DRD2 Antagonist Research Articles

Therapeutic strategies for colorectal cancer: antitumor efficacy of dopamine D2 receptor antagonists.

Colorectal cancer (CRC) is one of the leading causes of death, accounting for more than half a million deaths annually. Even worse, an increasing number of cancer cases are diagnosed yearly, and two and a half million new cancer cases are estimated to be diagnosed in 2035. Some antipsychotic drugs, especially those targeting dopamine receptor (DR) D2, demonstrated anticancer activity. Studies have revealed the potential of DRD2 antagonists as anticancer therapeutics, whether alone or as an adjuvant, in treating breast cancer, lung cancer, and others. Emerging evidences indicate DRD2 is involved in the CRC biology, and the association between DRD2 and CRC could be utilized in treating CRC. This study selected DRD2 antagonists with anticancer activity to elucidate the possibility of DRD2 antagonists as new therapeutics for treating CRC.

Selective DRD2 antagonist and ClpP agonist ONC201 in a recurrent non-midline H3 K27M-mutant glioma cohort.

Diffuse midline glioma, H3 K27-altered (H3 K27M-altered DMG) are invariably lethal, disproportionately affecting the young and without effective treatment besides radiotherapy. The 2016 World Health Organization (WHO) Central Nervous System (CNS) Tumors Classification defined H3 K27M mutations as pathognomonic but restricted diagnosis to diffuse gliomas involving midline structures by 2018. Dordaviprone (ONC201) is an oral investigational small molecule, DRD2 antagonist, and ClpP agonist associated with durable responses in recurrent H3 K27M-mutant DMG. Activity of ONC201 in non-midline H3 K27M-mutant diffuse gliomas has not been reported. Patients with recurrent non-midline H3 K27M-mutant diffuse gliomas treated with ONC201 were enrolled in 5 trials. Eligibility included measurable disease by Response Assessment in Neuro-Oncology (RANO) high-grade glioma, Karnofsky/Lansky performance score ≥60, and ≥90 days from radiation. The primary endpoint was overall response rate (ORR). Five patients with cerebral gliomas (3 frontal, 1 temporal, and 1 parietal) met inclusion. One complete and one partial response were reported by investigators. Blinded independent central review confirmed ORR by RANO criteria for 2, however, 1 deemed nonmeasurable and another stable. A responding patient also noted improved mobility and alertness. H3 K27M-mutant diffuse gliomas occasionally occur in non-midline cerebrum. ONC201 exhibits activity in H3 K27M-mutant gliomas irrespective of CNS location.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

Domperidone Exerts Antitumor Activity in Triple-Negative Breast Cancer Cells by Modulating Reactive Oxygen Species and JAK/STAT3 Signaling.

The lack of molecular targets hampers the treatment of triple-negative breast cancer (TNBC). In this study, we determined the cytotoxicity of domperidone, a dopamine D2 receptor (DRD2) antagonist in human TNBC BT-549 and CAL-51 cells. Domperidone inhibited cell growth in a dose- and time-dependent manner. The annexin V/propidium iodide staining showed that domperidone induced apoptosis. The domperidone-induced apoptosis was accompanied by the generation of mitochondrial superoxide and the down-regulation of cyclins and CDKs. The apoptotic effect of domperidone on TNBC cells was prevented by pre-treatment with Mito-TEMPO, a mitochondria-specific antioxidant. The prevention of apoptosis with Mito-TEMPO even at concentrations as low as 100 nM, implies that the generation of mitochondrial ROS mediated the domperidone-induced apoptosis. Immunoblot analysis showed that domperidone-induced apoptosis occurred through the down-regulation of the phosphorylation of JAK2 and STAT3. Moreover, domperidone downregulated the levels of D2-like dopamine receptors including DRD2, regardless of their mRNA levels. Our results support further development of DRD2 antagonists as potential therapeutic strategy treating TNBC.

