Diffuse midline glioma invasion and metastasis rely on cell-autonomous signaling.

BACKGROUND: Diffuse midline gliomas (DMG) are pediatric tumors with negligible 2-year survival after diagnosis characterized by their ability to infiltrate the central nervous system. In the hope of controlling the local growth and slowing the disease, all patients receive radiotherapy. However, distant progression occurs frequently in DMG patients. Current clues as to what causes tumor infiltration circle mainly around the tumor microenvironment, but there are currently no known determinants to predict the degree of invasiveness. METHODS: In this study, we use patient-derived glioma stem cells (GSCs) to create patient-specific 3D avatars to model interindividual invasion and elucidate the cellular supporting mechanisms. RESULTS: We show that GSC models in 3D mirror the invasive behavior of the parental tumors, thus proving the ability of DMG to infiltrate as an autonomous characteristic of tumor cells. Furthermore, we distinguished 2 modes of migration, mesenchymal and ameboid-like, and associated the ameboid-like modality with GSCs derived from the most invasive tumors. Using transcriptomics of both organoids and primary tumors, we further characterized the invasive ameboid-like tumors as oligodendrocyte progenitor-like, with highly contractile cytoskeleton and reduced adhesion ability driven by crucial over-expression of bone morphogenetic pathway 7 (BMP7). Finally, we deciphered MEK, ERK, and Rho/ROCK kinases activated downstream of the BMP7 stimulation as actionable targets controlling tumor cell motility. CONCLUSIONS: Our findings identify 2 new therapeutic avenues. First, patient-derived GSCs represent a predictive tool for patient stratification in order to adapt irradiation strategies. Second, autocrine and short-range BMP7-related signaling becomes a druggable target to prevent DMG spread and metastasis.

VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells.

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.

A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action. EXPERIMENTAL APPROACH: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats. KEY RESULTS: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective.

A snake toxin as a theranostic agent for the type 2 vasopressin receptor.

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2ß between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions.