Low concentrations of saracatinib promote definitive endoderm differentiation through inhibition of FAK-YAP signaling axis.

Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.

Dual Drug Repurposing: The Example of Saracatinib.

Saracatinib (AZD0530) is a dual Src/Abl inhibitor initially developed by AstraZeneca for cancer treatment; however, data from 2006 to 2024 reveal that this drug has been tested not only for cancer treatment, but also for the treatment of other diseases. Despite the promising pre-clinical results and the tolerability shown in phase I trials, where a maximum tolerated dose of 175 mg was defined, phase II clinical data demonstrated a low therapeutic action against several cancers and an elevated rate of adverse effects. Recently, pre-clinical research aimed at reducing the toxic effects and enhancing the therapeutic performance of saracatinib using nanoparticles and different pharmacological combinations has shown promising results. Concomitantly, saracatinib was repurposed to treat Alzheimer's disease, targeting Fyn. It showed great clinical results and required a lower daily dose than that defined for cancer treatment, 125 mg and 175 mg, respectively. In addition to Alzheimer's disease, this Src inhibitor has also been studied in relation to other health conditions such as pulmonary and liver fibrosis and even for analgesic and anti-allergic functions. Although saracatinib is still not approved by the Food and Drug Administration (FDA), the large number of alternative uses for saracatinib and the elevated number of pre-clinical and clinical trials performed suggest the huge potential of this drug for the treatment of different kinds of diseases.

Saracatinib prompts hemin-induced K562 erythroid differentiation but suppresses erythropoiesis of hematopoietic stem cells.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

Exploring the benefits of in-diet versus repeated oral dosing of saracatinib (AZD0530) in chronic studies: insights into pharmacokinetics and animal welfare.

Saracatinib/AZD0530 (SAR), a Src tyrosine kinase inhibitor, mitigates seizure-induced brain pathology in epilepsy models upon repeated oral dosing. However, repeated dosing is stressful and can be challenging in some seizing animals. To overcome this issue, we have incorporated SAR-in-Diet and compared serum pharmacokinetics (PK) and brain concentrations with conventional repeated oral dosing. Saracatinib in solution or in-diet was stable at room temperature for >4 weeks (97 ± 1.56%). Adult Sprague Dawley rats on SAR-in-Diet consumed ~1.7 g/day less compared to regular diet (16.82 ± 0.6 vs. 18.50 ± 0.5 g/day), but the weight gain/day was unaffected (2.63 ± 0.5 g/day vs. 2.83 ± 0.2 g/day). Importantly, we achieved the anticipated SAR dose range from 2.5-18.7 mg/kg of rat in response to varying concentrations of SAR-in-Diet from 54 to 260 ppm of feed, respectively. There was a strong and significant correlation between SAR-in-Diet dose (mg/kg) and serum saracatinib concentrations (ng/ml). Serum concentrations also did not vary significantly between SAR-in-Diet and repeated oral dosing. The hippocampal saracatinib concentrations derived from SAR-in-Diet treatment were higher than those derived after repeated oral dosing (day 3, 546.8 ± 219.7 ng/g vs. 238.6 ± 143 ng/g; day 7, 300.7 ± 43.4 ng/g vs. 271.1 ± 62.33 ng/g). Saracatinib stability at room temperature and high serum and hippocampal concentrations in animals fed on SAR-in-Diet are useful to titer the saracatinib dose for future animal disease models. Overall, test drugs in the diet is an experimental approach that addresses issues related to handling stress-induced variables in animal experiments.

Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-ß gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Evaluation of the Efficacy of Saracatinib-Loaded Nanoparticles in Lymphatic Metastases of HNSCC with the Aid of Bioluminescence Imaging.

Head and neck squamous cell carcinoma (HNSCC) remains a deadly disease despite concerted efforts to improve its diagnosis and treatment in recent decades. Metastasis of advanced HNSCC nearly always occurs first in neck lymph nodes before the development of distant metastasis. However, the development of preclinical animal models and therapeutic treatments for metastatic HNSCC is lagged from bench to clinic. In this protocol, we exemplify an orthotopic tongue tumor model that can recapitulate the cervical lymphatic metastases of HNSCC and the application to study the effect of novel saracatinib-loaded nanoparticles (Nano-Sar). By taking advantage of bioluminescence imaging (BLI), the present protocol reveals the strong anti-metastatic efficacy of Nano-Sar in the experimental setup. Collectively, the protocol with a novel metastatic mouse model shows great potential to evaluate treatments on metastatic diseases with the aid of bioluminescent technology.

Protocol paper: a multi-center, double-blinded, randomized, 6-month, placebo-controlled study followed by 12-month open label extension to evaluate the safety and efficacy of Saracatinib in Fibrodysplasia Ossificans Progressiva (STOPFOP).