Dopamine D2 receptor antagonist modulates rTMS-induced pain experiences and corticospinal excitability dependent on stimulation targets

Both the primary motor cortex (M1) and dorsolateral prefrontal cortex (DLPFC) rTMS have the potential to reduce certain chronic pain conditions. However, the analgesic mechanisms remain unclear, in which M1- and DLPFC-rTMS may have different impact on the release of dopamine receptor D2 neurotransmissions (DRD2). Using a double-blind, randomised, sham- and placebo-controlled design, this study investigated the influence of DRD2 antagonist on rTMS-induced analgesia and corticospinal excitability across the M1 and DLPFC. Healthy participants in each group (M1, DLPFC, or Sham) received an oral dose of chlorpromazine or placebo before the delivery of rTMS in two separate sessions. Heat pain and cortical excitability were assessed before drug administration and after rTMS intervention. DRD2 antagonist selectively abolished the increased heat pain threshold induced by DLPFC stimulation and increased pain unpleasantness. The absence of analgesic effects in DLPFC stimulation was not accompanied by plastic changes in the corticospinal pathway. In contrast, DRD2 antagonist increased corticospinal excitability and rebalanced excitation-inhibition relationship following motor cortex stimulation, although there were no clear changes in pain experiences. These novel findings together highlight the influence of dopaminergic neurotransmission on rTMS-induced analgesia and corticospinal excitability dependent on stimulation targets.

Dopamine receptors D1 and D2 show prognostic significance and potential therapeutic applications for endometrial cancer patients

ObjectiveCatecholaminergic signaling has been a target for therapy in different type of cancers. In this work, we characterized the ADRβ2, DRD1 and DRD2 expression in healthy tissue and endometrial tumors to evaluate their prognostic significance in endometrial cancer (EC), unraveling their possible application as an antitumor therapy. Methods109 EC patients were included. The expression of the ADRβ2, DRD1 and DRD2 proteins was evaluated by immunohistochemistry and univariate and multivariate analysis to assess their association with clinic-pathological and outcome variables. Finally, HEC1A and AN3CA EC cell lines were exposed to different concentrations of selective dopaminergic agents alone or in combination to study their effects on cellular viability. ResultsADRβ2 protein expression was not associated with clinico-pathological parameters or prognosis. DRD1 protein expression was reduced in tumors samples but showed a significant inverse association with tumor size and stage. DRD2 protein expression was significantly associated with non-endometrioid EC, high grade tumors, tumor size, worse disease-free survival (HR = 3.47 (95%CI:1.35–8.88)) and overall survival (HR = 2.98 (95%CI:1.40–6.34)). The DRD1 agonist fenoldopam showed a reduction of cellular viability in HEC1A and AN3CA cells. The exposure to domperidone, a DRD2 antagonist, significantly reduced cell viability compared to the control. Finally, DRD1 agonism and DRD2 antagonism combination induced a significant reduction in cell viability of the AN3CA cells compared to monotherapy, close to being an additive response than a synergistic effect (CI of 1.1 at 0.5% Fa). ConclusionDRD1 and DRD2 expression levels showed a significant association with clinico-pathological parameters. Both the combined activation of DRD1 and blockage of DRD2 may form an innovative strategy to inhibit tumor growth in EC.

The DRD2 antagonist haloperidol mediates autophagy-induced ferroptosis to increase temozolomide sensitivity by promoting endoplasmic reticulum stress in glioblastoma.

Temozolomide (TMZ) resistance remains a major obstacle in the treatment of glioblastoma (GBM). The combination of TMZ with another agent could offer an improved treatment option if it could overcome chemoresistance and prevent side effects. Here, we determined the critical drug that cause ferroptosis in GBM cells and elucidated the possible mechanism by which drug combination overcomes chemoresistance. Haloperidol/TMZ synergism was assessed in GBM cell lines with different DRD2 expression in vitro and in vivo. Inhibitors of ferroptosis, autophagy, endoplasmic reticulum stress and cAMP/PKA were used to validate the specific mechanisms by which haloperidol and TMZ induce ferroptosis in GBM cells. In the present work, we demonstrate that the dopamine D2 receptor (DRD2) level is increased by TMZ in a time-dependent manner and is inversely correlated with TMZ sensitivity in GBM. The DRD2 antagonist haloperidol (HP), a butylbenzene antipsychotic, markedly induces ferroptosis and effectively enhances TMZ efficacy in vivo and in vitro. Mechanistically, HP suppressed the effect of TMZ on cAMP by antagonizing DRD2 receptor activity, and the increases in cAMP/PKA triggered endoplasmic reticulum stress, which led to autophagy and ferroptosis. Furthermore, elevated autophagy mediates downregulation of FTH1 expression at the posttranslational level in an autophagy-dependent manner and ultimately leads to ferroptosis. Our results provide experimental evidence for repurposing HP as an effective adjunct therapy to inhibit adaptive TMZ resistance in order to enhance the efficacy of chemoradiotherapy in GBM, a strategy that may have broad prospects for clinical application.

ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

Rasd2 Mediates Acute Fasting-Induced Antidepressant-Like Effects via Dopamine D2 Receptor Activation in Ovariectomized Mice.

Previous studies have shown that estrogen and acute fasting for 9 hours have antidepressant-like effects by reducing immobility time in the forced swimming test. Estrogen and acute fasting share a common regulatory gene, Rasd2. RASD2 regulates dopamine D2 receptor (DRD2) transmission, but the role of Rasd2 in the DRD2-mediated antidepressant-like effect of acute fasting has not been examined. In this study, open field test, forced swimming test, tail suspension test and sucrose preference test were used for behavioral assessments. RNA-seq, western blot, enzyme-linked immunosorbent assay, and co-immunoprecipitation were used to explore the role of Rasd2 in a depression model induced by ovariectomy and the antidepressant-like effects of 9-hour fasting. The RNA seq results showed that acute fasting induced a significant change in Rasd2 gene expression. Depression-like behaviors induced by ovariectomy were associated with decreased RASD2 and DRD2 protein levels in the hippocampus, and Rasd2 overexpression in the hippocampus alleviated depression-like behaviors and increased DRD2 expression. Nine-hour fasting had antidepressant-like effects in ovariectomized mice by upregulating the protein levels of RASD2, DRD2, CREB-BDNF, Akt, and estrogen receptor beta, and these effects can be blocked by DRD2 antagonists. Our results suggest that Rasd2 and DRD2 play pivotal roles in depression-like behavior induced by ovariectomy. Rasd2 regulates DRD2-mediated antidepressant-like effects of acute fasting in ovariectomized mice. Rasd2 can therefore be postulated to be a potential therapeutic target for depression and perhaps also a potential predictive marker for depression.

CTNI-61. CLINICAL EFFICACY AND PREDICTIVE BIOMARKERS OF ONC201 IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Patients with H3K27M-mutated diffuse midline glioma (DMG) have no proven effective therapies beyond radiation. ONC201, a DRD2 antagonist and mitochondrial ClpP agonist, has shown promise in this population. Clinical and genetic variables associated with ONC201 response in H3K27M-mutant DMG continue to be investigated. A combined clinical and genetic study evaluated patients with H3K27M-DMG treated with single-agent ONC201 at the established phase 2 dose. Clinical outcomes of patients treated on two recently completed multi-site clinical studies (NCT03416530 and NCT03134131, n = 75) were compared with historical control data from patients with confirmed H3K27M-DMG (n = 391 total, n = 119 recurrent). Patients treated with ONC201 monotherapy following initial radiation, but prior to recurrence, demonstrated a median overall survival (OS) of 25.6 months from diagnosis and recurrent patients demonstrated a median OS of 16.2 months from recurrence, both of these more than doubling historical outcomes. Using a Cox model to correct for age, gender and tumor location, OS of ONC201-treated patients with H3K27M-mutant tumors remained significantly better than non-ONC201-treated historical controls (p = 0.0001). A survival and radiographic analysis based on tumor location, revealed stronger responses in thalamic patients. In patients with thalamic tumors treated after initial radiation (n = 16), median OS was not reached with median follow up of 22.1 months (historical control median OS of 12.5 months, n = 83, p = 0.0001). Significant correlations were found between baseline cerebral blood flow (CBF) on perfusion imaging and OS (Pearson’s r = 0.75, p = 0.003) and between nrCBF and PFS (r = 0.77, p = 0.002). Baseline tumor sequencing from treated patients (n = 20) demonstrates EGFR mutation (n = 3) and high EGFR expression as a marker of resistance and improved response in tumors with MAPK-pathway alterations (n = 5). In conclusion, ONC201 demonstrates unprecedented clinical and radiographic efficacy in H3K27M-mutant DMG with outcomes enriched in patients with thalamic tumors, treatment prior to recurrence, MAPK-pathway alterations, and patients with relatively high CBF.

