Dopamine Receptor D2 Antagonism Research Articles

Dopamine receptor D2 antagonism normalizes profibrotic macrophage-endothelial crosstalk in non-alcoholic steatohepatitis

Currently there is no effective treatment for liver fibrosis, which is one of the main histological determinants of non-alcoholic steatohepatitis (NASH). While Hippo/YAP (Yes-associated protein) signaling is essential for liver regeneration, its aberrant activation frequently leads to fibrosis and tumorigenesis. Unravelling "context-specific" contributions of YAP in liver repair might help selectively bypass fibrosis and preserve the pro-regenerative YAP function in hepatic diseases. We used murine liver fibrosis and minipig NASH models, and liver biopsies from patients with cirrhosis. Single-cell RNA-sequencing (scRNA-Seq) was performed, and a G-protein-coupled receptor (GPCR) ligand screening system was used to identify cell-selective YAP inhibitors. YAP levels in macrophages are increased in the livers of humans and mice with liver fibrosis. The increase in type I interferon and attenuation of hepatic fibrosis observed in mice specifically lacking Yap1 in myeloid cells provided further evidence for the fibrogenic role of macrophage YAP. ScRNA-Seq further showed that defective YAP pathway signaling in macrophages diminished a fibrogenic vascular endothelial cell subset that exhibited profibrotic molecular signatures such as angiocrine CTGF and VCAM1 expression. To specifically target fibrogenic YAP in macrophages, we utilized a GPCR ligand screening system and identified a dopamine receptor D2 (DRD2) antagonist that selectively blocked YAP in macrophages but not hepatocytes. Genetic and pharmacological targeting of macrophage DRD2 attenuated liver fibrosis. In a large animal (minipig) NASH model recapitulating human pathology, the DRD2 antagonist blocked fibrosis and restored hepatic architecture. DRD2 antagonism selectively targets YAP-dependent fibrogenic crosstalk between macrophages and CTGF+VCAM1+ vascular niche, promoting liver regeneration over fibrosis in both rodent and large animal models. Fibrosis in the liver is one of the main histological determinants of non-alcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes. Our study demonstrates that a macrophage-specific deficiency in Yes-associated protein (YAP) attenuates liver fibrosis. Dopamine receptor D2 (DRD2) antagonism selectively blocks YAP in macrophages and thwarts liver fibrosis in both rodent and large animal models, and thus holds potential for the treatment of NASH.

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

BackgroundONC201 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.MethodsCell 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.ResultsIncreasing 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.ConclusionONC201 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.

DDRE-33. MOLECULAR DIFFERENTIATION OF IMIPRIDONES ONC201 AND ONC206

Abstract ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) and agonist of ClpP in oncology. In clinical trials, the small molecule has induced durable tumor regressions and clinical benefit in H3 K27M-mutant glioma patients while being well tolerated. ONC206 is a chemical derivative of ONC201 with nanomolar anti-cancer potency. In this study, we describe receptor pharmacology, gene expression profiling, acquired resistance and biodistribution studies that suggest ONC206 exhibits distinct therapeutic properties relative to ONC201. ONC206 exhibited a nanomolar Ki for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. Six residues were critical for ONC201 and ONC206, however the impact varied between the two compounds and one allosteric residue was exclusive to ONC206. Structural mapping revealed that some ONC206-critical allosteric residue interactions are located at the interface of TM-IV and –V that mediates the DRD2 homodimer interface. Gene expression profiling revealed ONC206 and ONC201 induce distinct signatures in U87 glioblastoma cells, further supporting distinct functional effects. Similarly, T98G glioblastoma cells with acquired resistance to ONC201 or ONC206 reveal partial cross resistance. Finally, rat biodistribution studies revealed nanomolar CSF concentrations that exceed therapeutic thresholds, unlike ONC201. In summary, ONC206 exhibits increased non-competitive DRD2 antagonism, nanomolar potency, distinct biodistribution, differentiated gene expression and disruption of DRD2 dimers relative to ONC201. Thus, ONC206 may be uniquely poised to address tumors that are not addressed by ONC201 or have developed acquired resistance.

