Single Agent ONC201 Research Articles

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.

Window-of-opportunity study of ONC201 in pediatric patients with diffuse intrinsic pontine glioma (DIPG) and thalamic glioma.

TPS2082 Background: H3 K27M-mutant diffuse midline glioma is a universally fatal malignancy primarily affecting children and young adults; while radiotherapy (RT) provides transient benefit, no effective systemic therapy is currently available. ONC201, a first-in-class imipridone, is an oral, blood-brain barrier penetrating, selective small molecule antagonist of dopamine receptor D2/3 and agonist of the mitochondrial protease ClpP. Previously, ONC201 monotherapy demonstrated durable objective responses in adults with recurrent H3 K27M-mutant glioma. This phase 1 trial was designed to evaluate ONC201±RT in pediatric patients with H3 K27M-mutant midline glioma DIPG. Methods: This multicenter, open-label, dose escalation and expansion phase 1 study of ONC201 is comprised of eight arms that will evaluate the recommended phase 2 dose (RP2D) of ONC201, biomarkers, and pharmacokinetics (PK) of ONC201±RT in various treatment settings. Arm G previously defined the RP2D for ONC201 administered twice weekly on consecutive days in patients with H3 K27M-mutant glioma who had completed radiotherapy. Arm H, for which enrollment is ongoing, will estimate the influence of tumor location and blood-brain barrier integrity on PK and intratumoral ONC201 exposure in biopsy-eligible pediatric tumors (DIPG or contrast-enhancing thalamic glioma). Patients eligible for Arm H will be aged 2-≤19 years, ≥2 weeks from last RT administration, and have a Karnofsky/Lansky performance score ≥50; prior confirmation of H3 K27M mutation is not required. In Arm H, single-agent ONC201 administration will occur on two consecutive days each week during each 21-day cycle at the RP2D defined in Arm G. Evidence of disease progression is not required; as such, ONC201 may be administered in the maintenance setting or for recurrent disease. Arm H has a planned enrollment of 27 patients. Each patient will undergo biopsy at a single prespecified biopsy window, which will be assigned at enrollment (Table); plasma for PK analysis will be collected from all patients at all time points shown in the Table, with additional collection pre-dose and 0.5 h post first dose. Clinical trial information: NCT03416530. [Table: see text]

Phase I dose escalation and expansion trial of single agent ONC201 in pediatric diffuse midline gliomas following radiotherapy.

ONC201, a dopamine receptor D2 (DRD2) antagonist and caseinolytic protease P (ClpP) agonist, has induced durable tumor regressions in adults with recurrent H3 K27M-mutant glioma. We report results from the first phase I pediatric clinical trial of ONC201. This open-label, multi-center clinical trial (NCT03416530) of ONC201 for pediatric H3 K27M-mutant diffuse midline glioma (DMG) or diffuse intrinsic pontine glioma (DIPG) employed a dose-escalation and dose-expansion design. The primary endpoint was the recommended phase II dose (RP2D). A standard 3 + 3 dose escalation design was implemented. The target dose was the previously established adult RP2D (625 mg), scaled by body weight. Twenty-two pediatric patients with DMG/DIPG were treated following radiation; prior lines of systemic therapy in addition to radiation were permitted providing sufficient time had elapsed prior to study treatment. The RP2D of orally administered ONC201 in this pediatric population was determined to be the adult RP2D (625 mg), scaled by body weight; no dose-limiting toxicities (DLT) occurred. The most frequent treatment-emergent Grade 1-2 AEs were headache, nausea, vomiting, dizziness and increase in alanine aminotransferase. Pharmacokinetics were determined following the first dose: T 1/2, 8.4 h; T max, 2.1 h; C max, 2.3 µg/mL; AUC0-tlast, 16.4 hµg/mL. Median duration of treatment was 20.6 weeks (range 5.1-129). Five (22.7%) patients, all of whom initiated ONC201 following radiation and prior to recurrence, were alive at 2 years from diagnosis. The adult 625 mg weekly RP2D of ONC201 scaled by body weight was well tolerated. Further investigation of ONC201 for DMG/DIPG is warranted.

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.

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.