SYST-14 CLINICAL EFFICACY OF ONC201 IN RECURRENT H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA (DMG) PATIENTS

Abstract BACKGROUND H3 K27M-mutant DMG predominantly affects children and young adults; no effective therapy is known. ONC201 is a first-in-class, anti-cancer DRD2 antagonist and ClpP agonist. METHODS Fifty pediatric and adult patients with recurrent H3 K27M DMG who received oral ONC201 monotherapy in clinical trials and expanded access were selected for a planned efficacy analysis. Eligibility criteria included measurable contrast-enhancing disease by RANO-HGG criteria (excluding pontine and spinal cord tumors), KPS/LPS≥60, ≥90 days from prior radiation, and adequate washout from prior anti-cancer therapy. The primary endpoint was overall response rate (ORR) by RANO-HGG criteria. Secondary endpoints included duration of response, time to response, progression-free survival (PFS), overall survival (OS), corticosteroid response rate, performance status response rate, and ORR by RANO-LGG criteria. Radiographic endpoints were assessed by dual-reader blinded independent central review. Data cutoff was May 31, 2021. RESULTS ORR was 20.0% (95%CI, 10.0–33.7) by RANO-HGG criteria. Median duration of response was 11.2 months (95%CI, 3.8–not reached) and median time to response was 8.3 months (range, 1.9–15.9). PFS at 6 months was 35.1% (95%CI, 21.2–49.3). The ORR was 26.0% (95%CI, 14.6–40.3) by RANO-LGG criteria. Fifteen patients (30.0%; 95%CI, 17.9–44.6) achieved an objective response by RANO-HGG and/or RANO-LGG criteria. Of 15 patients receiving ≥4 mg daily dexamethasone at baseline, 7 (46.7%; 95%CI, 21.3–73.4) achieved ≥50% confirmed reduction in dose. Of 34 patients with baseline KPS/LPS <80, 7 (20.6%; 95%CI, 8.7–37.9) achieved a confirmed performance status improvement. With a median follow-up of 18.8 months, median OS was 13.7 months (95%CI, 8.0–20.3) and OS at 24 months was 34.7% (95%CI, 20.7–49.2). Twenty-five patients had serious adverse events with one possibly related to ONC201 by investigator assessment. CONCLUSIONS ONC201 monotherapy exhibits durable and clinically meaningful efficacy in recurrent H3 K27M-mutant DMG.

Inhibition of astrocytic DRD2 suppresses CNS inflammation in an animal model of multiple sclerosis.

Astrocyte activation is associated with progressive inflammatory demyelination in multiple sclerosis (MS). The molecular mechanisms underlying astrocyte activation remain incompletely understood. Recent studies have suggested that classical neurotransmitter receptors are implicated in the modulation of brain innate immunity. We investigated the role of dopamine signaling in the process of astrocyte activation. Here, we show the upregulation of dopamine D2 receptor (DRD2) in reactive astrocytes in MS brain and noncanonical role of astrocytic DRD2 in MS pathogenesis. Mice deficient in astrocytic Drd2 exhibit a remarkable suppression of reactive astrocytes and amelioration of experimental autoimmune encephalomyelitis (EAE). Mechanistically, DRD2 regulates the expression of 6-pyruvoyl-tetrahydropterin synthase, which modulates NF-κB activity through protein kinase C-δ. Pharmacological blockade of astrocytic DRD2 with a DRD2 antagonist dehydrocorybulbine remarkably inhibits the inflammatory response in mice lacking neuronal Drd2. Together, our findings reveal previously an uncharted role for DRD2 in astrocyte activation during EAE-associated CNS inflammation. Its therapeutic inhibition may provide a potent lever to alleviate autoimmune diseases.