Abstract 5688: IND-enabling characterization of ONC206 as the next bitopic antagonist for oncology

Abstract ONC201 is the first clinical bitopic antagonist of dopamine receptor D2 (DRD2), that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 that shares the imipridone core structure, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, high specificity, nanomolar potency, and disruption of DRD2 homodimers. ONC206 exhibited a Ki of ~320nM for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. Six residues were critical for ONC201 and ONC206, however the impact varied between the two compounds and one allosteric residue was exclusive to ONC206 located at the region that mediates the DRD2 homodimer interface. Gene expression profiling revealed ONC206 and ONC201 (upon 200nM treatment, 72 h) induce distinct signatures in U87 glioblastoma cells, further supporting distinct functional effects. Cell lines resistant to ONC201 and ONC206 are being generated to profile acquired-resistance mechanisms. Broad nanomolar efficacy of ONC206 (GI50 <78-889nM, 72h) was observed in >1,000 GDSC cancer cell lines with the highest sensitivity in cell lines exhibiting a DRD2+/DRD5- RNA expression signature. ONC206 reduced the viability of normal human fibroblasts at higher doses (GI50 > 5µM), suggesting a wide therapeutic window. Antitumor efficacy without body weight loss was observed with 50 mg/kg weekly oral ONC206 in a dopamine-secreting HuCCT1 cholangiocarcinoma subcutaneous xenograft model. Oral ONC206 at 50mg/kg exhibited a ~12 µM plasma Cmax and ~6 hours terminal half-life in Sprague-Dawley rats. Additionally, 5-10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma. Nanomolar concentrations were also observed in the CSF above DRD2 antagonism thresholds, unlike ONC201. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs revealed no dose-limiting toxicities. Mild and reversible body weight changes were observed at the highest evaluated dose in both species. The no observed adverse effect level was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats that exceed efficacious doses. A 50 mg starting dose of ONC206 was selected for the first-in-human clinical trial in biomarker-enriched adult recurrent CNS tumors. In summary, ONC206 is poised for clinical introduction as the next imipridone bitopic DRD2 antagonist for oncology that exhibits differentiated target engagement, signaling, and biodistribution profiles. Citation Format: Varun Vijay Prabhu, Sara Morrow, Caroline A. Cuoco, Abed R. Kawakibi, Jinkyu Jung, Neel Madhukar, Matthew J. Garnett, Ultan McDermott, Cyril H. Benes, Robert Wechsler-Reya, Lakshmi Anantharaman, Neil Charter, Joseph B. Rucker, Benjamin J. Doranz, Joel Basken, Olivier Elemento, R. Benjamin Free, David R. Sibley, Martin Stogniew, Wolfgang Oster, Mark R. Gilbert, Sharon DeMorrow. IND-enabling characterization of ONC206 as the next bitopic antagonist for oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5688.

Abstract 3173: Predictive biomarker evaluation for the anti-cancer imipridone ONC201

Abstract Small molecule imipridone ONC201 is a selective, mixed competitive and non-competitive antagonist of dopamine receptor D2 (DRD2), a GPCR that is overexpressed in a broad range of malignancies (Madhukar et al, Nature Communications, 2019). ONC201 was recently found to also act as an allosteric agonist of the mitochondrial protease ClpP that degrades members of the electron transport chain and other mitochondrial substrates (Ishizawa et al, Cancer Cell 2019). ONC201 is currently being tested in several Phase II clinical trials with evidence of clinical activity in DRD2-dysregulated tumor types, including high-grade glioma, endometrial cancer, mantle cell lymphoma and adrenal tumors. Previously, we reported that DRD2 and DRD5 expression correlates with ONC201 efficacy in the NCI60 and GDSC panels (>1000 cancer cell lines) (Prabhu et al, Clinical Cancer Research, 2019). In this study, we explored additional predictive biomarkers of response for ONC201, including ClpP and known downstream effects of ONC201 across the NCI60 and GDSC cancer cell line panels. Elevated ClpP expression emerged as a top predictive biomarker for ONC201 anti-cancer efficacy (low IC50) in the NCI60 and GDSC (p = 0.0066 and p = 0.00071, respectively) panels. Upregulation of HIF1 expression is known to drive cancer cells toward glycolysis; conversely, its loss shunts cellular energetics toward oxidative phosphorylation as the primary mechanism of ATP generation (Clark et al, Cell, 2019) that is inhibited by ONC201 via ClpP activation. Accordingly, decreased HIF1a expression correlated with increased anti-cancer efficacy in the NCI60 (p = 0.058) and GDSC (p = 0.0021) panels. ONC201 is known to cause downstream activation of the integrated stress response pathway, triggering induction of ATF4 (Ishizawa et al, Science signaling, 2016). High baseline ATF4 expression correlated with lower ONC201 IC50s in the GDSC panel (p = 0.027). Proteasomal degradation of c-myc by ONC201 has been reported in association with DRD2 antagonism and inhibition of oxidative phosphorylation in GBM (Ishida et al Clinical Cancer Research, 2018). High c-myc expression correlated with lower ONC201 IC50s in the GDSC panel (p = 2.5e-6). Ongoing studies are investigating biomarker combinations and identifying tumor types that are enriched for these predictive biomarkers. In conclusion, a series of target and downstream signaling biomarkers can be used to predict innate ONC201 tumor cell sensitivity that may provide guidance for patient and tumor type selection in future ONC201 clinical trials. Citation Format: Sara Morrow, Varun Vijay Prabhu, Joshua E. Allen, Wolfgang Oster. Predictive biomarker evaluation for the anti-cancer imipridone ONC201 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3173.