BACKGROUND: Fibrodysplasia Ossificans Progressiva (FOP) is a genetic, progressive and devastating disease characterized by severe heterotopic ossification (HO), loss of mobility and early death. There are no FDA approved medications. The STOPFOP team identified AZD0530 (saracatinib) as a potent inhibitor of the ALK2/ACVR1-kinase which is the causative gene for this rare bone disease. AZD0530 was proven to prevent HO formation in FOP mouse models. The STOPFOP trial investigates the repositioning of AZD0530, originally developed for ovarian cancer treatment, to treat patients with FOP. METHODS: The STOPFOP trial is a phase 2a study. It is designed as a European, multicentre, 6-month double blind randomized controlled trial of AZD0530 versus placebo, followed by a 12-month trial comparing open-label extended AZD0530 treatment with natural history data as a control. Enrollment will include 20 FOP patients, aged 18-65 years, with the classic FOP mutation (ALK2 R206H). The primary endpoint is objective change in heterotopic bone volume measured by low-dose whole-body computer tomography (CT) in the RCT phase. Secondary endpoints include 18F NaF PET activity and patient reported outcome measures. DISCUSSION: Clinical trials in rare diseases with limited study populations pose unique challenges. An ideal solution for limiting risks in early clinical studies is drug repositioning - using existing clinical molecules for new disease indications. Using existing assets may also allow a more fluid transition into clinical practice. With positive study outcome, AZD0530 may provide a therapy for FOP that can be rapidly progressed due to the availability of existing safety data from 28 registered clinical trials with AZD0530 involving over 600 patients. TRIAL REGISTRATION: EudraCT, 2019-003324-20. Registered 16 October 2019, https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-003324-20/NL . CLINICALTRIALS: gov , NCT04307953 . Registered 13 March 2020.

An overview of the active clinical trials for Parkinson's disease psychosis.

Tweetable abstract An overview of the active clinical trials for Parkinson's disease psychosis. In this article, we review the drugs currently undergoing clinical testing for Parkinson's disease psychosis and offer some perspectives on the treatment of the condition.

Gut dysbiosis following organophosphate, diisopropylfluorophosphate (DFP), intoxication and saracatinib oral administration.

Organophosphate nerve agents (OPNAs) act as irreversible inhibitors of acetylcholinesterase and can lead to cholinergic crisis including salivation, lacrimation, urination, defecation, gastrointestinal distress, respiratory distress, and seizures. Although the OPNAs have been studied in the past few decades, little is known about the impact on the gut microbiome which has become of increasing interest across fields. In this study, we challenged animals with the OPNA, diisopropylfluorophosphate (DFP, 4mg/kg, s.c.) followed immediately by 2mg/kg atropine sulfate (i.m.) and 25mg/kg 2-pralidoxime (i.m.) and 30 minutes later by 3mg/kg midazolam (i.m.). One hour after midazolam, animals were treated with a dosing regimen of saracatinib (SAR, 20mg/kg, oral), a src family kinase inhibitor, to mitigate DFP-induced neurotoxicity. We collected fecal samples 48 hours, 7 days, and 5 weeks post DFP intoxication. 16S rRNA genes (V4) were amplified to identify the bacterial composition. At 48 hours, a significant increase in the abundance of Proteobacteria and decrease in the abundance of Firmicutes were observed in DFP treated animals. At 7 days there was a significant reduction in Firmicutes and Actinobacteria, but a significant increase in Bacteroidetes in the DFP groups compared to controls. The taxonomic changes at 5 weeks were negligible. There was no impact of SAR administration on microbial composition. There was a significant DFP-induced reduction in alpha diversity at 48 hours but not at 7 days and 5 weeks. There appeared to be an impact of DFP on beta diversity at 48 hours and 7 days but not at 5 weeks. In conclusion, acute doses of DFP lead to short-term gut dysbiosis and SAR had no effect. Understanding the role of gut dysbiosis in long-term toxicity may reveal therapeutic targets.

Differential Impact of Severity and Duration of Status Epilepticus, Medical Countermeasures, and a Disease-Modifier, Saracatinib, on Brain Regions in the Rat Diisopropylfluorophosphate Model.

Acute organophosphate (OP) toxicity poses a significant threat to both military and civilian personnel as it can lead to a variety of cholinergic symptoms including the development of status epilepticus (SE). Depending on its severity, SE can lead to a spectrum of neurological changes including neuroinflammation and neurodegeneration. In this study, we determined the impact of SE severity and duration on disease promoting parameters such as gliosis and neurodegeneration and the efficacy of a disease modifier, saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor. Animals were exposed to 4 mg/kg diisopropylfluorophosphate (DFP, s.c.) followed by medical countermeasures. We had five experimental groups: controls (no DFP), animals with no continuous convulsive seizures (CS), animals with ∼20-min continuous CS, 31-60-min continuous CS, and > 60-min continuous CS. These groups were then assessed for astrogliosis, microgliosis, and neurodegeneration 8 days after DFP exposure. The 31-60-min and > 60-min groups, but not ∼20-min group, had significantly upregulated gliosis and neurodegeneration in the hippocampus compared to controls. In the piriform cortex and amygdala, however, all three continuous CS groups had significant upregulation in both gliosis and neurodegeneration. In a separate cohort of animals that had ∼20 and > 60-min of continuous CS, we administered saracatinib for 7 days beginning three hours after DFP. There was bodyweight loss and mortality irrespective of the initial SE severity and duration. However, in survived animals, saracatinib prevented spontaneous recurrent seizures (SRS) during the first week in both severity groups. In the ∼20-min CS group, compared to the vehicle, saracatinib significantly reduced neurodegeneration in the piriform cortex and amygdala. There were no significant differences in the measured parameters between the naïve control and saracatinib on its own (without DFP) groups. Overall, this study demonstrates the differential effects of the initial SE severity and duration on the localization of gliosis and neurodegeneration. We have also demonstrated the disease-modifying potential of saracatinib. However, its' dosing regimen should be optimized based on initial severity and duration of CS during SE to maximize therapeutic effects and minimize toxicity in the DFP model as well as in other OP models such as soman.