Abstract 1156: The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines

Abstract First-in-class small molecule ONC201 is an anti-cancer agent that activates the TRAIL pathway through ATF4 and the integrated stress response (ISR). ONC201 demonstrated durable tumor regressions and prolonged disease stabilization in some patients with histone H3K27M-mutated diffuse midline glioma (DMG). The H3K27M-mutation reduces H3K27 methylation. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. We hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of the H3K27M-mutation, particularly in tumor types beyond DMG. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, glioblastoma, and DMG with single agent ONC201, EZH2 inhibitor EPZ-6438 or PF-06821497, or the combination of ONC201 plus EPZ-6438 or ONC201 plus PF-06821497. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activation widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4. We determined TRAIL pathway activity by analysis of DR5, and apoptosis by analysis of PARP cleavage. The effect of EPZ-6438 or PF-06821497on the target was validated by immunoblotting of H3K27me3. We knocked down ATF4 with siRNA to evaluate the role of ISR in cell death. Our results demonstrate that ONC201 synergistically reduced cell viability in combination with EZH2i, induced integrated stress response and apoptosis in combination with EZH2i. There was greater induction of ATF4 and DR5 in combinatorial therapy-treated tumor cells versus monotherapy. Knockdown of ATF4 reduced the apoptosis induced by combination therapy. EPZ-6438 or PF-06821497 decreased H3K27 tri-methylation. Our study results unravel potent synergy between ONC201 and EZH2i, highlight the role of ISR in the synergy, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Varun V. Prabhu, Joshua E. Allen, Attila A. Seyhan, Wafik S. El-Deiry. The integrated stress response (ISR) is involved in the synergistic combinatorial efficacy of ONC201 and epigenetic modulators in brain tumor cell lines [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 1156.

Single agent activity of ONC201 in non-midline H3 K27M-mutant diffuse gliomas.

e14037 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, though 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.

ONC201 induces the unfolded protein response (UPR) in high- and low-grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity.

ObjectivesTreatment of both platinum resistant high grade (HG) and low‐grade (LG) ovarian cancer (OVCA) poses significant challenges as neither respond well to conventional chemotherapy leading to morbidity and mortality. Identification of novel agents that can overcome chemoresistance is therefore critical. Previously, we have demonstrated that OVCA has basal upregulated unfolded protein response (UPR) and that targeting cellular processes leading to further and persistent upregulation of UPR leads to cell death. ONC201 is an orally bioavailable Dopamine Receptor D2 inhibitor demonstrating anticancer activity and was found to induce UPR. Given its unique properties, we hypothesized that ONC201 would overcome platinum resistance in OVCA.MethodsCisplatin sensitive and resistant HG OVCA and two primary LG OVCA cell lines were studied. Cell viability was determined using MTT assay. Cell migration was studied using wound healing assay. Apoptosis and mitochondrial membrane potential were investigated using flow cytometry. Analysis of pathway inhibition was performed by Western Blot. mRNA expression of UPR related genes were measured by qPCR. In vivo studies were completed utilizing axillary xenograft models. Co‐testing with conventional chemotherapy was performed to study synergy.ResultsONC201 significantly inhibited cell viability and migration in a dose dependent manner with IC50’s from 1‐20 µM for both cisplatin sensitive and resistant HG and LG‐OVCA cell lines. ONC201 lead to upregulation of the pro‐apoptotic arm of the UPR, specifically ATF‐4/CHOP/ATF3 and increased the intrinsic apoptosispathway. The compensatory, pro‐survival PI3K/AKT/mTOR pathway was downregulated. In vivo, weekly dosing of single agent ONC201 decreased xenograft tumor size by ~50% compared to vehicle. ONC201 also demonstrated significant synergy with paclitaxel in a highly platinum resistant OVCA cell‐line (OV433).ConclusionsOur findings demonstrate that ONC201 can effectively overcome chemoresistance in OVCA cells by blocking pro‐survival pathways and inducing the apoptotic arm of the UPR. This is a promising, orallybioavailable therapeutic agent to consider in clinical trials for patients with both HG and LG OVCA.

