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).

Multimodal imaging of biological tissues using combined MALDI and NAPA-LDI mass spectrometry for enhanced molecular coverage.

Mass spectrometry imaging (MSI) is a powerful analytical technique that enables detection, discovery, and identification of multiple classes of biomolecules, while simultaneously mapping their spatial distributions within a sample (e.g., a section of biological tissue). The limitation in molecular coverage afforded by any single MSI platform has led to the development of multimodal approaches that incorporate two or more techniques to obtain greater chemical information. Matrix-assisted laser desorption ionization (MALDI) is a preeminent ionization technique for MSI applications because the wide range of available matrices allows some degree of enhancement with respect to the detection of particular molecular classes. Nonetheless, MALDI has a limited ability to detect and image several classes of molecules, e.g., neutral lipids, in complex samples. Laser desorption ionization from silicon nanopost arrays (NAPA-LDI or NAPA) has been shown to offer complementary coverage with respect to MALDI by providing improved detection of neutral lipids and some small metabolites. Here, we present a multimodal imaging method in which a single tissue section is consecutively imaged at low and high laser fluences, generating spectra that are characteristic of MALDI and NAPA ionization, respectively. The method is demonstrated to map the distributions of species amenable to detection by MALDI (e.g., phospholipids and intermediate-mass metabolites) and NAPA (e.g., neutral lipids such as triglycerides and hexosylceramides, and small metabolites) in mouse brain and lung tissue sections.

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays.

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix-assisted laser desorption ionization (MALDI) is recognized as the method-of-choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix-free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI-MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227-fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295-fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na+ . Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49-fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19-fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.

Matrix-free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays.

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix-assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA-MSI is compared to that of MALDI-MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI-MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA- and MALDI-MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.