Abstract 5314: A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Abstract Objectives: Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for patients with homologous recombination (HR) deficient tumors. While HR mutations are rare in endometrial cancers (ECs), emerging data suggests that loss of function of the tumor suppressor gene Phosphatase and Tensin homolog (PTEN), which is mutated in 80% of endometrioid ECs, may also sensitize tumors to PARP inhibition. The imipridone ONC206, a chemical derivative of ONC201, is an orally bioavailable dopamine receptor D2 (DRD2) antagonist that has anti-tumorigenic effects in EC via induction of apoptosis and activation of the integrated stress response. ONC206 exhibits enhanced non-competitive DRD2 antagonism, potency, and bioavailability relative to ONC201. Both olaparib and imipridones (i.e. ONC201) are being evaluated in EC clinical trials, but have yet to be explored in combination. Thus, we sought to examine the effects of olaparib in combination with ONC206 in human endometrioid EC cell lines. Methods: The ECC-1 and HEC-1 EC cell lines were used. Cell proliferation was assessed after exposure to varying doses of olaparib (MedChemExpress) and ONC206 (Oncoceutics) via the MTT assay. Synergy between olaparib and ONC206 was assessed by the combination index (CI) method of Chou-Talalay. Cellular stress was evaluated using the DCFH-DA assay. Apoptosis was assessed using the cleaved caspase-3 assay. Western immunoblotting was performed to determine effects of olaparib and ONC206 on apoptosis and the mTOR pathway. Results: Both drugs inhibited cell proliferation in a dose-dependent manner (IC50 for olaparib of 15 uM for ECC-1 and 25 uM for HEC-1; IC50 for ONC206 of 3.3 uM for ECC-1 and 0.9 uM for HEC-1). Simultaneous exposure of OC cells to ONC206 and olaparib at low doses resulted in synergistic anti-proliferative effects (CI<1). Combination treatment with ONC206 and olaparib at low doses increased cellular stress and apoptosis in both cell lines (p=0.05-0.001), compared to either drug alone. Dual therapy with olaparib and ONC206 decreased expression of the anti-apoptotic protein Bcl-2 and decreased phosphorylation of S6, a downstream target of the mTOR pathway, with greater effects compared to either drug alone. Conclusions: Olaparib and ONC206 had synergistic anti-proliferative effects in human endometrioid EC cell lines, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in EC. Citation Format: Sarah E. Paraghamian, Gabrielle M. Hawkins, Wenchuan Sun, Yali Fan, Xin Zhang, Hongyan Suo, Varun Vijay Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria Bae-Jump. A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5314.

Abstract 4808: ONC201 decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, intregrated stress response and tumor cell death

Abstract Brain tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy and temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SU-DIPG-IV and SU-DIPG-36) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy and temozolomide up to 100 μM. We observed synergy between ONC201 and radiation and temozolomide by multiple assays with the best combination index of 0.31 under the treatment of 0.625 μM ONC201, 25 μM TMZ and 4 Gy irradiation in cell viability assay. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. Mitochondrial dysfunction induced by ONC201 was greatly reflected in reduction of maximal cell respiration in brain tumor cell lines. ClpXP is a proteolytic machine in mitochondria comprising of the protein chaperone ClpX and protease ClpP, a recently described binding target of ONC201. We have observed that ONC201 induced cytotoxicity depends on ClpP and knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated (but not other drugs, e.g. temozolomide) cytotoxicity. The ClpX protein level decreased dramatically upon ONC201 treatment. We are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers. The results implicate ClpX and ClpP in downstream cell death induction following ONC201 treatment of brain tumor cells. Citation Format: Lanlan Zhou, Howard P. Safran, Michael J. Glantz, Wafik S. El-Deiry. ONC201 decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, intregrated stress response and tumor cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4808.