DIPG-52. PHASE I CLINICAL TRIAL OF ONC201 IN PEDIATRIC H3 K27M-MUTANT GLIOMA OR NEWLY DIAGNOSED DIPG

H3 K27M-mutant gliomas often manifest as midline gliomas, have a dismal prognosis, and have no effective treatments. ONC201 efficacy has been shown in high-grade glioma preclinical models and durable responses with single agent ONC201 have been reported in adults with recurrent H3 K27M-mutant gliomas. These observations led to a Phase I pediatric clinical trial of ONC201 dosed by body weight. This multi-center, open-label, 3 + 3 dose-escalation and dose-expansion clinical trial (NCT03416530) for H3 K27M-mutant glioma or non-biopsied DIPG has 6 arms: arms A and E determine the RP2D in pediatric post-radiation (recurrent or not-recurrent) H3 K27M-mutant glioma patients with ONC201 administered as an oral capsule as well as a liquid formulation, respectively. Both arms have completed accrual. The study is currently enrolling newly diagnosed DIPG patients to determine the RP2D for ONC201 in combination with radiation (arm B). Dedicated assessment of intratumoral ONC201 concentrations in midline gliomas patients (arm C) and the effects of ONC201 in H3K27M DNA levels in circulating CSF (arm D) are currently enrolling patients. ONC201 as a single agent in patients with progressive H3K27M mutant tumors following irradiation (excluding DIPG/spinal cord tumors) was recently opened (arm F). Once the RP2D is confirmed, there is a dose-expansion cohort to confirm the safety, radiographic efficacy and survival with ONC201. The primary endpoints of arms A, B, and E have been established with the RP2D of 625mg scaled by body weight as a capsule or liquid formulation administered alone or in combination with radiation without incidence of dose-limiting toxicity.

HGG-18. CLINICAL EFFICACY OF ONC201 IN THALAMIC H3 K27M-MUTANT GLIOMA

ONC201, a bitopic DRD2 antagonist and allosteric ClpP agonist, has shown encouraging efficacy in H3 K27M-mutant glioma. Given that the thalamus has the highest extra-striatal expression of DRD2, we performed an integrated preclinical and clinical analysis of ONC201 in thalamic H3 K27M-mutant glioma. ONC201 was effective in mouse intra-uterine electroporation (IUE)-generated H3 K27M-mutant gliomas, with an in vitro IC50 of 500 nM and 50% prolongation of median survival in vivo (p=0.02, n=14). We analyzed thalamic H3 K27M-mutant glioma patients treated with ONC201 on active clinical trials as of 5/22/19 enrollment (n=19 recurrent and 10 post-radiation, non-recurrent; 5–70 years old). As of 12/18/2019, PFS6 and OS12 are 26.3% and 36.8%, respectively, in the recurrent group. For non-recurrent patients, with median follow up of 21.9 months (8.6–26.6) from diagnosis, median PFS or OS have not been reached. This surpasses historical OS of 13.5 months. Best response by RANO includes 1 CR, 3 PR, 4 SD, 8 PD for recurrent patients and 2 PR, 4 SD, 1 PD for non-recurrent patients (4 on-trial patients experienced regressions that are yet unconfirmed responses). Median duration of response for recurrent patients is 14.0 months (2.0–33.1). Furthermore, H3 K27M cell-free tumor DNA in plasma and CSF correlated with MRI response. In summary, single agent ONC201 administered at recurrence, or adjuvantly following radiation, demonstrates promising clinical efficacy in thalamic H3 K27M-mutant glioma patients who currently have no effective treatments following radiation. Investigations are ongoing to assess whether micro-environmental DRD2 expression explains the early exceptional responses in thalamic H3 K27M-mutant glioma.

CTNI-37. EFFICACY OF ONC201 IN PATIENTS WITH ONC201 FOR RECURRENT H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA

Abstract H3 K27M-mutant diffuse midline gliomas (DMG) have a dismal prognosis. We report an integrated analysis for ONC201, a DRD2 antagonist and ClpP agonist, in patients with recurrent H3 K27M DMG administered as monotherapy in 3 clinical studies (NCT03295396, n=20; NCT02525692, n=6; or expanded access, n=4) by independent, central radiology review among 30 patients. At baseline, patients had measurable contrast-enhancing disease by RANO criteria, KPS>60, and were >90 days from prior radiation. Patients with primary lesions that involved the pons or spinal cord or had evidence of leptomeningeal or cerebrospinal fluid dissemination were excluded. ONC201 was orally administered at 625 mg (scaled by body weight for patients < 18 years old) weekly in 29 patients and every 3 weeks in 1 patient. Median age was 31 years (range 8–70; two patients < 18). There were 16 women and 14 men. Tumors were predominantly thalamic (73%), with other locations including the cerebellum (5%), brainstem (non-pontine) (3%), basal ganglia (3%), and midbrain (3%). Median time from prior radiation was 7.5 months. The most frequent drug-related adverse events were low grade nausea (10%) and fatigue (10%). Nine patients (30%) had >50% regression of T1 contrast enhancement and 11 (36.7%) patients had regression of T2/FLAIR. Objective response rate and progression-free survival by RANO criteria, as well overall survival will be reported. Among 8 patients with sustained radiographic regressions, 6 were tapered off dexamethasone and 4 had improvement in KPS. Five patients (16.7%) remain on treatment with a median of 9.5 months (range 7.3–12). In conclusion, single agent ONC201 is well tolerated and clinically active in recurrent H3 K27M DMG patients.

