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Supplementary Figure 3 shows the gating strategy and absolute numbers of CD8 cell subsets in the blood, spleen, and tumor to generate the heatmaps in figure 3

Supplementary Table 1

Supplementary Table 1 provides a list of the antibodies used in this study

Supplementary Figures 1-6 and Supplementary Methods

Supplementary Table 6

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance.Significance:mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813

Supplementary File 1 lists the most differentially expressed genes from the RNAseq analysis

Immune checkpoint blockade (ICB) therapy, which targets T cell–inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy.Significance:These data demonstrate for the first time that age determines the T cell–inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required.This article is highlighted in the In This Issue feature, p. 1143

Supplementary Table 3

Supplementary Table 2

Supplementary Table 4

Supplementary Table 5

ONC201 is the first bitopic dopamine receptor D2 (DRD2) antagonist and allosteric mitochondrial protease ClpP agonist. Downstream of target engagement, ONC201 activates the integrated stress response pathway resulting in selective tumor cell death. ONC201 is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 currently in Phase I trials for adult and pediatric brain tumors, is also a bitopic DRD2 antagonist and ClpP agonist with nanomolar potency. We further evaluated in vitro efficacy and mechanism of action specific to ClpP for ONC206 and investigated acquired resistance to ONC201 and ONC206. FITC-casein and peptide degradation assays confirmed that ONC206 acts as an agonist of human ClpP with enhanced potency relative to ONC201. Broad efficacy in the nanomolar range for ONC206 (GI50 Citation Format: Andrew Lee, Cristina Maranto, Scott Foster, Sara Morrow, Joshua E. Allen, Randall Lanier, Phiroze Sethna, Varun V. Prabhu. Role of ClpP in the anti-cancer effects of imipridone ONC201 and ONC206. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4914.

ONC201 was originally discovered as TNF-Related Apoptosis Inducing Ligand (TRAIL)-inducing compound TIC10. ONC201 appears to act as a selective antagonist of the G protein coupled receptor (GPCR) dopamine receptor D2 (DRD2), and as an allosteric agonist of mitochondrial protease caseinolytic protease P (ClpP). Downstream of target engagement, ONC201 activates the ATF4/CHOP-mediated integrated stress response leading to TRAIL/Death Receptor 5 (DR5) activation, inhibits oxidative phosphorylation via c-myc, and inactivates Akt/ERK signaling in tumor cells. This typically results in DR5/TRAIL-mediated apoptosis of tumor cells; however, DR5/TRAIL-independent apoptosis, cell cycle arrest, or antiproliferative effects also occur. The effects of ONC201 extend beyond bulk tumor cells to include cancer stem cells, cancer associated fibroblasts and immune cells within the tumor microenvironment that can contribute to its efficacy. ONC201 is orally administered, crosses the intact blood brain barrier, and is under evaluation in clinical trials in patients with advanced solid tumors and hematological malignancies. ONC201 has single agent clinical activity in tumor types that are enriched for DRD2 and/or ClpP expression including specific subtypes of high-grade glioma, endometrial cancer, prostate cancer, mantle cell lymphoma, and adrenal tumors. Synergy with radiation, chemotherapy, targeted therapy and immune-checkpoint agents has been identified in preclinical models and is being evaluated in clinical trials. Structure-activity relationships based on the core pharmacophore of ONC201, termed the imipridone scaffold, revealed novel potent compounds that are being developed. Imipridones represent a novel approach to therapeutically target previously undruggable GPCRs, ClpP, and innate immune pathways in oncology.

Drawing on folklore, oral history, songs, historic ethnography, literature, and archaeology, this study investigates the economic, social, and material evidence of traditional late nineteenth- to early twentieth-century wakes along coastal Ireland through the lens of an archaeological case study on the island of Inishark. We analyze material practices of “good” and “bad” deaths, and explore the myriad ways that islanders used everyday material culture in mortuary rituals, particularly wakes and funerals. We argue that material culture could hold multiple symbolic valences throughout its history, especially everyday items used in ritual contexts. Finally, we describe the material residues associated with mourning practices on Inishark.

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

Imipridone ONC201 is an investigational agent in phase II clinical trials for H3 K27M-mutant diffuse midline glioma with evidence of durable objective responses and clinical symptom improvements as a single agent. The systemic pharmacokinetics of ONC201 and its ability to achieve target concentrations in supratentorial glioblastoma have been demonstrated in clinical trials; however, its clearance and distribution in midline structures and other areas of the central nervous system (CNS) haven’t been evaluated. A quantitative whole-body autoradiography study was conducted in Long-Evans rats with a single dose of [14C]-ONC201. [14C]-ONC201-related material was rapidly distributed throughout the body, with concentrations peaking at 1h for most tissues. The endocrine, metabolic/excretory, ocular and gastrointestinal tract tissues contained the highest distribution of [14C]-ONC201-derived radioactivity. Importantly, [14C]-ONC201 material was distributed evenly across brain substructures, including the meninges and midline structures of the brain; all tissues had a half-life of 1.4–7.9h, with the exception of the meninges (2397.3h). Given the uniform distribution throughout the CNS, we evaluated the permeability and efflux potential of ONC201 in bidirectional transport assays using human Caco-2 cell monolayers. ONC201 displayed high permeability in the apical to basolateral direction with apparent permeability values of 23–31×10–6 cm/s at 7–700μM. In the basolateral to apical direction, apparent permeability values were 11–24 × 10–6 cm/s. The efflux ratio values were 0.46–0.79 for ONC201, suggesting that ONC201 is not a substrate of efflux transporters. However, ONC201 exerted inhibitory potential on MDR1- (90.6%) and BCRP-mediated (81.6%) transport at 200mM. In summary, ONC201 exhibits passive diffusion without being effluxed, which may enable its rapid and wide distribution throughout the CNS. This distribution profile suggests that the compound may achieve therapeutic concentrations throughout the CNS with oral administration and that investigation of additional CNS tumors will not be hindered by drug delivery to specific anatomic structures.