Genotoxicity and carcinogenicity risk assessment of prucalopride, a selective 5-hydroxytryptamine 4 receptor agonist

Prucalopride, a high affinity, selective serotonin type 4 (5-HT4) receptor agonist, was associated with increased neoplasia incidence (in endocrine tissues and liver) in 2-year rodent bioassays, without evidence of a genotoxic mechanism of action. Proposed mechanisms of action involve prolactin and the constitutive androstane receptor (CAR). Epigenetic mechanisms and their relevance to humans are discussed. Data from in vitro and in vivo rodent studies demonstrated that prucalopride-related stimulation of prolactin secretion (via dopamine receptor D2 antagonism at high doses) is a rodent-specific, non-genotoxic mechanism for inducing hyperplasia and neoplasia in prolactin receptor-expressing endocrine tissues. Additional data demonstrated that CAR-mediated liver enzyme induction underlies the observed hepatocellular adenomas and thyroid follicular adenomas in rodents. A 12-month neonatal mouse carcinogenicity study confirmed the lack of a genotoxic mechanism of action. Furthermore, tumors were observed only at very high exposures (200 and 63 fold higher in mice and rats, respectively, than human exposure after a daily therapeutic dose of 2 mg). The studies indicate that non-genotoxic, rodent-specific, epigenetic mechanisms that are considered clinically irrelevant are responsible for the increased incidence of neoplasias associated with very high exposure to prucalopride in rodents, and that prucalopride does not pose a carcinogenic safety risk to humans.

DDIS-20. IMIPRIDONE STRUCTURE ACTIVITY RELATIONSHIP UNCOVERS ONC206 AS THE NEXT BITOPIC DRD2 ANTAGONIST FOR ONCOLOGY WITH DIFFERENTIATED RECEPTOR PHARMACOLOGY

Abstract ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) in oncology clinical trials, which have shown that the small molecule is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC201 possesses a unique tri-heterocyclic core structure that prompted a medicinal chemistry exploration following its successful clinical translation. Chemical derivatization of the ONC201 pharmacophore from an angular to a linear isomer ablated DRD2 antagonist activity and anti-cancer activity, indicating the core structure integrity is critical. A series of analogs that share the same core structure, called imipridones, were profiled for modulation of β-arrestin recruitment to GPCRs and anti-cancer efficacy. The profiled imipridones exhibited a heterogeneous spectrum of GPCR agonist/antagonist activity that was exclusive to Class A GPCRs. The addition of electron withdrawing groups to one of two peripheral benzyl rings enhanced the potency of GPCR engagement and anti-cancer effects, while derivatization of the other benzyl ring was inactivating. Among the GPCR hits identified, maximal variance in imipridone GPCR antagonism was identified for DRD2/DRD3. ONC206 emerged as the most selective and potent antagonist for DRD2/DRD3 with a Ki of ~320nM for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. Six residues were critical for ONC201 and ONC206, however the impact varied between the two compounds and one allosteric residue was exclusive to ONC206. Structural mapping revealed that some ONC206-critical allosteric residue interactions are located at the interface of TM-IV and –V that mediates the DRD2 homodimer interface. Thus, ONC206 is a bitopic DRD2 antagonist that may be poised to address oncogenic DRD2 monomers or dimers.

EXTH-57. PRECLINICAL COMBINATION OF ONC201 WITH RADIOTHERAPY OR TEMOZOLOMIDE IN GBM, DIPG AND ATRT CELL LINES RESULTS IN DOPAMINE RECEPTOR ANTAGONISM, ATF4 INDUCTION AND CELL DEATH

Abstract CNS tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy or temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SF8628) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy or temozolomide up to 100 μM. We observed synergy between ONC201 and radiation or temozolomide by multiple assays. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. We are evaluating the relevance of mitochondrial protease ClpP, a recently described binding target of ONC201, in ONC201-induced glioma cell death. We have observed that knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated cytotoxicity. Thus, we are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers.

