phenotypic, transcriptomic, and epigenetic underpinnings of CRPC-NEPCs are defined. Short-term culture in tumor-expressed ECM differentially regulated DNA methylation and mobilized genes in CRPC-NEPCs. The ECM type distinctly regulates the response to small-molecule inhibitors of epigenetic targets and Dopamine Receptor D2 (DRD2), the latter being an understudied target in neuroendocrine tumors. In vivo patient-derived xenograft in immunocompromised mice showed strong anti-tumor response when treated with a DRD2 inhibitor. Finally, we demonstrate that therapeutic response in CRPC-NEPCs under drug-resistant ECM conditions can be overcome by first cellular reprogramming with epigenetic inhibitors, followed by DRD2 treatment. The synthetic organoids suggest the regulatory role of ECM in therapeutic response to targeted therapies in CRPC-NEPCs and enable the discovery of therapies to overcome resistance.

ONC201/TIC10 Is Empowered by 2-Deoxyglucose and Causes Metabolic Reprogramming in Medulloblastoma Cells in Vitro Independent of C-Myc Expression.

The purpose of this study was to examine whether the imipridone ONC201/TIC10 affects the metabolic and proliferative activity of medulloblastoma cells in vitro. Preclinical drug testing including extracellular flux analyses (agilent seahorse), MTT assays and Western blot analyses were performed in high and low c-myc-expressing medulloblastoma cells. Our data show that treatment with the imipridone ONC201/TIC10 leads to a significant inihibitory effect on the cellular viability of different medulloblastoma cells independent of c-myc expression. This effect is enhanced by glucose starvation. While ONC201/TIC10 decreases the oxidative consumption rates in D458 (c-myc high) and DAOY (c-myc low) cells extracellular acidification rates experienced an increase in D458 and a decrease in DAOY cells. Combined treatment with ONC201/TIC10 and the glycolysis inhibitor 2-Deoxyglucose led to a synergistic inhibitory effect on the cellular viability of medulloblastoma cells including spheroid models. In conclusion, our data suggest that ONC201/TIC10 has a profound anti-proliferative activity against medulloblastoma cells independent of c-myc expression. Metabolic targeting of medulloblastoma cells by ONC201/TIC10 can be significantly enhanced by an additional treatment with the glycolysis inhibitor 2-Deoxyglucose. Further investigations are warranted.

Highly potent dopamine receptor D2 antagonist ONC206 demonstrates anti-tumorigenic activity in endometrial cancer.

Endometrial cancer (EC) is a highly obesity-driven cancer, with limited treatment options. ONC201 is an imipridone that selectively antagonizes the G protein-coupled receptors dopamine receptor D2 and D3 (DRD2/3) and activates human mitochondrial caseinolytic protease P (ClpP). It is a promising first-in-class small molecule that has been reported to have anti-neoplastic activity in various types of cancer through induction of the integrated stress response (ISR) as well as through stimulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and subsequent induction of apoptosis. ONC201 is being evaluated in Phase II clinical trials for solid tumors and hematological malignancies, including EC. ONC206 is an analog of ONC201 with nanomolar potency in Phase I clinical trials. This study evaluated the anti-tumor efficacy of ONC206 in EC cell lines and the Lkb1fl/flp53fl/fl genetically engineered mouse model of endometrioid EC. ONC206 revealed greater potency than ONC201 in the inhibition of proliferation in EC cell lines, with IC50 concentration ranges of 0.21-0.32 µM for ONC026 versus 2.14-3.53 µM for ONC201. ONC206 induced cellular stress, apoptosis and cell cycle G1 arrest, accompanied by inhibition of the AKT/mTOR/S6 pathways in EC cells. Diet-induced obesity accelerated tumor growth in Lkb1fl/flp53fl/fl mice. ONC206 inhibited EC tumor size and weight in both obese and lean mice after 4 weeks of treatment. Treatment with ONC206 led to a decrease in expression of Ki67, BCL-XL and phosphorylation of S6, as well as an increase in ClpP in endometrial tumors under both obese and lean conditions. Overall, the pre-clinical efficacy of ONC206 is promising and worthy of further exploration in clinical trials for endometrioid EC.

Combination of ONC201 and TLY012 induces selective, synergistic apoptosis in vitro and significantly delays PDAC xenograft growth in vivo.

The five-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained a dismal 9% for approximately 40 years with an urgent need for novel therapeutic interventions. ONC201 is the founding member of the imipridone class, comprised of orally bioavailable small molecules that have shown efficacy in multiple tumor types both in animal models and in Phase I/II clinical trials. ONC201 is a potent inducer of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. TRAIL is an innate immune mechanism which induces programmed cell death of cancer cells. We observed that PDAC cells upregulated ATF4, CHOP, and DR5 after treatment with ONC201. This occurred in cell lines that are susceptible to ONC201-induced apoptosis and in ones that are not. In response to ONC201, PDAC cells downregulated anti-apoptotic proteins including c-FLIP, BclXL, XIAP, cIAP1, and survivin. We hypothesized that TRAIL receptor agonists might induce selective, synergistic apoptosis in pancreatic cancer cell lines treated with ONC201. We screened 7 pancreatic cancer cell lines and found synergy with ONC201 and rhTRAIL or the novel TRAIL receptor agonist TLY012 in 6 of the 7 cell lines tested. In vivo experiments using BxPC3 and HPAFII xenograft models showed that the combination of ONC201 plus TLY012 significantly delays tumor growth as compared to controls. Immunohistochemical analysis of the tumors after three doses of the combination showed significantly increased cleavage of caspase 3 in vivo as compared to controls. Taken together, the preclinical efficacy of ONC201 and TLY012 represents a novel therapeutic option for further testing in pancreatic cancer patients. This combination showed marked efficacy in tumor cells that are both sensitive and resistant to the pro-apoptotic effects of ONC201, providing rationale to further investigate the combination of ONC201 plus TLY012 in patients with pancreatic cancer.

