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RAS GTPase-activating protein-binding protein 1 (G3BP1) is a component of the RAS signaling pathway and a phosphorylation-dependent RNA/DNA endoribonuclease that links signal transduction and RNA metabolism. We identified G3BP1 as a nuclear interactor of platelet-derived growth factor receptor-β, forming a complex with BAF155, a component of the SWI/SNF chromatin remodeling complex, as well as with the transcription factor STAT3. Depletion of G3BP1 in human primary fibroblasts AG1523 reduced PDGF-BB-induced activation of STAT3 while increasing mRNA levels of FOS, MYC, and CCND1, which encode proteins involved in growth stimulation. Both STAT1 and cyclin D1 mRNA and protein levels were elevated upon G3BP1 knockdown, identifying G3BP1 as a negative regulator of STAT1 and cyclin D1 expression. G3BP1 depletion did not abolish PDGF-BB-induced proliferation of human primary fibroblasts. Thus, G3BP1 interacts with the SWI/SNF chromatin remodeling complex in the nucleus and regulates cell growth pathways by modulating STAT signaling and transcription of growth-associated genes.

Background

Platelet-derived growth factor receptor-beta (PDGFR beta) is a canonical marker of pericytes and stromal cells in most tissues. It is present on cancer associated fibroblasts (CAFs) in the tumor microenvironment and has thus been proposed as a potential therapeutic target both for anti-cancer drugs as well as for radioligand therapy (RLT). ATH001 is a novel Affibody molecule-based radiopharmaceutical in clinical development for targeting of PDGFR beta. We hypothesize that dimerization of ATH001 could improve affinity to PDGFR beta, leading to better characteristics for in vivo targeting. The dimeric construct, named ATH022, was generated by conjugation of two chemically synthesized ATH001 binders to a tri-functional linker comprising a DOTA chelator. Here, we present a comprehensive in vitro and in vivo evaluation of Gallium-68 and Indium-111 labeled ATH022, in direct comparison with ATH001.

Results

DOTA-ATH022 acted as a PDGFR beta antagonist and competed dose-dependently with the endogenous ligand PDGF-BB. Affinity of the dimer was improved approximately tenfold compared to DOTA-ATH001, mainly due to strongly decreased off-rate. Radiolabeled DOTA-ATH022 demonstrated higher specific binding and longer retention to U87 cells in vitro. Radiolabeled DOTA-ATH022 also exhibited elevated binding in PDGFR beta-positive tissues spleen and U87 tumors, that could be blocked by ATH001 in excess. Binding of DOTA-ATH022 in PDGFR beta avid and well-perfused spleen was 3 times higher than for DOTA-ATH001, but tumor binding was lower. In vivo retention in spleen was 5 times longer for Indium-111 labeled ATH022, in agreement with its in vitro binding characteristics.

Conclusions

DOTA-ATH022 is a novel dimerized version of ATH001, with significantly improved affinity towards PDGFR beta. Retention of radiolabeled DOTA-ATH022 in PDGFR beta-avid tissues and tumors was increased, however uptake in solid tumor tissue was not improved.

Background: Activated hepatic stellate cells (aHSCs) are the key cell population in the injured liver driving fibrogenesis. aHSCs express platelet-derived growth factor receptor beta (PDGFRβ), which is absent from quiescent HSCs. PDGFRβ is therefore an attractive target of PET tracers for imaging of fibrogenesis. Here, we present the pharmacological characterization of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 in preparation for clinical translation and further confirm PDGFRβ as a biomarker of activated HSCs in liver disease by single cell sequencing.

Methods: The expression of PDGFRβ in subpopulations of HSCs was evaluated in scRNAseq datasets from both a mouse and human liver samples. DOTA-Cys-ATH001 was evaluated for affinity and mechanism of binding to PDGFRβ. [⁶⁸Ga]Ga-DOTA-Cys-ATH001 was evaluated for binding in vitro in mouse and human liver biopsies.The in vivo stability, biodistribution, pharmacokinetics, dosimetry and microdosing toxicology were evaluated in rats and pigs.

Results: PDGFRβ expression was specifically upregulated in activated HSCs. [⁶⁸Ga] Ga-DOTA-Cys-ATH001 could differentiate fibrotic liver from healthy liver.The binding co-localized with tissue areas positive for collagen deposition and PDGFRβ immunostaining. Based on the microdosing toxicology study the no observed adverse effect level was at least 1000 μg/kg, suggesting that the intended clinical PET scan dose is safe for use. Dosimetry calculations of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 predicted an effective dose in human amenable to repeated examinations.

