Achondroplasia is the most common form of human dwarfism caused by mutations in the FGFR3 receptor tyrosine kinase. Current therapy begins at 2 years of age and improves longitudinal growth but does not address the cranial malformations including midface hypoplasia and foramen magnum stenosis, which lead to significant otolaryngeal and neurologic compromise. A recent clinical trial found partial restoration of cranial defects with therapy starting at 3 months of age, but results are still inconclusive. The benefits of achondroplasia therapy are therefore controversial, increasing skepticism among the medical community and patients. We used a mouse model of achondroplasia to test treatment protocols aligned with human studies. Early postnatal treatment (from day 1) was compared with late postnatal treatment (from day 4, equivalent to ~5 months in humans). Animals were treated with the FGFR3 inhibitor infigratinib and the effect on skeleton was thoroughly examined. We show that premature fusion of the skull base synchondroses occurs immediately after birth and leads to defective cranial development and foramen magnum stenosis in the mouse model to achondroplasia. This phenotype appears significantly restored by early infigratinib administration when compared with late treatment, which provides weak to no rescue. In contrast, the long bone growth is similarly improved by both early and late protocols. We provide clear evidence that immediate postnatal therapy is critical for normalization of skeletal growth in both the cranial base and long bones and the prevention of sequelae associated with achondroplasia. We also describe the limitations of early postnatal therapy, providing a paradigm-shifting argument for the development of prenatal therapy for achondroplasia.

• MeSH
• achondroplazie * patologie   farmakoterapie   MeSH
• lebka patologie   účinky léků   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• myši MeSH
• receptor fibroblastových růstových faktorů, typ 3 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

• MeSH
• eukaryotický iniciační faktor 4F * metabolismus   genetika   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosfatasa 6 s dvojí specificitou metabolismus   genetika   MeSH
• GTP-fosfohydrolasy * metabolismus   genetika   MeSH
• lidé MeSH
• MAP kinasový signální systém * genetika   MeSH
• melanom * genetika   metabolismus   patologie   MeSH
• membránové proteiny * metabolismus   genetika   MeSH
• mutace * MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protoonkogenní proteiny B-Raf * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

• MeSH
• aktivinové receptory typu I antagonisté a inhibitory   metabolismus   MeSH
• aktivinové receptory typu II * metabolismus   antagonisté a inhibitory   MeSH
• inhibitory proteinkinas farmakologie   chemie   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• molekulární sondy chemie   MeSH
• myši MeSH
• objevování léků MeSH
• pyrazoly chemie   farmakologie   chemická syntéza   MeSH
• signální transdukce účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The FGF system is the most complex of all receptor tyrosine kinase signaling networks with 18 FGF ligands and four FGFRs that deliver morphogenic signals to pattern most embryonic structures. Even when a single FGFR is expressed in the tissue, different FGFs can trigger dramatically different biological responses via this receptor. Here we show both quantitative and qualitative differences in the signaling of one of the FGF receptors, FGFR1c, in response to different FGFs. We provide an overview of the recent discovery that FGFs engage in biased signaling via FGFR1c. We discuss the concept of ligand bias, which represents qualitative differences in signaling as it is a measure of differential ligand preferences for different downstream responses. We show how FGF ligand bias manifests in functional data in cultured chondrocyte cells. We argue that FGF-ligand bias contributes substantially to FGF-driven developmental processes, along with known differences in FGF expression levels, FGF-FGFR binding coefficients and differences in FGF stability in vivo.