Potent preclinical sensitivity to imipridone-based combination therapies in oncohistone H3K27M-mutant diffuse intrinsic pontine glioma is associated with induction of the integrated stress response, TRAIL death receptor DR5, reduced ClpX and apoptosis.

The H3K27M oncohistone mutation, identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), is a potential target for therapy. Imipridone ONC201/TIC10 (TRAIL-Inducing Compound #10) induces apoptosis of cancer cells, and has clinical efficacy against H3K27M-mutant DIPG. We demonstrate synergy between ONC201, ONC206 and ONC212, and targeted therapies with known preclinical activity against DIPG. We hypothesized that imipridone combinations with HDAC or proteasome inhibitors may be superior to single agent ONC201 treatment in H3K27M mutant DIPG. Six patient-derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36) were exposed to imipridones alone or combinations with histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide. Dose-dependent response to imipridones was observed in DIPG cells with half-maximal inhibitory concentration (IC50) of 1.46 µM, 0.11 µM, and 0.03 µM, for ONC201, ONC206, and ONC212, respectively. Upon treatment with the imipridones, DIPG cell lines engaged CLpP/CLPX, the integrated stress response with ATF4 activation, and TRAIL death receptor 5 (DR5) induction. Strong synergy was identified between ONC201 and HDACi panobinostat (combination index [CI] 0.01), romidepsin (CI 0.08) and proteasome inhibitor marizomib (CI 0.19). Synergy was demonstrated between ONC201 and etoposide (CI 0.54), although to a lesser degree than with panobinostat, romidepsin, and marizomib. ONC206 and ONC212 showed similar synergistic effects with panobinostat, romidepsin, and marizomib. Induction of apoptosis was demonstrated with imipridones and panobinostat or romidepsin combinations. Our results suggest increased sensitivity of H3K27M-mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is synergistic cell death with combination of imipridones and panobinostat, romidepsin, or marizomib, which may be further tested in vivo and in clinical trials.

Correction: Discovery and clinical introduction of first-in-class imipridone ONC201.

[This corrects the article DOI: 10.18632/oncotarget.11814.].

Pharmacological Characterization of the Imipridone Anticancer Drug ONC201 Reveals a Negative Allosteric Mechanism of Action at the D2 Dopamine Receptor.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated ß-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

Targeting dopamine receptor D2 as a novel therapeutic strategy in endometrial cancer.

BACKGROUND: ONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer. METHODS: Cell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1fl/flp53fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer. RESULTS: Increasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1fl/fl/p53fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice. CONCLUSION: ONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.

Epidermal growth factor receptor as a molecular determinant of glioblastoma response to dopamine receptor D2 inhibitors.

BACKGROUND: There are ongoing clinical trials exploring the efficacy of dopamine receptor D2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. METHODS: The Cancer Genome Atlas glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and epidermal growth factor receptor (EGFR) expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient-derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201. RESULTS: Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD2 mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, variant (v)III, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (0.037). CONCLUSIONS: High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

ONC206, an Imipridone Derivative, Induces Cell Death Through Activation of the Integrated Stress Response in Serous Endometrial Cancer In Vitro.

ONC206 (Oncoceutics) is an imipiridone with nanomolar potency and analogue of ONC201, a selective dopamine receptor D2 (DRD2) antagonist currently being investigated in phase II clinical trials for serous endometrial cancer (SEC). This study investigated the anti-proliferative efficacy of ONC206 in SEC cell lines as well as its impact on cellular stress and adhesion/invasion. ONC206 inhibited cellular proliferation in a dose-dependent manner and was more potent than ONC201 in the ARK1 (IC50 = 0.33µM vs. IC50 = 1.59uM) and SPEC-2 (IC50 = 0.24uM vs. IC50 = 0.81uM) cell lines. Treatment with ONC206 resulted in induction of ROS production and reduction of mitochondrial membrane potential, accompanied by an increase in cleaved caspase-3 and caspase-9 activity (p < 0.01). ONC206 also significantly inhibited cellular adhesion and migration in both cell lines (p < 0.01). Pretreatment with the stress inhibitor N-acetylcysteine (NAC) significantly attenuated the efficacy of ONC206 on cell proliferation, ROS production and cellular invasion. ONC206 demonstrates nanomolar potency for the inhibition of proliferation in SEC cells. Specifically, ONC206 utilizes ISR activation as a significant pathway in the propagation of its anti-proliferative and anti-metastatic effects. Thus, ONC206 may be a promising agent in future SEC clinical trials as was its predecessor ONC201.