Conclusions: The data presented here suggests that PDGFRβ PET imaging with [⁶⁸Ga] Ga-DOTA-Cys-ATH001 has potential for non-invasive detection of activated HSCs. Clinical translation of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 is ongoing.

The transcription factor ZEB2 is essential for early embryonic development. Using CRISPR/Cas9, we generated a ZEB2 deficient human iPSC cell line (KICRi002A-4), carrying a homozygous 790 bp deletion in ZEB2 that involves part of exon 5, intron 5 and part of exon 6. The deletion leads to markedly reduced levels of a truncated ZEB2 transcript. No ZEB2 protein was detected by immunopreciptation. The iPSC line expressed pluripotency markers and showed a capacity to differentiate into three germ layers in vitro. . Assessment of genomic integrity revealed a normal karyotype without detectable OFF-target editing. The iPSC line KICRi002A-4 thus a valuable resource to study the role of ZEB2 for the commitment and differentiation of various human cell lineages.

Platelet-derived growth factor (PDGF)-induced signalling via PDGF receptor β (PDGFRβ) leads to activation of downstream signalling pathways which regulate multiple cellular responses. It is unclear how PDGFRβ is degraded; both lysosomal and proteasomal degradation have been suggested. In this study, we have characterised the proteolytic cleavage of ligand-activated PDGFRβ, which results in two fragments: a larger fragment containing the extracellular domain, the transmembrane segment, and a part of the intracellular juxtamembrane region with a molecular mass of ∼130 kDa, and an intracellular ∼70 kDa fragment released into the cytoplasm. The proteolytic processing did not take place without internalisation of PDGFRβ. In addition, chelation of intracellular Ca²⁺ inhibited proteolytic processing. Inhibition of the proteasome affected signal transduction by increasing the phosphorylation of PDGFRβ, PLCγ, and STAT3 while reducing it on Erk1/2 and not affecting Akt. The proteolytic cleavage was observed in fibroblasts or cells that had undergone epithelial-mesenchymal transition.

Background

The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to α- and β-tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively). SUMOylation is an important posttranslational modification (PTM) which regulates protein stability, localization, activation and protein interactions. A mass spectrometry screen has demonstrated SUMOylation of PDGFRα. However, the functional role of SUMOylation of PDGFRα has remained unknown.

Results

In the present study, we validated that PDGFRα is SUMOylated on lysine residue 917 as was previously reported using a mass spectrometry approach. Mutation of lysine residue 917 to arginine (K917R) in PDGFRα substantially decreased SUMOylation, indicating that this amino acid residue is a major SUMOylation site. Whereas no difference in the stability of wild-type and mutant receptor was observed, the K917R mutant PDGFRα was less ubiquitinated than wild-type PDGFRα. The internalization and trafficking of the receptor to early and late endosomes were not affected by the mutation, neither was the localization of the PDGFRα to Golgi. However, the K917R mutant PDGFRα showed delayed activation of PLC-γ and enhanced activation of STAT3. Functional assays showed that the mutation of K917 of PDGFRα decreased cell proliferation in response to PDGF-BB stimulation.

Conclusions

SUMOylation of PDGFRα decreases ubiquitination of the receptor and affects ligand-induced signaling and cell proliferation.

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor beta (PDGFR beta) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFR beta did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGR beta lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

The active hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D-3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1 alpha,25-dihydroxyvitamin D-3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1 alpha,25-dihydroxyvitamin D-3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indi-cated suppression of p70-S6 kinase levels by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G cells, whereas the levels of PLC gamma, a target for phospholipid signaling, was slightly increased. Activation of STAT3, an important regulator of malignancy, was suppressed by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1 alpha,24-dihydroxyvitamin D-3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumor-igenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.

Platelet-derived growth factor (PDGF) is a family of growth factors with mitogenic and chemotactic activity. However, uncontrolled and overactivated PDGF signaling has been implicated in a variety of diseases, such as cancers and atherosclerosis. In this context, inhibition of PDGF-PDGFR signaling is of paramount importance in progression of such diseases. The purpose of the current study was to identify novel PDGF-B inhibitors using virtual screening methods. To this end, a combination of molecular modeling techniques such as molecular docking and dynamics simulation, as well as drug likeness filtering criteria, was applied to select anti-PDGF peptidomimetic candidates based on crystallography solved structure of an anti-PDGF-B monoclonal antibody named, MOR8457. In vitro biological assays of the selected compounds revealed two of them being active at micromolar IC50, concentrations. The presented work can provide a framework for systematic peptidomimetic identification for anti-PDGF-B agents from large chemical libraries.