Abstract 1856: Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206

Abstract ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving agonist activity against mitochondrial ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may compete with ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, lung cancer, GBM, and DIPG with dopamine for 12 hours, 24 hours or 48 hours, respectively, followed by treatment of ONC201 or ONC206 for 72hours. Cell viability was determined by the CellTiter Glo assay. Single treatment with dopamine did not increase cell viability. We observed that 48 hours of pre-treatment with dopamine at 50 μM, 100 μM or 200 μM impaired the cell viability suppression effect of ONC201 at 4 μM and 8 μM, while ONC206 at 0.5 μM, 1 μM and 2 μM led to similar protection for loss of cell viability in pancreatic cancer cells. The impairment of the anti-cancer effect by dopamine was also observed in colorectal cancer cells with 48 hours pre-treatment with dopamine at 50 μM, 100 μM, 200 μM or 400 μM and treatment with ONC201 at 4 μM and 8 μM, or ONC206 at 0.5 μM, 1 μM, or 2 μM. In addition, we treated pancreatic cancer and colorectal cancer cells with ONC212 after 48 hours pre-treatment with dopamine and observed a similar impairment of the anti-cancer effect, even though ONC212 is not a DRD2 antagonist. We pre-treated pancreatic cancer and colorectal cancer cells with specific DRD2 agonist, sumanirole, for 48 hours followed by treatment of ONC201, ONC206, or ONC212 for 72 hours, and no impairment was seen. We pre-treated multiple cancer cell lines with 2 μM or 10 μM dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine at 10 μM impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer. Pre-treatment with dopamine at 2 μM or 10 μM impaired the anti-tumor effect of ONC201 in GBM cells. By contrast to the cell viability result, no impairment of ONC212 effect from dopamine was seen in pancreatic cancer cells. Western blotting was conducted to investigate whether dopamine pre-treatment impacted signaling pathways reported to be affected by ONC201. Dopamine pre-treatment did not impact changes induced by ONC201 in ATF4, CHOP, DR5, ClpX, clpP, pERK, pAkt and pS6. We are pursuing whether the protective effect of dopamine is due to the agonism of DRD2 or alternative pro-survival mechanisms. Citation Format: Yiqun Zhang, Wafik S. El-Deiry. Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1856.

Continuous Activation of Dopamine Receptors Alleviates LPS-Induced Liver Injury in Mice via β-arrestin2 Dependent Akt/NF-κB Pathway.

Many studies showed that dopamine receptors (DRs) agonists have anti-inflammatory effects. Rotigotine, a non-ergot dopamine receptor agonist, mainly actives DRD2/DRD3/DRD1. Rotigotine extended-release microspheres (RoMS) are a sustained-release formulation that can release sustainably rotigotine for more than 7 days after a single dose of RoMS. This study aimed to investigate whether RoMS can attenuate the lipopolysaccharide (LPS)-induced liver injury of mice. The liver injury was evaluated by assaying serum transaminase and observing histopathological changes. The levels of pro-inflammatory cytokines in serum were also detected. Western blot was employed to assay the expression of proteins in the Akt/NF-κB pathway. The results showed that pre-administration with a single dose of RoMS could inhibit the increase of serum transaminase induced by LPS, alleviate the pathological damage of liver tissue, and decrease the levels of tumor necrosis factor-α and interleukin-6. In addition, RoMS decreased Toll-like receptor 4 protein expression in liver tissue. RoMS mitigated liver injury by activating DRs and negatively regulating the β-arrestin2-dependent Akt/NF-κB signaling pathway. The effects of RoMS could be weakened or abolished by the specific DRD2 antagonist, R121. In conclusion, activation of DRs inhibited the releases of pro-inflammatory cytokines and alleviated the immune-mediated liver injury induced by LPS in mice. The anti-inflammatory mechanism of RoMS may be related to the regulation of the β-arrestin2-dependent Akt/NF-κB signaling pathway.