CTNI-15. CLINICAL EFFICACY OF ONC201 IN NEWLY DIAGNOSED DIPG AND IN PREVIOUSLY IRRADIATED PEDIATRIC H3 K27M-MUTANT GLIOMAS

Abstract ONC201, an anti-cancer DRD2 antagonist and ClpP agonist, is in Phase II trials for adult H3 K27M-mutant diffuse midline gliomas. In adults, the recommended phase 2 dose (RP2D) of 625mg ONC201 once a week has been established as a biologically active dose that is well tolerated. Radiographic regressions with single agent ONC201 have been reported in adult recurrent H3 K27M-mutant glioma patients. This multi-arm, dose-escalation and dose-expansion trial determined the pediatric RP2D of ONC201 administered as an oral capsule (Arm A) or liquid formulation (Arm E) in post-radiation H3 K27M-mutant glioma (Arm A) or in newly diagnosed DIPG (Arm B) patients. Molecular assessments include intratumoral ONC201 concentrations (Arm C) and CSF H3 K27M DNA levels (Arm D). Enrollment as of April 30, 2020 is complete in Arm A (22) and Arm E (26) and continues in Arm B (18/24), Arm C (5/12), and Arm D (22/24). The RP2D of weekly 625mg ONC201 scaled by body weight was confirmed when administered as a capsule or a liquid formulation as a single agent or in combination with radiation without dose-limiting toxicity. The most frequent adverse events regardless of attribution to the drug were predominantly low grade: ONC201 capsule alone was headache (54.5%), nausea (36.4%), and fatigue (36.4%); ONC201 liquid formulation was vomiting (31.8%), headache (22.7%), VIth nerve disorder (22.7%); ONC201 capsules in combination with radiation (Arm B) was headache (47.1%), vomiting (52.9%), nausea (41.2%). Pharmacokinetic analysis in plasma of Arm A patients revealed T1/2: 8.4h; Tmax: 2.1h; Cmax: 2.3ug/mL; AUC0-tlast: 16.4ug/mL, with similar exposure across body weights. In conclusion, when scaled by body weight the ONC201 capsule or liquid formulation alone or in combination with radiation were associated with safety and pharmacokinetic profiles in pediatric H3 K27M-mutant diffuse midline glioma patients that are similar to the experience in adults.

Abstract 5407: Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG)

Abstract Malignant glioma including diffuse midline glioma (DMG) is the leading cause of cancer related death in children. Recent advances in sequencing have identified the oncohistone H3K27M mutation in ~80% of pediatric DMG, which represents a target for new therapeutics. ONC201 is a first-in-class small molecule which upregulates TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic receptor DR5, independent of p53, selecting for apoptosis/growth arrest of many different tumor types including H3K27M mutant DMG. While single agent ONC201 along with RT is currently being tested in clinical trials for newly diagnosed and refractory DMG, there is an urgent need to evaluate ONC201 and other imipridone analogs in combination with other agents. Patient derived H3K27M mutant DMG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36, and SF8628), were grown in culture and exposed to imipridones (ONC201, ONC206 and ONC212) alone, and in combination with other chemotherapeutic agents that have demonstrated pre-clinical activity against DMG (HDAC inhibitors and etoposide). Response to treatment was determined by Cell Titer-Glo cell viability assays as well as Western Immunoblotting for evidence of apoptosis and integrated stress response (ISR) activation. All H3K27M-mutant DMG cell lines tested to date exhibit a dose-dependent response to imipridone therapy. Mean IC50 values were significantly lower with the imipridone analogs ONC206 (0.11 µM) and ONC212 (0.03 µM) as compared to ONC201 (1.46 µM) when used as a single agent. ONC206 and ONC212 were associated with robust induction of apoptosis evidenced by increased expression of cleaved PARP, and ISR by increased expression ATF4. In the SU-DIPG-IV cell line, combination studies of ONC201 and panobinostat demonstrated a synergistic effect of both agents, as calculated by CompuSyn software. Combination indices below one were observed at concentrations of 0.003 - 0.05 µM of panobinostat with ONC201 at 0.156 - 5 µM, with the best combination index of 0.22. We similarly observed synergy between either ONC201 or ONC206 with Etoposide. Additional combination testing and synergy analysis is ongoing and will be presented. Our findings suggest that H3K27M DMG cells demonstrate increased sensitivity to imipridones ONC206 and ONC212 as single agents and combinational strategies with HDAC inhibitors and etoposide, and provide insights into novel therapies for further clinical testing. Citation Format: Robyn Borsuk, Lanlan Zhou, Rishi Lulla, Wafik El-Deiry. Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG) [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 5407.

