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

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).

Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro.

Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro. LAY SUMMARY: Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules - called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.

Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols.

The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.