CTNI-27. SINGLE AGENT ACTIVITY OF ONC201 IN NON-MIDLINE H3 K27M-MUTANT DIFFUSE GLIOMAS

Abstract BACKGROUND H3 K27M-mutant diffuse midline glioma is an invariably lethal form of brain cancer that disproportionately affects children and young adults and has no effective treatment following front-line radiation. The initial disease definition in the 2016 WHO Classification of Tumors of the Central Nervous System regarded the H3 K27M mutation as pathognomonic, though the definition was updated in 2018 restricting the diagnosis to histologically diffuse gliomas that involve midline CNS structures (cIMPACT-NOW update 2). ONC201 is an investigational anti- cancer small molecule, DRD2 antagonist and ClpP agonist that has induced durable tumor regressions by RANO-HGG criteria in a registration cohort of recurrent diffuse midline glioma, H3 K27M-mutant patients treated with single agent ONC201. METHODS We present 7 patients with H3 K27M-mutant diffuse gliomas were enrolled in ONC201 clinical studies, excluded from the registration cohort due to involvement of non-midline CNS structures, all within the cerebral hemispheres (3 frontal, 1 temporal, 1 frontotemporal, 1 parietal, and 1 corona radiata). RESULTS Two of the 7 patients underwent objective responses by RANO-HGG criteria as assessed by investigator, which was associated with clinical benefit that included increased mobility and level of alertness. CONCLUSIONS These results demonstrate that H3 K27M-mutant diffuse gliomas occur outside of midline CNS structures, and suggest that ONC201 has single agent activity in H3 K27M-mutant gliomas irrespective of CNS location.

EXTH-30. HARNESSING CELLULAR STRESS FOR IMMUNE TARGETING OF DIPGS

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor. An almost ubiquitous dominant negative mutation at lysine (K)-27 in genes encoding histone genes HIST1H3B and H3F3A found in patient tumors is a driver of DIPG development. ONC201, a small molecule DRD2 antagonist and ClpP agonist developed by Chimerix Inc, targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling. It is under clinical investigation in patients with recurrent H3K27M DMGs. In adults, single agent studies have shown durable objective responses when administered orally. A multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) for pediatric patients with H3K27M DMGs is open and accruing. Preliminary results suggest that the drug has a favorable safety profile and holds promise for patients with DIPGs and other midline gliomas. However, the mechanism of action of ONC201 against DIPGs warrants further study. Here, we show that ONC201 is cytotoxic to DIPGs in vitro and in vivo. RNA Seq analyses revealed cell context specific deployment of PERK-activated UPR and calcium signaling-associated RON tyrosine kinase-macrophage stimulating protein (MSP) signaling in DIPGs. Single cell proteomic assays revealed substantial heterogeneity in the sensitivity of DIPG cells to ONC201, and identified stem-like sub-populations of H3K27M DIPGs with intrinsic insensitivity to the drug. ONC201 treatment, which induces cellular stress, also sensitized DIPG cells to cytolytic activity by ex-vivo expanded and activated innate immune cells, in vitro. Ongoing in vivo experiments are expected to support a novel investigational study in patients with midline gliomas.

CTNI-36. SAFETY OF ONC201 ADMINISTERED TWO CONSECUTIVE DAYS PER WEEK IN PEDIATRIC H3 K27M-MUTANT GLIOMA PATIENTS

Abstract ONC201, an anti-cancer DRD2 antagonist and ClpP agonist, is in Phase II trials for adult H3 K27M-mutant diffuse midline gliomas. The recommended phase 2 dose (RP2D) of 625mg ONC201 once a week has been established as a biologically active dose that is well tolerated in adult and pediatric populations. Radiographic regressions with single agent ONC201 have been reported in recurrent H3 K27M-mutant glioma patients. In another study, twice/week dosing was explored in adult patients and deemed to be safe (no DLTs observed). This warranted exploration of twice/week dosing in pediatric patients and will be discussed in this presentation. This multi-arm, dose-escalation and dose-expansion trial (ONC014; NCT03416530) determined the pediatric RP2D of ONC201 administered once per week and twice per week on two consecutive days. ONC201 was orally administered and scaled by body weight. Dose escalation was performed by a 3 + 3 design beginning with two 125mg capsules less than the adult RP2D equivalent. Twelve children (8 females; 4 males) with H3 K27M-mutant gliomas (pons: 8; thalamus: 2; spinal cord: 2) aged 4-19 years have been treated post-radiation: 3 at dose level -1; 3 at dose level 1; 6 as part of the dose expansion cohort on dose level 2. Median KPS was 90 (range 70-100). One treatment cycle was 21 days (6 doses), which also defined the DLT window. Patients were on-treatment for a median length of 4 cycles (range: 2-11). Twice weekly dosing of ONC201 was tolerated well, as observed with weekly dosing, with no instance of DLT. A total of 4 SAEs were reported, none of which were related to the study drug. The most common AEs (regardless of relatedness) included headache, facial nerve disorder, abducens nerve disorder, nausea, fatigue and ataxia. Additional safety data, PK, and clinical outcomes from this arm will be reported.

Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

Simple SummaryThe present study showed that a prescribed psychotropic medicine paliperidone inhibits GBM growth and prolongs survival in mouse brain tumor model and decreased the programmed death ligand 1 expression. Using the 3D co-culture also found that dopamine receptor D2 regulates the interaction of GBM-macrophage-induced PD-L1 expression in GBMs. In addition, the expression of DRD2 and PD-L1 in GBM modulates tumor-associated macrophage polarization. Our results also indicated that there is a contact-independent mechanism of PD-L1 induction in GBM upon interaction between GBM and monocytes. The present study also found that the interaction of GBM-macrophage-enhanced PD-L1 expression in GBM occurred by modulating the ERK and STAT3 signaling pathways. In addition, the inhibition of DRD2 reduces the upregulation of PD-1 expression, and it is regulating signaling in GBM.A previous study from our group reported that monocyte adhesion to glioblastoma (GBM) promoted tumor growth and invasion activity and increased tumor-associated macrophages (TAMs) proliferation and inflammatory mediator secretion as well. The present study showed that prescribed psychotropic medicine paliperidone reduced GBM growth and immune checkpoint protein programmed death ligand (PD-L)1 expression and increased survival in an intracranial xenograft mouse model. An analysis of the database of patients with glioma showed that the levels of PD-L1 and dopamine receptor D (DRD)2 were higher in the GBM group than in the low grade astrocytoma and non-tumor groups. In addition, GFP expressing GBM (GBM-GFP) cells co-cultured with monocytes-differentiated macrophage enhanced PD-L1 expression in GBM cells. The enhancement of PD-L1 in GBM was antagonized by paliperidone and risperidone as well as DRD2 selective inhibitor L741426. The expression of CD206 (M2 phenotype marker) was observed to be markedly increased in bone marrow-derived macrophages (BMDMs) co-cultured with GBM. Importantly, treatment with paliperidone effectively decreased CD206 and also dramatically increased CD80 (M1 phenotype marker) in BMDMs. We have previously established a PD-L1 GBM-GFP cell line that stably expresses PD-L1. Experiments showed that the expressions of CD206 was increased and CD80 was mildly decreased in the BMDMs co-cultured with PD-L1 GBM-GFP cells. On the other hands, knockdown of DRD2 expression in GBM cells dramatically decreased the expression of CD206 but markedly increased CD80 expressions in BMDMs. The present study suggests that DRD2 may be involved in regulating the PD-L1 expression in GBM and the microenvironment of GBM. Our results provide a valuable therapeutic strategy and indicate that treatments combining DRD2 antagonist paliperidone with standard immunotherapy may be beneficial for GBM treatment.