Abstract 4042: Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors

Abstract ONC201 is a promising anti-cancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. It has broad spectrum anti-tumor activity and achieved tumor regression and prolonged disease stabilization in clinical trials, especially in patients with H3K27M mutant gliomas. Based on the fact that the H3K27M mutation reduces the level of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that cancer cells could be sensitized to ONC201 by epigenetic modulators that impact on histone methylation or acetylation. We tested the synergy between epigenetic modulators and ONC201 in tumor cell lines. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. We also treated cancer cell lines originating from colorectal cancer, breast cancer, gastric cancer, prostate cancer, GBM and DIPG with combination of histone deacetylase inhibitor (HADCi), vorinostat, and ONC201. Cell viability was determined with the Cell Titer-Glo assay. Apoptosis was evaluated through flow cytometry analysis of cell distribution. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Cytotoxic TRAIL pathway signaling was evaluated using Western blot analysis of DR5. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response, cytotoxic TRAIL pathway signaling and apoptosis in combination with epigenetic modulators. There was greater induction of ATF4 and DR5 in combination therapy-treated tumor cells versus monotherapy. Immunoblotting analysis showed that combination of ONC201 and EZH2 inhibitor EPZ-6438 reduced H3K27me3 and EZH2. ONC201 is an antagonist of dopamine receptor D2 (DRD2). Dopamine receptor D5 (DRD5) is a dopamine receptor family member that opposes DRD2 signaling. The DRD2+DRD5- biomarker signature is associated with enhanced ONC201 tumor cell sensitivity. In order to investigate the role of dopamine receptors in anti-tumor effect of ONC201, we also tested the mRNA level of dopamine receptors by RT-PCR and observed that both vorinostat and EPZ-6438 upregulated DRD5 more than DRD2. Thus, we suspect other target genes may be involved in sensitization to ONC201 due to epigenetic modulation. We are currently investigating the interplay between H3K27 methylation versus acetylation in ONC201 sensitivity with or without additional therapeutic modulation by epigenetic drugs. Our results unravel potent synergy between ONC201 and EZH2 inhibitors or HDAC inhibitors and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Michael Glantz, Howard Safran, Attila Seyhan, Wafik S. El-Deiry. Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors [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 4042.

ONC201 in previously irradiated pediatric H3 K27M-mutant glioma or newly diagnosed DIPG.