ONC206 is a DRD2 antagonist and ClpP agonist that is a chemical derivative of ONC201, which is in Phase II clinical trials for H3 K27M-mutant glioma. We have previously reported that dopamine receptor expression correlates with ONC201 and ONC206 efficacy. Here, we evaluated additional predictive biomarkers for both agents in the GDSC panel using RNA-seq expression data. ClpP emerged as a strong predictor of efficacy for ONC206 (IC50: p = 1.83E-4, AUC: 9.92E-13). ClpP activation enhances degradation of its substrates, including electron transport chain members responsible for oxidative phosphorylation (OXPHOS). Imipridones activate the integrated stress response involving ATF4 and cause proteasomal degradation of c-myc, which also reduces OXPHOS. Accordingly, high ATF4 (IC50: p = 4.92E-5, AUC: p=2.84E-9) and elevated c-myc correlated with ONC206 efficacy (IC50: p = 6.42E-6, AUC: 3.31E-12). EGFR expression is inversely correlate with DRD2 expression in glioma and its activation is associated with glycolysis. Low EGFR expression correlated with increased ONC206 efficacy (IC50: p = 4.32E-7, AUC: p = 6.44E-20). Loss of HIF1 shunts cellular metabolism toward OXPHOS. Low HIF1 expression correlated with increased anticancer efficacy for ONC206 (IC50: p = 1.96E-3, AUC: p = 1.56E-11). Expression of each of the five markers was significantly different in cell lines that achieved an IC90 with ONC206 (~10%) versus those that did not. A similar analysis for ONC201 revealed that ClpP, MYC and EGFR are more predictive of efficacy relative to ATF4 and HIF1. Combinatorial biomarker analyses revealed MYC/EGFR as the most significant predictor of IC50 for both agents. ClpP/MYC and ClpP/HIF1 were the most significant predictors of AUC for ONC201 and ONC206, respectively. Ongoing studies are further investigating tumor type enrichment of biomarkers. Prediction of innate imipridone sensitivity using biomarkers identified in this study may guide patient and tumor type selection in clinical trials.

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.

Immune checkpoint blockade (ICB) therapy, which targets T cell–inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy. Significance: These data demonstrate for the first time that age determines the T cell–inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required. This article is highlighted in the In This Issue feature, p. 1143

ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) and allosteric mitochondrial protease ClpP agonist, that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, nanomolar potency, and disruption of DRD2 homodimers. In these studies, a FITC-casein degradation assay revealed that ONC206 also acts as an agonist of human ClpP and has a 3-fold improved potency. GEPIA database analysis showed ClpP mRNA was overexpressed in glioblastoma cells relative to normal cells. 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 high ClpP and/or DRD2+/DRD5- RNA expression signatures. Among solid tumors, nervous system related cell lines were particularly sensitive. 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 subcutaneous xenograft model of DRD2-overexpressing, dopamine-secreting tumor cells. 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. 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 open label, dose escalation, and food effect Phase I study in biomarker-enriched adult recurrent primary CNS tumors.

Imipridone ONC201 is an investigational agent in phase II clinical trials for high grade gliomas as a single agent once weekly. Systemic and tumor exposure to ONC201 exceeds therapeutic thresholds consistently; however, exposure is variable despite fasted conditions and does not strongly covary with body weight or body surface area. Clinical trials with ONC201 have restricted the use of concomitant medication that induce or inhibit cytochrome P450 (CYP) enzymes; however, several supportive medications for glioma patients such as corticosteroids and anti-seizure medications can affect these enzymes. Studies in pooled human liver microsomes revealed that ONC201 is not an inducer of seven major drug-metabolizing human CYP enzymes: CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. However, ONC201 inhibits multiple CYPs IC50 values ranging from 34.9 to 428.6 μM and is substrate of CYP3A4 and, to lesser extent, other CYPs. Given potential metabolism of ONC201 by these enzymes, we conducted metabolic profiling in human hepatocytes that revealed at least 13 metabolites; however, only one was >10% abundance. The major metabolite, termed ONC207, that lacks a benzyl moiety of ONC201 was assayed by LC-MS-MS in plasma samples obtained from 16 ONC201-treated H3 K27M-mutant glioma patients. Peak plasma concentrations of ONC207 relative to ONC201 was 22% (range: 6%-48%) and its abundance was not correlated with ONC201, suggesting that variable exposure to ONC201 may not be associated with variable metabolism. ONC207 does not inhibit dopamine receptor D2 or cancer cell viability in contrast to the parent compound. In summary, ONC201 is an inhibitor and substrate, but not an inducer, of multiple CYP enzymes. The major metabolite of ONC207 appears to be biologically inactive and is unlikely to explain variable ONC201 exposure or its prolonged biological activity that outlives its systemic presence.

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.

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 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.

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.