Dopamine receptor D2 on CD4+ T cells is protective against neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disease. Recently, neuroinflammation driven by CD4+ T cells has been involved in PD pathophysiology. Human and murine lymphocytes express all the five subtypes of dopamine receptors (DRs), DRD1 to DRD5. However, roles of DRs particularly DRD2 expressed on CD4+ T cells in PD remain elucidated. Global Drd1- or Drd2-knockout (Drd1−/− or Drd2−/−) mice or CD4+ T cell-specific Drd2-knockout (Drd2fl/fl/CD4Cre) mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD with the different mutants. On the 7th day following MPTP injection, mice were assessed for dopaminergic neurodegeneration, locomotor impairments, microglial activation, as well as CD4+ T-cell differentiation and function. Furthermore, in vitro CD4+ T cells were exposed to DRD2 agonist and antagonist and then differentiation and function of the cells were determined. MPTP induced dopaminergic neuronal loss in the nigrostriatal system, motor coordinative and behavioral impairments, microglial activation, and CD4+ T-cell polarization to pro-inflammatory T-helper (Th)1 and Th17 phenotypes. Importantly, either Drd2−/− or Drd2fl/fl/CD4Cre mice manifested more severe dopaminergic neurodegeneration, motor deficits, microglial activation, and CD4+ T-cell bias towards Th1 and Th17 phenotypes in response to MPTP, but Drd1−/− did not further alter MPTP intoxication. DRD2 agonist sumanirole inhibited shift of CD4+ T cells obtained from MPTP-intoxicated mice to Th1 and Th17 phenotypes and DRD2 antagonist L-741,626 reversed sumanirole effects. These findings suggest that DRD2 expressed on CD4+ T cells is protective against neuroinflammation and neurodegeneration in PD. Thus, developing a therapeutic strategy of stimulating DRD2 may be promising for mitigation of PD.

Abstract 1370: ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201

Abstract Imipridone ONC201 is a first-in-class DRD2 antagonist and ClpP agonist that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients with once weekly dosing. We evaluated the toxicology, absorption, distribution, metabolism and excretion of ONC201 in animals. Repeat-dose 28-day studies with weekly oral ONC201 in rats and dogs revealed NOAELs (75 and 60 mg/kg, respectively) that represent higher dose levels compared to the 625 mg clinical dose. Genotoxicity (AMES, in vitro and in vivo micronucleus assays) assays demonstrated that ONC201 is not mutagenic at exposures above the RP2D and 2µM average Cmax in patients. The 3T3 neural red uptake assay showed ONC201 is not a phototoxin (photo-irritant factor 1.1). In embryofetal development studies, ONC201 did not lead to maternal effects in rats or rabbits and caused fetal malformations in 3/61 rabbit fetuses with daily dosing (from gestation day 7-17 or 19) at 62.5 and 25 mg/kg, respectively. A quantitative whole-body autoradiography study conducted in rats with one dose of [14C]-ONC201 showed rapidly tissue distribution peaking at 1h in most tissues and exhibiting a plasma terminal half-life of 5.6h. The endocrine, metabolic/excretory, ocular and GI tract tissues contained the highest distribution of [14C]ONC201. [14C]-ONC201 was distributed evenly across CNS substructures, including the midline region. Consistent with the uniform distribution, ONC201 displayed high permeability (23-31×10-6 cm/s, 7-700 μM) and was not a substrate of efflux transporters in Caco-2 cells (efflux ratio 0.46-0.79). ONC201 exerted inhibitory potential on MDR1- and BCRP-mediated transport at 200μM. Clinical trials with ONC201 restrict use of concomitant medications that induce or inhibit CYP enzymes, but several supportive medications can affect these enzymes. In vitro studies revealed that ONC201 is not an inducer of 7 major human CYP enzymes but does inhibit and is a substrate of CYP 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 (IC50 20-90µM). Analysis of dexamethasone, a known inducer of CYP3A4, did not show a correlation between baseline dose level and ONC201 plasma concentrations in H3 K27M-mutant glioma patients (n=22). Metabolic profiling in human hepatocytes revealed only one metabolite, ONC207, that was >10% in abundance. ONC207 did not achieve an IC50 in cancer cell viability and DRD2 antagonism assays. Assaying by LC-MS/MS of plasma samples obtained from ONC201-treated H3 K27M-mutant glioma patients (n=20) confirmed the presence of ONC207 as the major metabolite. A mass balance study conducted in rats with one dose of [14C]-ONC201 showed that the main route of excretion was biliary (61.18%) via the feces (69.78%), with urine (24.99%) also contributing to overall recovery. Together, these data inform the administration of ONC201 to advanced cancer patients who often require a range of comedications and comorbidities. Citation Format: Sara Morrow, Rohinton Tarapore, Ailan Guo, Magdalena Nej, Yunlan Fang, Gina Theerman, Leah Lake, Martin Stogniew, Varun V. Prabhu, Joshua E. Allen. ADME and toxicology profiles of first-in-class DRD2/ClpP-targeted imipridone ONC201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1370.