3619 Background: ONC201 is a first-in-class DRD2 antagonist and ClpP agonist that has demonstrated promising activity in high-grade glioma preclinical models and radiographic regressions with single agent ONC201 in recurrent H3 K27M-mutant glioma patients . The recommended phase 2 dose (RP2D) of 625mg ONC201 orally once a week has been established in adult patients as well tolerated and biologically active. ONC201 efficacy has been shown in high-grade glioma preclinical models and radiographic regressions with single agent ONC201 have been reported in adult recurrent H3 K27M-mutant glioma patients. We report results from the first Phase I pediatric clinical trial of ONC201. Methods: This open-label, multi-center trial for pediatric H3 K27M-mutant glioma or non-biopsied DIPG employed a 3+3 dose-escalation and dose-expansion design with 6 arms. Arms A and E, which have completed accrual, determined the RP2D of ONC201 using oral capsule and liquid formulations in post-radiation pediatric H3 K27M-mutant glioma patients ONC201, respectively. Arm B aims to determine the RP2D for ONC201 in combination with radiotherapy in patients with newly diagnosed DIPG. Arms C and D aim to measure intratumoral ONC201 concentrations in midline glioma patients and the impact of ONC201 on H3 K27M DNA levels in CSF, respectively. Arm F was recently opened to study ONC201 as a single agent in patients with progressive H3 K27M-mutant tumors (excluding DIPG and spinal cord tumors) following radiotherapy. After determining the RP2D, a dose-expansion cohort will evaluate the safety, radiographic response, and activity of ONC201. Results: An RP2D of weekly 625mg ONC201 scaled by body weight as a capsule or in liquid formulation was established in the primary endpoints of arms A, B and E alone or in combination with radiation, without incidence of dose-limiting toxicity (DLT). Pharmacokinetic profiles were similar to those observed in adults (T1/2: 8.4h; Tmax: 2.1h; Cmax: 2.3ug/mL; AUC0-tlast: 16.4ug/mL), with similar exposure across body weights. Conclusions: ONC201 was well tolerated without DLTs at the same adult RP2D scaled by body weight as monotherapy or in combination with radiotherapy in pediatric H3 K27M-mutant glioma patients. Further investigation of ONC201 to treat H3 K27M-mutant glioma and DIPG is warranted. Clinical trial information: NCT03416530 .

Single-agent ONC201 in recurrent H3 K27M-mutant diffuse midline glioma.

3615 Background: Recurrent H3 K27M-mutant diffuse midline glioma is a lethal brain tumor that predominantly affects children and young adults and has no effective therapy. ONC201 is a first-in-class orally administered, anti-cancer small molecule that selectively antagonizes the dopamine receptors DRD2/DRD3 and agonizes ClpP, a mitochondrial protease. Prior studies have indicated dysregulated dopamine receptor expression and enhanced ONC201 sensitivity among H3 K27M-mutant gliomas. Methods: Adults with midline H3 K27M-mutant glioma patients were enrolled to a dedicated Phase II clinical trial (NCT03295396), a multi-arm Phase II clinical trial (NCT02525692), and expanded access protocols under the Sponsor’s IND. Results were pooled among patients treated with ONC201 monotherapy through any of these trial with H3 K27M confirmed glioma, progressive and measurable disease by RANO, > 90 days from completion of prior radiation, no evidence of leptomeningeal dissemination, midline location other than primarily pons or spinal cord, and baseline KPS > 60. Using an enrollment cutoff of February 15, 2019 and data cutoff of July 31, 2019, there were 20 patients (NCT03295396, 12; NCT02525692, 7; expanded access, 1). Dosage was 625 mg weekly in 19 and once every 3 weeks in 1. Results: No DLTs or treatment discontinuations due to toxicity occurred. Midline gliomas can exhibit minimal contrast enhancement or exhibit a mixture of contrast-enhancing and non-contrast enhancing regions in the tumor. As a result, blinded independent central review (BICR) of tumor response by MRI was assessed by RANO-HGG and RANO-LGG for each patient to capture contrast-enhancing lesions by T1 post-contrast and non-contrast-enhancing assessments by T2/FLAIR, respectively, in the object response rate. The best response by RANO-HGG or RANO-LGG is 30% (95% CI, 11.9-54.3%). Duration of response by RANO-HGG is median 52.7 weeks (range 15.9-138.3). One patient with stable disease as of this data cutoff has continued on treatment beyond 12 months and recently underwent an investigator-reported PR by RANO-HGG that is pending confirmation. Conclusions: Single agent ONC201 is well tolerated and clinically active in recurrent H3 K27M-mutant diffuse midline glioma patients. Clinical trial information: NCT03295396, NCT02525692 .

Clinical efficacy of ONC201 in thalamic H3 K27M-mutant glioma.