• MeSH
• chondrocyty metabolismus   MeSH
• fibroblastové růstové faktory * metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• receptor fibroblastových růstových faktorů, typ 1 * metabolismus   MeSH
• receptory fibroblastových růstových faktorů * metabolismus   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• lidé MeSH
• růst a vývoj * MeSH
• vývojová biologie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Non-canonical DNA structures in regulatory regions of the human genome, such as DNA i-motifs, DNA G-quadruplexes, and mismatch/abasic sites containing double stranded DNAs, are important therapeutic targets for design and development of anti-cancer drugs. Currently established procedures for the identification of DNA targets and development of DNA-binding ligands/drugs are either based on data that abstracts from the influence of the specific intracellular microenvironment of cancer cells or on data that do not connect the ligand-induced biological phenotype to the direct action of the ligand on the desired DNA target. These shortcomings in the process of the therapeutic target identification as well as in the process of the drug development constitute significant source of bias being in part responsible for: i) high failure-rate of potential drugs in pre-clinical trials, ii) undesirable side-affects of drugs encountered in clinical practice, and iii) clinical manifestations of cancer cells’ drug insensitivity/resistance.

Nekanonické struktury DNA vyskytující se v regulačních oblastech lidského genomu, jako jsou DNA i-motivy, DNA G-kvadruplexy a dvou-řetězcové úseky DNA obsahující nepárující se báze či abazická místa, jsou důležitými cíli pro vývoj protinádorových léčiv. Současné postupy pro identifikaci cílových struktur DNA a vývoj DNA vazebných ligandů (léčiv) nezohledňují vliv specifického vnitrobuněčného prostředí nádorových buněk a neumožňují jednoznačně určit, zda-li je léčivem vyvolaný fenotyp následkem přímé interakce ligandu s vybranou cílovou strukturou DNA. Tyto nedostatky v procesu identifikace cílů protinádorové terapie a vývoje příslušných léčiv mají za následek: i) vysoký podíl potenciálních léčiv, které neuspějí v preklinických testech, ii) nežádoucí vedlejší účinky již zavedených léčiv v klinické praxi a iii) klinické projevy necitlivosti/resistence rakovinných buněk k danému léčivu.

• Klíčová slova
• non-B DNA, in-cell NMR spectroscopy, léčiva vazající DNA, interakce léčivo-DNA in vivo, DNA binding drugs, non-B DNA, in-cell NMR spectroscopy, DNA-ligand interactions in vivo,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) account for the most prevalent form of dwarfism in humans, the achondroplasia. Although achondroplasia is considered a curable condition, a clinical need for safe and effective FGFR3 inhibitor in this disorder is presently unmet. Moreover, the therapeutics evaluated at present are likely to have a limited effect due to the patient resistance, calling for development of novel approaches for FGFR3 inhibition in the growing bone. Our preliminary data demonstrate an efficient inhibition of FGFR3 signaling in achondroplasia models by RNA aptamer molecule designed to neutralize the FGFR3’s cognate ligand FGF2. Although this proofs the concept, a direct aptamer-based FGFR3 targeting is necessary to restrict the effect only to cartilage-expressed FGFR3 but not to the other FGFRs. In this project, we will develop an RNA aptamer molecule designed to inhibit FGFR3 signaling in skeletal dysplasia via preventing its dimerization.

Aktivující mutace v receptoru 3 pro růstový faktor fibroblastů (FGFR3) způsobují nejrozšířenější formu trpasličího vzrůstu u lidí, achondroplázii. I když je achondroplázie považovaná za léčitelné onemocnění, v současnosti žádná léčba neexistuje. Navíc právě vyvíjené léky mají spíše omezený efekt kvůli vzniku rezistence, což vede k nutnosti vyvinout nové přístupy. Naše prvotní data ukazují účinnou inhibici FGFR3 signalizace u modelů achondroplazie pomocí RNA aptametru, malé molekuly vyvinuté in vitro selekcí. Vazba aptameru na FGF2, ligand pro FGFR3, zabraňuje aktivaci FGFR3 a dokazuje tak použitelnost RNA aptameru jako inhibitoru FGFR signalizace. Pro terapeutické účely je však nutné vyvinout aptamer cílící přímo na receptor FGFR3, tak aby nedocházelo k omezení funkce jiných FGF receptorů. V tomto projektu budeme in vitro selekcí vyvíjet RNA aptamer, který zabraní aktivaci FGFR3, a bude využitelný jako lék pro léčbu růstových poruch.