EXTH-33. RECEPTOR PHARMACOLOGY OF ONC201: THE FIRST BITOPIC DRD2 ANTAGONIST FOR CLINICAL NEURO-ONCOLOGY

Abstract ONC201 has exhibited durable tumor regressions in midline high grade glioma patients. G protein-coupled receptor (GPCR) dopamine receptor D2 (DRD2) is overexpressed in high grade gliomas, controls several oncogenic mechanisms, and its antagonism causes tumor cell apoptosis. Using bioinformatic and GPCR screens, we previously demonstrated selective DRD2/3 antagonism by ONC201 at therapeutic concentrations that translates into a wide therapeutic window, unlike antipsychotics that also target other dopamine receptors and GPCRs (Prabhu et al. CCR 2019 and Madhukar et al. Nat Comm in press). Here, we identify hallmarks of non-competitive DRD2 antagonism by ONC201 in β-arrestin, cAMP and radioligand competition assays. ONC201 caused both a shift in the IC50 and repression of Emax in the dopamine-dose-response curves for DRD2. Schild analysis and fitting these data to an operational model of allostery were consistent with non-competitive antagonism. Alanine scanning mutagenesis of DRD2 identified 6 orthosteric and allosteric residues that are critical for ONC201-mediated antagonism. The residues identified were predominantly conserved in DRD2/3 and were not relevant for antagonism by antipsychotics further explaining the unique selectivity of ONC201. Molecular docking revealed orthosteric interactions at TM-II and an extended binding pocket into an allosteric site. Other critical residues were clustered at an allosteric area at the interface of TM-IV and –V that has been shown to mediate the DRD2 homodimer interface. Point mutations at either site decreased the affinity of ONC201 in competition assays with radiolabeled methyl-spiperone, suggesting cooperativity between these topographically distinct sites. In summary, ONC201 causes a mixed non-competitive and competitive antagonism of DRD2 consistent with a bitopic mechanism of action that may explain its unique selectivity, safety, and anti-cancer activity in clinical trials as the first compound to target this receptor for clinical neuro-oncology. Non-competitive DRD2 antagonism may be critical for ONC201 anti-tumor efficacy in dopamine-rich microenvironments such as midline gliomas.

Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201. The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies. DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes. These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

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

Abstract 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-naïve 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 <.05). Furthermore, a missense DRD5 mutation was identified in cancer cells with acquired resistance to ONC201. Resistance could be recapitulated with overexpression of the mutant or wild-type DRD5 gene. A significant induction of serum prolactin, a surrogate biomarker of target engagement, was detected upon ONC201 administration to recurrent glioblastoma patients. Intratumoral drug concentrations surpassed therapeutic levels, ranging from ~0.6-10µM at 24 hours post-dose. Immune effector levels in the serum correlated with the kinetics of a durable objective response observed in a patient with an H3.3 K27M glioma. Among the 15 available archival tumor tissue specimens, all had expression of DRD2 and 8/17 patients had low expression of DRD5. Patients with PFS>5 month had no detectable expression of DRD5, unlike those with PFS<5 months. In addition, 4/8 DRD2+DRD5- and 0/7 DRD2+DRD5+ patients are still alive with a median follow-up of 47.4 weeks. Conclusion: The dopamine receptor pathway is a novel therapeutic target that is dysregulated in glioblastoma and provides predictive and pharmacodynamic biomarkers of tumor sensitivity to ONC201. 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.