3617 Background: Diffuse midline gliomas, H3 K27M-mutant are associated with a poor prognosis compared to H3 wild-type gliomas and have no effective therapy following first-line radiation. ONC201 is a bitopic DRD2 antagonist and allosteric ClpP agonist that has shown encouraging single agent efficacy in recurrent H3 K27M-mutant gliomas located in various midline structures of the brain. In addition to tumor and immune cells, the pharmacodynamics of ONC201 extend to stromal cells that can mediate a bystander antitumor response in preclinical models. Given this observation and that the thalamus has the highest extrastriatal expression of DRD2, we report the clinical experience of ONC201 in a subgroup of H3 K27M-mutant glioma patients with primary tumors located in the thalamus. Methods: We analyzed 29 thalamic H3 K27M-mutant glioma patients treated with ONC201 in clinical trials enrolled as of 5/22/19. Nineteen enrolled with recurrent disease whereas 10 enrolled following radiation prior to recurrence. Twelve patients enrolled on NCT03295396, 10 NCT03416530, 4 NCT02525692, and 3 expanded access. Median age was 22 years old (range: 5-70) and baseline KPS was 80 (range: 60-90). Median time from radiation to start of ONC201 was 1.8 months (range: 0.2-8.7) for non-recurrent patients and 7.2 months (range: 1.4-102.0) for recurrent patients. Results: As of 12/18/2019, PFS6 and OS12 measured relative to initiation of ONC201 are 26.3% and 36.8%, respectively, in the recurrent group. For patients initiating ONC201 post-radiation prior to recurrence, median PFS or OS have not been reached with a median follow up of 21.9 months (8.6-26.6) from diagnosis, which surpass historical OS of 13.5 months. Best response for evaluable recurrent patients by RANO: 1 CR, 3 PR, 4 SD, 8 PD, 3 not reported; for non-recurrent patients: 2 PR, 4 SD, 1 PD, 3 not reported. Median duration of response for recurrent patients is 14.0 months (2.0-33.1). ONC201 was well tolerated and no dose-limiting toxicities or treatment discontinuations due to toxicity occurred. Furthermore, H3 K27M cell-free tumor DNA in plasma and CSF correlated with MRI response. Conclusions: In summary, single agent ONC201 administered at recurrence or following radiation, demonstrates promising clinical efficacy in thalamic H3 K27M-mutant glioma patients. Investigations are ongoing to assess whether micro-environmental DRD2 expression correlates with responses of thalamic H3 K27M-mutant glioma to ONC201. Clinical trial information: NCT03295396, NCT03416530, NCT02525692 .

Clinical experience of ONC201 in patients with recurrent H3 K27M-mutant spinal cord glioma.

2563 Background: High-grade gliomas of the spinal cord are a rare and understudied entity, representing < 5% of all spinal cord tumors. Reported median survival times range from 10-16 months. Up to 53% of tumors harbor the H3 K27M mutation, which is associated with an unfavorable prognosis. Postsurgical treatment often includes radiation ± temozolomide, although the role of chemotherapy has not been conclusively established. At recurrence, there are no effective therapies and most clinical studies exclude patients with spinal cord tumors. We report our clinical experience with ONC201, a small molecule DRD2 antagonist and caseinolytic protease P agonist, in patients with recurrent H3 K27M-mutant diffuse gliomas of the spinal cord (scDG). Methods: Adults and children with recurrent H3 K27M-mutant scDG received ONC201 in two Phase II clinical trials enrolling adult recurrent H3 K27M-mutant glioma patients (NCT02525692; NCT03295396) and in one Phase I clinical trial enrolling pediatric patients (NCT03416530). Adult patients received ONC201 at the RP2D dose of 625 mg weekly and pediatric patients received the RP2D of 625 mg weekly, scaled by body weight. All patients began ONC201 as a single agent until disease progression. Five patients continued ONC201 combined with bevacizumab beyond progression. Results: As of January 15, 2020, 12 evaluable patients (adult n = 8, pediatric n = 4) received ONC201. The median age was 20.9 (range: 7-72) years. The median follow-up time for the single agent ONC201 group was 5.4 (range 1.3-9.7) months while that of the combination group is 7.4 (range 6.2-25.1) months. The median number of ONC201 doses was 10 (range: 5-39) for the ONC201 single agent group and 34 (range: 21-100) for the combination group. Five of 7 patients remain alive in the ONC201 single agent group while 3 of 5 patients remain alive in the combination group. Three patients in the ONC201 single agent group and 2 patients in the combination group continue on treatment. There were no drug-related toxicities requiring dose reduction or discontinuation. Conclusions: Treatment with ONC201 alone or combined with bevacizumab is well tolerated in patients with recurrent H3 K27M-mutant scDG and a subset of patients experiences prolonged survival that exceeds historical outcomes. Clinical trial information: NCT02525692; NCT03295396; NCT03416530 .

