Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.

A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway.

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

Negative Regulation of FGFR (Fibroblast Growth Factor Receptor) Signaling.

FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling.

The Effect of Mutations in the TPR and Ankyrin Families of Alpha Solenoid Repeat Proteins.

Protein repeats are short, highly similar peptide motifs that occur several times within a single protein, for example the TPR and Ankyrin repeats. Understanding the role of mutation in these proteins is complicated by the competing facts that 1) the repeats are much more restricted to a set sequence than non-repeat proteins, so mutations should be harmful much more often because there are more residues that are heavily restricted due to the need of the sequence to repeat and 2) the symmetry of the repeats in allows the distribution of functional contributions over a number of residues so that sometimes no specific site is singularly responsible for function (unlike enzymatic active site catalytic residues). To address this issue, we review the effects of mutations in a number of natural repeat proteins from the tetratricopeptide and Ankyrin repeat families. We find that mutations are context dependent. Some mutations are indeed highly disruptive to the function of the protein repeats while mutations in identical positions in other repeats in the same protein have little to no effect on structure or function.

Cancer Mutations in FGFR2 Prevent a Negative Feedback Loop Mediated by the ERK1/2 Pathway.

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties.

Protein Tyrosine Phosphatase Receptor Type G (PTPRG) Controls Fibroblast Growth Factor Receptor (FGFR) 1 Activity and Influences Sensitivity to FGFR Kinase Inhibitors.

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.

Enhanced liver fibrosis (ELF) test in obese children with ultrasound-proven liver steatosis.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) in obese children is a diagnostic challenge. The enhanced liver fibrosis test (ELF) based on the combination of serum concentration of hyaluronic acid (HA), aminoterminal propeptide of type III procollagen (PIIINP), tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) was developed as a noninvasive diagnostic tool for estimation of degree of liver fibrosis. The aim of our study was to investigate the performance of ELF test in obese children with ultrasound-proven steatosis in order assess the possibility of early detection of fibrotic changes in liver structure. MATERIAL AND METHODS: 58 obese (BMI >95th percentile) children, 27 male (mean age 13.9±2.65 years) and 31 female (mean age 13.82±2.64 years). Based on the liver ultrasound (US) examination results two groups of obese children were studied: group with steatosis (N=20, 8/12 M/F, mean age 14.2±1.90 years, BMI 32.9±5.60 kg/m2) and group with normal liver US (n=38, 19/19 M/F, mean age 13.7±2.94 years, BMI 30.4±4.67 kg/m2). Serum activity of aminotransferases (AST, ALT) and lactate dehydrogenase (LDH), and γ-glutamyl transpeptidase (GGT), and ELF test (HA, PIIINP, and TIMP-1 were analyzed. RESULTS: Children with liver steatosis presented with significantly higher AST (34.1 vs. 25.6 U/L), ALT (43.4 vs. 32 U/L), LDH (427.5 vs. 361.3 U/L), GGT (30.7 vs. 18.9 U/L). The ELF test value was also significantly higher in that group (8.98 vs. 8.49). Nevertheless no combination of measured parameters with ELF test value show better diagnostic value for differentiation between children with and without steaotosis. CONCLUSION: ELF test cannot be used for assessment of steatosis in obese children.

Interpretation of hormone levels in older patients: points for consideration.

Blood hormone and tumor marker concentrations are usually determined by immunochemical methods which are based on an unique reaction between antigen and assay capture antibody. Despite the speed and simplicity of assays performance on automatic immunochemistry platforms, the interpretation of final results requires a deep knowledge of method fallibility. General lack of immunoassays standardization, presence of cross-reacting substances in patient's sample, limitation of free hormones measurement due to abnormal analyte binding protein concentrations, assay interferences due to patient's autoantibodies, and heterophilic antibodies, as well as proper interpretation of very low- and very high-sample analyte levels, are the main points discussed in respect to hormones and tumor markers measurement in geriatric population.