DDIS-08. THE SMALL MOLECULE IMIPRIDONE ONC201 IS ACTIVE IN GLIOBLASTOMA WITH DRD2 PATHWAY DYSREGULATION

ONC201, an imipridone that is a selective antagonist of the G protein-coupled receptors dopamine receptor D2 (DRD2) and D3 (DRD3), has exhibited tumor shrinkage and an exceptional safety profile in a phase II recurrent glioblastoma clinical trial (Arrillaga et al, 2017). In vitro and in vivo studies have previously demonstrated single agent ONC201 efficacy in glioblastoma models (Allen et al 2013). In vitro efficacy profiling of ONC201 in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines confirmed broad anti-cancer efficacy with high sensitivity in human brain cancer cell lines. DRD2 is overexpressed in glioblastoma and DRD2 antagonism induces tumor cell apoptosis via the same signaling pathways that respond to ONC201. Investigation of The Cancer Genome Atlas (TCGA) revealed that DRD2 is highly expressed in glioblastoma relative to other dopamine receptor family members and that genetic aberrations were rare. High expression of DRD2 occurred in primary, rather than secondary, glioblastoma and was associated with a poor prognosis. Immunohistochemistry analyses 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. A significant induction of serum prolactin, a surrogate biomarker of target engagement, was detected in ONC201-treated glioblastoma patients. 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 <.05). Furthermore, a missense DRD5 mutation was identified in cancer cells with acquired resistance to ONC201. Resistance could be recapitulated with overexpression of the mutant DRD5 gene or, to a lesser extent, with the wild-type gene. In conclusion, the DRD2 pathway is a therapeutic target that is dysregulated in glioblastoma and contains biomarkers of tumor sensitivity to ONC201 that are under clinical evaluation.

Abstract 2792: The small molecule imipridone ONC201 is active in tumor types with dysregulation of the DRD2 pathway

Abstract ONC201 is the lead small molecule of the imipridone class of anti-cancer compounds that is currently being evaluated in phase I/II advanced cancer clinical trials. ONC201 is a highly selective antagonist of the G protein-coupled receptor dopamine receptor D2 (DRD2) that has exhibited promising anti-cancer efficacy and an exceptional safety profile. In the current study, we evaluated the influence of the DRD2 pathway on the responsiveness of tumors to ONC201 in preclinical and clinical studies. In vitro and in vivo studies have previously demonstrated robust ONC201 efficacy in glioblastoma (Allen et al 2013) and lymphoma (Ishizawa et al 2016) models. ONC201 Phase I trials have also revealed evidence of clinical benefit in endometrial cancer (Stein et al 2016). In vitro efficacy profiling of ONC201 in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines confirmed broad-spectrum anti-cancer efficacy with particularly high sensitivity in lymphoma, neuroblastoma, endometrial and brain cancer. DRD2 is overexpressed in many cancers and DRD2 antagonism kills cancer cells via the same signaling pathways that are altered in response to ONC201. Results from the Project Achilles screen indicate that anti-cancer effects of DRD2 knockdown in various tumor types correlated with overall ONC201 efficacy. In particular, we noted that lymphoma cells are highly sensitive to DRD2 knockdown- a tumor type where ONC201 performs well. Gene expression analysis of samples in the Cancer Genome Atlas (TCGA) revealed high DRD2 expression in ONC201-sensitive tumor types, such as lymphoma and glioblastoma, and that high expression of DRD2 in glioma was associated with a poor prognosis. High DRD2 expression was also observed in neuroendocrine prostate cancer relative to other prostate cancer subtypes. In immunohistochemistry analyses of patient-derived tumor tissue microarrays, DRD2 overexpression was particularly noted in endometrial cancer, neuroblastoma and pheochromocytoma relative to normal tissues. The anti-cancer activity of ONC201 in pheochromocytoma and neuroendocrine prostate cancer was confirmed in cell viability assays. In ONC201-treated patients, ELISA was used to quantitate serum prolactin levels, a clinical biomarker of DRD2 antagonism. A 2-fold mean induction of prolactin, was detected in the serum of ONC201-treated patients, in accordance with physiological DRD2 antagonism. Interestingly, expression of DRD5 (a D1-like dopamine receptor), which counteracts DRD2 signaling, was significantly negatively correlated with ONC201 in vitro potency in the NCI60 and GDSC dataset (P &amp;lt;.05). Furthermore, a missense DRD5 mutation was identified in cancer cells with acquired resistance to ONC201. In conclusion, the DRD2 pathway is expressed in ONC201-sensiive tumors and may provide biomarkers of response. Citation Format: Neel Madhukar, Varun Vijay Prabhu, Etienne Dardenne, Faye Doherty, Alexander VanEngelenburg, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Wolfgang Oster, Wafik El-Deiry, Mark Stein, David Rickman, Joshua Allen, Olivier Elemento. The small molecule imipridone ONC201 is active in tumor types with dysregulation of the DRD2 pathway [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 2792. doi:10.1158/1538-7445.AM2017-2792