Imipridones affect tumor bioenergetics and promote cell lineage differentiation in diffuse midline gliomas

Abstract

Background: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs.

Methods: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq.

Results: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state.

Conclusion: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).

Keywords: ClpP; ONC201; ONC206; diffuse midline glioma (DMG); integrated stress response (ISR).

© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Figures

Fig. 1

High-grade pediatric CNS tumors upregulate ClpP, and imipridone-mediated ClpP hyperactivation reduces DMG cell viability. CPTAC/CBTTC datasets were quarried to assess ClpP expression across pediatric CNS cancers. (A) ClpP protein expression correlated with mRNA expression across pediatric brain tumors (P < .05). (B) ClpP mRNA and protein abundance significantly associated with tumor grade (P < .01). (C) ClpP protein is upregulated in HGG (P < .001), and M (P < .001) compared to LGG. HGG, High-grade glioma/astrocytoma; LGG, Low-grade glioma/astrocytoma; M, medulloblastoma. (D) ClpP RNA expression (highest 50% vs lowest 50%; RSEM TPM quantification) is significantly (p = 0.01) associated with lower overall survival in pediatric (4–14 years old) DMG patients (clinical data NCT02274987). (E, F) Human primary DMG cell viability is reduced in a dose-dependent manner following treatment with ONC201 (E) or ONC206 (F), summarized in Table (SD = Standard Deviation). (G) Dose-response treatment of DMG cells with ONC201-ONC206 combination reveled drug synergy (ZIP and Bliss score >10) or drug additivity (Loewe score –10 to 10).

Fig. 2

ONC201 and ONC206 directly bind to ClpP, and ClpP is essential for imipridone response in DMGs. (A) Left Panel: ChemPLP docking algorithm predicted a higher binding affinity of ClpP by ONC206 (P = .042), contrary to the Goldscore algorithm (P = .93). Right Panel: Binding energies displayed enhanced binding for ONC206 compared to ONC201. (B) ClpP protein stability increased in the presence of ONC201 or ONC206 (CETSA). Melting curves (left panel) are based on ClpP protein expression (right panel) relative to the intensity at 55°C; NDP, nondenatured protein. (C) CLPP knockout (KO) DMG cells were resistant to ONC201 and ONC206. Control DMG cells transduced with nontargeting control (NTC) showed no difference in drug response. Mean ±SD viable cells after 96 hrs treatment.

Fig. 3

Imipridone-induced ClpP hyperactivation leads to mitochondrial damage and impaired mitochondrial metabolism in DMGs. (A) Protein expression of ClpP and ClpX (top panel), NDUFA12 (middle panel), and respiratory chain complexes I, II, IV components (bottom panel) decreased in ONC201 and ONC206 treated cells (B) ONC201 and ONC206 treatment (24 hrs IC50) decreased mitochondrial membrane potential, with a larger reduction in ONC206 treated cells. scale bar 50 µm. (C) ONC206 resulted in greater (top panel, 24 hrs treatment) and earlier (bottom panel, 4 vs 24 hrs treatment at IC50) production of mitochondrial ROS. (D) Superoxide production in mitochondria was increased after 24 hrs treatment. Scale bar 50 µm. (E) TEM revealed mitochondrial morphological abnormalities in DMG cells treated with ONC201 (24 hrs, IC50) where a temporal sequence of events was observed including cristae disintegration and cristae whirl formation (1), mitochondrial swelling (2-3), and possible encapsulation/vacuolation of mitochondria (4). Control DMSO-treated cells showed healthy mitochondria with round, dense morphologies, and an absence of cristae whirl formation. Scale bar 1 µm.

Fig. 4

ONC201 and ONC206 activate the integrated stress response (ISR) leading to DMG cell apoptosis. (A) Protein expression of ISR biomarkers ATF4, DR5, CHOP, and p-eIF2α increased, while ClpX decreased, after 72 hrs at indicated doses. (B) Protein expression of apoptosis biomarkers cleaved caspase-7, PARP, cleaved-PARP and XIAP after 72 hrs at indicated doses. (C) ONC201 or ONC206 treated subcutaneous DMG tumor model in mice revealed a higher expression of ATF4 and DR5, and lower expression of ClpX and NDUFA12. Representative tumor sections, scale bar 50 µm and 20 µm in inset. (D) Median survival significantly increased in DMG PDX models treated with ONC201 (117 days, P = .01) or ONC206 (113 days, P = .01) compared to vehicle control (100 days). Drug combination (50mg/kg each) increased median survival (125 days) when compared to untreated (P = .01), single therapy with ONC201 (P = .03), or ONC206 (P = .02). Mice (n = 3) were treated (IP, weekly) for 6 weeks, starting at 14 days post-tumor implantation. (E) Histological analysis of DMG PDX tumors at necropsy showed ClpX downregulation in the combination treatment compared to untreated control.

Fig. 5

ONC201 and ONC206 target DMG bioenergetics, and combination of imipridones with glycolysis suppression potently inhibits tumor cell metabolism. (A and B) Mitochondrial respiration function, measured through oxygen consumption rate (OCR), was significantly (P < .0001) decreased after ONC201 and ONC206 treatment (IC50) as single and combination agents. Arrows indicate addition of oligomycin (1), FCCP (2) and Rotenone/ AA (3). (C) Glycolysis, as measured by extracellular acidification rate (ECAR), was significantly reduced by ONC201 and combination treatment, whereas ONC206 showed a slight increase (IC50). 2-deoxyglucose (2-DG) reduced glycolysis in all conditions. For (A-C): OCR/ECAR were measured 4 times with 5 replicates for each condition.

Fig. 6

ONC201 and ONC206 single and combination treatments induce transcriptional reprogramming and cell lineage shifts. (A) DMG cells were treated with ONC201/6 as single or in combination (48 hrs), followed by bulk RNA sequencing. Differential gene expression (volcano plot) of cells treated with monotherapy (ONC201, top left; ONC206, top right), and combination therapy (bottom) indicated differential gene expression induced by each drug. (B) Combination treatment resulted in significant differential gene expression (relative to DMSO control). Functional enrichment analyses (Gene Ontology, KEGG) identfied molecular pathways uniquely activated (red) and inhibited (blue) in each condition. (C) Left panel: Single-cell RNA sequencing of DMG cells treated with ONC201 (96 hrs) showed a decrease in cycling and oligodendrocyte-like progenitor cells (OPC), and an increase in differentiated astrocytic-like (AC) cells. PCA plot (left) and bar graph (right) show the proportion of cells (AC, OPC, oligodendrocyte-like, cycling) in control (DMSO) and in treated conditions. Right panel: Visual representation of cell lineage shift from OPC-like to AC-like cells in response to imipridone treatment. Arrows from OPC towards AC or oligodendrocyte lineages indicate developmental hierarchy; arrow encircling cycling cells indicates self-renewal and replenishment of OPC pool. Area taken up by a given cell type is respective to its observed proportion in the left panel.