Abstract LB-C10: Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic

Abstract ONC201, the founding member of the imipridone class of small molecules, has broad spectrum anti-tumor activity and achieved tumor regressions and prolonged disease stability in clinical trials, especially in patients with histone H3 K27M mutated glioma. Levels of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3) are reduced globally in H3 K27M patient samples. EZH2 is the catalytic component of the Polycomb Repressor Complex 2 (PRC2), a conserved multi-subunit that represses gene transcription by methylating lysine 27 on Histone H3 (H3K27). EZH2 is frequently overexpressed or mutated (activating) in numerous human malignancies and EZH2 inhibitors represent a promising therapeutic strategy. We hypothesized that cancer cells could be sensitized to ONC201 by EZH2 inhibition through suppression of H3K27 methylation that may mimic tumors such as DIPG with H3K27M mutations which are known to respond to ONC201 in the clinic and are defective in H3K27 methylation. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. Cell viability was determined with the Cell Titer-Glo assay. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response and apoptosis in combination with EZH2 inhibitor EPZ-6438. There was greater induction of ATF4 in combination therapy-treated tumor cells versus monotherapy. Intriguingly, U-251 GBM cells that were resistant to ONC201 under hypoxia could be sensitized to ONC201 by EZH2 inhibitor EPZ-6438. Our findings provide a preclinical rationale for developing ONC201 and EZH2 inhibitors as a novel synergistic combinatorial regimen with the potential for clinical translation in the treatment of several human malignancies including GBM, pancreatic cancer, colorectal cancer, and breast cancer. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Robyn Borsuk, Howard P. Safran, Attila A. Seyhan, Wafik S. El-Deiry. Preclinical synergistic anti-tumor effect against multiple cancer types by EZH2 inhibitor EPZ-6438 plus imipridone ONC201 to mimic H3K27M mutation observed in DIPG tumors that respond to ONC201 in the clinic [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C10. doi:10.1158/1535-7163.TARG-19-LB-C10

ACTR-34. SINGLE AGENT ONC201 IN PREVIOUSLY-TREATED, PROGRESSIVE ADULT H3 K27M-MUTANT GLIOMA

Abstract H3 K27M-mutant gliomas often manifest as midline gliomas, have a dismal prognosis, and have no established or effective treatments at recurrence. ONC201 is the first clinical bitopic DRD2 antagonist/ClpP agonist and is under evaluation in Phase II trials for gliomas and other cancers. We previously reported in vitro studies suggesting dysregulated dopamine receptor expression and enhanced ONC201 sensitivity among H3 K27M-mutant gliomas. Following these observations, adults with midline H3 K27M-mutant glioma patients were enrolled to a dedicated Phase II clinical trial (NCT03295396), a multi-arm Phase II trial (NCT0252569), and expanded access protocols under the Sponsor’s IND. An integrated radiographic analysis with an objective response rate primary endpoint in patients who received ONC201 monotherapy with confirmed H3 K27M-mutant glioma (not primarily in the pons or spinal cord and without leptomeningeal spread) that was progressive and measurable disease by RANO criteria, >90 days from completion of prior radiation, and had KPS >60. As of December 15, 2018, 15 patients have received single agent ONC201 who meet these criteria (n=9 NCT03295396; n=5 NCT0252569; n=1 expanded access). ONC201 was orally administered at 625 mg weekly, except for one patient dosed once every 3 weeks. As midline gliomas can exhibit a mixture of contrast-enhancing and non-contrast-enhancing disease, objective response was assessed by blinded independent central review using RANO-HGG and RANO-LGG criteria for each patient. Best response to date by RANO-HGG criteria is at least 27%: 1 CR, 3 PR, 7 SD, and 4 PD; by RANO-LGG is at least 36%: 1 CR, 1 PR, 3 minor response (MR), 4 SD, 5 PD, 1 unevaluable. By RANO-HGG, median onset of response is 2.6 months (range 1.3–3.4); median duration of response has not been reached with a median follow-up of 7.7 months (range 1.8–29.8). Updated radiographic response, pharmacodynamics, safety, and other clinical outcomes will be reported.