• Klíčová slova
• léčba, treatment, aptamer, achondroplasie, FGFR3, aptamer, achondroplasia, FGFR3, kostní dysplázie, skeletal dysplasia,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Primary cilia are dynamic compartments that regulate multiple aspects of cellular signaling. The production, maintenance, and function of cilia involve more than 1000 genes in mammals, and their mutations disrupt the ciliary signaling which manifests in a plethora of pathological conditions-the ciliopathies. Skeletal ciliopathies are genetic disorders affecting the development and homeostasis of the skeleton, and encompass a broad spectrum of pathologies ranging from isolated polydactyly to lethal syndromic dysplasias. The recent advances in forward genetics allowed for the identification of novel regulators of skeletogenesis, and revealed a growing list of ciliary proteins that are critical for signaling pathways implicated in bone physiology. Among these, a group of protein kinases involved in cilia assembly, maintenance, signaling, and disassembly has emerged. In this review, we summarize the functions of cilia kinases in skeletal development and disease, and discuss the available and upcoming treatment options.

• MeSH
• cilie metabolismus   MeSH
• ciliopatie * genetika   patologie   MeSH
• homeostáza MeSH
• polydaktylie * genetika   MeSH
• proteiny genetika   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Achondroplasia (ACH) is one of the most prevalent genetic forms of short-limbed skeletal dysplasia, caused by gain-of-function mutations in the receptor tyrosine kinase FGFR3. In August 2021, the C-type natriuretic peptide (CNP) analog vosoritide was approved for the treatment of ACH. A total of six other inhibitors of FGFR3 signaling are currently undergoing clinical evaluation for ACH. This progress creates an opportunity for children with ACH, who may gain early access to the treatment by entering clinical trials before the closure of their epiphyseal growth plates and cessation of growth. Pathophysiology associated with the ACH, however, demands a long observational period before admission to the interventional trial. Public patient registries can facilitate the process by identification of patients suitable for treatment and collecting the data necessary for the trial entry. RESULTS: In 2015, we established the prospective ACH registry in the Czechia and the Slovak Republic ( http://www.achondroplasia-registry.cz ). Patient data is collected through pediatric practitioners and other relevant specialists. After informed consent is given, the data is entered to the online TrialDB system and stored in the Oracle 9i database. The initial cohort included 51 ACH children (average age 8.5 years, range 3 months to 14 years). The frequency of selected neurological, orthopedic, or ORL diagnoses is also recorded. In 2015-2021, a total of 89 measurements of heights, weights, and other parameters were collected. The individual average growth rate was calculated and showed values without exception in the lower decile for the appropriate age. Evidence of paternal age effect was found, with 58.7% of ACH fathers older than the general average paternal age and 43.5% of fathers older by two or more years. One ACH patient had orthopedic limb extension and one patient received growth hormone therapy. Low blood pressure or renal impairment were not found in any patient. CONCLUSION: The registry collected the clinical information of 51 pediatric ACH patients during its 6 years of existence, corresponding to ~ 60% of ACH patients living in the Czechia and Slovak Republic. The registry continues to collect ACH patient data with annual frequency to monitor the growth and other parameters in preparation for future therapy.

• MeSH
• achondroplazie * epidemiologie   genetika   MeSH
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mutace MeSH
• předškolní dítě MeSH
• prospektivní studie MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   MeSH
• registrace MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Slovenská republika MeSH

Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFβ/BMP signaling pathway that included increased canonical TGFβ and noncanonical BMP signaling. In this study, the role of FLNB in the TGFβ/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFβ/BMP signaling and that loss of FLNB produces increased TGFβ receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFβ/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFβ/BMP pathway activity restored Flnb-/- IVD morphology. These most effective improvements resulted from specific inhibition of TGFβ and p38 signaling activation. FLNB acts as a bridge for TGFβ/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFβ/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

• Publikační typ
• časopisecké články MeSH