Biology subjects

Frenster, J. D.

Publications and source records attributed to Frenster, J. D..

7 recordsLinked to original sources

bioRxiv2025

N2B27 media formulations influence gastruloid development

Gastruloids are 3D aggregates of pluripotent stem cells grown in suspension culture that mimic many aspects of gastrulation and early axial elongation. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27) with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In this study we examined whether these formulations resulted in significant differences in gastruloid development. Our results reveal that while both media enable the standard gastruloid elongation, HM-N2B27 results in gastruloids that start the elongation process earlier, have higher number of cells and an increased anterior domain. RNAseq analysis showed significant differences in cell fate specification, with HM-N2B27 gastruloids exhibiting higher expression of spinal cord-related genes, while NDiff227 favours mesodermal differentiation. Furthermore, differential gene enrichment analysis suggests that changes in key signalling pathways underline the differences between HM-N2B27 and NDiff227 gastruloids. These findings highlight the importance of basal media composition for gastruloid development, underscoring the need for careful media selection during in vitro engineering of stem cell-based embryo models. Summary statementIn this work we explored the cellular and molecular sensitiveness of the mouse gastruloid model system to variations in N2B27 media formulations.

developmental biology↗

bioRxiv2025

A mosaic gastruloid model highlights the developmental stage-specific restriction of cell competition in mammalian pre-gastrulation

In early mammalian embryogenesis, the selective elimination of suboptimal cells is critical for developmental integrity. Cell competition (CC) is a cell non-autonomous quality control in which "winner" cells eliminate viable but suboptimal "loser" cells based on their relative difference in fitness. Due to its central role in fitness perception, loss of p53 results in the emergence of "supercompetitor" cells, which stand at the apex of cell competition and induce apoptosis in neighboring wild-type (WT) cells. Here, we investigate CC dynamics using mosaic 3D mouse gastruloids, an embryonic stem cell (ESC)-based in vitro model of gastrulation, composed of defined numbers of WT and p53-KO cells. In mosaic gastruloids, even low numbers of p53-KO cells robustly outcompete WT cells, and introduction of as few as two p53-KO cells is sufficient to measurably impair neighboring WT cell growth. CC in gastruloids is independent of cell proliferation rates, nutrient availability, or reactive oxygen species (ROS), and not influenced by Nodal and ERK signaling. However, we observe that Wnt and BMP signaling protect from CC, which is exclusively driven by intrinsic apoptosis, as indicated by Bcl2-mediated complete rescue of WT cells. During gastruloid development, CC is temporally restricted to a window of 48-96 hours after aggregation, mirroring embryonic days E5.5-E7.5 in the mouse. Heterochronic mosaic gastruloid experiments demonstrate that relative differences in pluripotency levels are neither necessary nor sufficient to cause supercompetition, but that CC is contingent on both competitors residing within the developmental window permissive to CC. Neither pluripotent mosaic 3D aggregates, nor 3D EpiGastruloids, which model more advanced developmental processes, display any competition, supporting the hypothesis that developmental CC is specific to the onset of gastrulation. Our findings offer insights into the mechanisms of cell fitness evaluation in mammalian embryogenesis and establish gastruloids as a powerful 3D model for investigating developmental stage-specific cell competition.

developmental biology↗

bioRxiv2023

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their functional role in gliomagenesis and their potential as treatment targets. To identify therapeutically targetable opportunities among aGPCR family members in unbiased fashion, we analyzed expression levels of all aGPCRs in GBM and non-neoplastic brain tissue. Using bulk and single cell transcriptomic and proteomic data, we show that CD97 (ADGRE5), an aGPCR previously implicated in GBM pathogenesis, is the most promising aGPCR target in GBM, by virtue of its abundance in all GBM tumors and its de novo expression profile in GBM compared to normal brain tissue and neural progenitors. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGC) in vitro and in vivo. Transcriptomic and metabolomic data from PDGCs suggest that CD97 promotes glycolytic metabolism. The oncogenic and metabolic effects of CD97 are mediated by the MAPK pathway. Activation of MAPK signaling depends on phosphorylation of the cytosolic C-terminus of CD97 and recruitment of {beta}-arrestin. Using single-cell RNA-sequencing and biochemical assays, we demonstrate that THY1/CD90 is the most likely CD97 ligand in GBM. Lastly, we show that targeting of PDGCs with an anti-CD97 antibody-drug conjugate in vitro selectively kills tumor cells but not human astrocytes or neural stem cells. Our studies identify CD97 as an important regulator of tumor metabolism in GBM, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it for therapeutic purposes.

cancer biology↗

bioRxiv2022

PTK7 is a positive allosteric modulator of GPR133 (ADGRD1) signaling in GBM

GPR133 (ADGRD1), an adhesion G protein-coupled receptor, supports growth of glioblastoma, a brain malignancy. We demonstrated that GPR133 is intramolecularly cleaved, and that dissociation of its N-terminal and C-terminal fragments (NTF and CTF) at the plasma membrane correlates with increased receptor signaling. However, how the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity purification and mass spectrometry to identify extracellular binding partners of GPR133 in patient-derived glioblastoma cells. We show that the transmembrane protein PTK7 binds the GPR133 NTF and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7s anchoring in the plasma membrane. The GPR133-PTK7 interaction facilitates orthosteric activation of GPR133 by soluble peptide mimicking the endogenous tethered Stachel agonist, suggesting PTK7 binding allosterically enhances accessibility of GPR133s orthosteric Stachel binding pocket. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this novel ligand-receptor interaction is relevant to the pathogenesis of glioblastoma, as well as physiological processes in several tissues.

cell biology↗

bioRxiv2021

Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

cell biology↗

bioRxiv2021

The H3K36me2 writer-reader dependency in H3K27M-DIPG

The lysine-to-methionine mutation at residue 27 of histone H3 (H3K27M) is a driving mutation in Diffuse Intrinsic Pontine Glioma (DIPG), a highly aggressive form of pediatric brain tumor with no effective treatment and little chance of survival. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity, the placement of methylation at lysine 27 of histone H3 (H3K27me2/3), promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation in vitro and tumorigenesis in vivo, and disrupts tumor-promoting gene expression programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers that mediate the pro-tumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Together, our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG and suggest a possibility to target this pathway for therapeutic interventions.

cancer biology↗

bioRxiv2020

Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

GPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation. Highlights- GPR133 is intramolecularly cleaved in patient-derived GBM cultures - Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant - The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane - Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signaling eTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

cell biology↗