Assessing a Novel 3D Assay System for Drug Screening against OS Metastasis.

Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.

Saracatinib Fails to Reduce Alcohol-Seeking and Consumption in Mice and Human Participants.

More effective treatments to reduce pathological alcohol drinking are needed. The glutamatergic system and the NMDA receptor (NMDAR), in particular, are implicated in behavioral and molecular consequences of chronic alcohol use, making the NMDAR a promising target for novel pharmacotherapeutics. Ethanol exposure upregulates Fyn, a protein tyrosine kinase that indirectly modulates NMDAR signaling by phosphorylating the NR2B subunit. The Src/Fyn kinase inhibitor saracatinib (AZD0530) reduces ethanol self-administration and enhances extinction of goal-directed ethanol-seeking in mice. However, less is known regarding how saracatinib affects habitual ethanol-seeking. Moreover, no prior studies have assessed the effects of Src/Fyn kinase inhibitors on alcohol-seeking or consumption in human participants. Here, we tested the effects of saracatinib on alcohol consumption and craving/seeking in two species, including the first trial of an Src/Fyn kinase inhibitor to reduce drinking in humans. Eighteen male C57BL/6NCrl mice underwent operant conditioning on a variable interval schedule to induce habitual responding for 10% ethanol/0.1% saccharin. Next, mice received 5 mg/kg saracatinib or vehicle 2 h or 30 min prior to contingency degradation to measure habitual responding. In the human study, 50 non-treatment seeking human participants who drank heavily and met DSM-IV criteria for alcohol abuse or dependence were randomized to receive 125 mg/day saracatinib (n = 33) or placebo (n = 17). Alcohol Drinking Paradigms (ADP) were completed in a controlled research setting: before and after 7-8 days of treatment. Each ADP involved consumption of a priming drink of alcohol (0.03 mg%) followed by ad libitum access (3 h) to 12 additional drinks (0.015 g%); the number of drinks consumed and craving (Alcohol Urge Questionnaire) were recorded. In mice, saracatinib did not affect habitual ethanol seeking or consumption at either time point. In human participants, no significant effects of saracatinib on alcohol craving or consumption were identified. These results in mice and humans suggest that Fyn kinase inhibition using saracatinib, at the doses tested here, may not reduce alcohol consumption or craving/seeking among those habitually consuming alcohol, in contrast to reports of positive effects of saracatinib in individuals that seek ethanol in a goal-directed manner. Nevertheless, future studies should confirm these negative findings using additional doses and schedules of saracatinib administration.

ActivinA Induced SMAD1/5 Signaling in an iPSC Derived EC Model of Fibrodysplasia Ossificans Progressiva (FOP) Can Be Rescued by the Drug Candidate Saracatinib.

Balanced signal transduction is crucial in tissue patterning, particularly in the vasculature. Heterotopic ossification (HO) is tightly linked to vascularization with increased vessel number in hereditary forms of HO, such as Fibrodysplasia ossificans progressiva (FOP). FOP is caused by mutations in the BMP type I receptor ACVR1 leading to aberrant SMAD1/5 signaling in response to ActivinA. Whether observed vascular phenotype in human FOP lesions is connected to aberrant ActivinA signaling is unknown. Blocking of ActivinA prevents HO in FOP mice indicating a central role of the ligand in FOP. Here, we established a new FOP endothelial cell model generated from induced pluripotent stem cells (iECs) to study ActivinA signaling. FOP iECs recapitulate pathogenic ActivinA/SMAD1/5 signaling. Whole transcriptome analysis identified ActivinA mediated activation of the BMP/NOTCH pathway exclusively in FOP iECs, which was rescued to WT transcriptional levels by the drug candidate Saracatinib. We propose that ActivinA causes transcriptional pre-patterning of the FOP endothelium, which might contribute to differential vascularity in FOP lesions compared to non-hereditary HO.

Post-status epilepticus treatment with the Fyn inhibitor, saracatinib, improves cognitive function in mice.

BACKGROUND: Status epilepticus (SE) is a life-threatening neurological disorder. The hippocampus, as an important area of the brain that regulates cognitive function, is usually damaged after SE, and cognitive deficits often result from hippocampal neurons lost after SE. Fyn, a non-receptor Src family of tyrosine kinases, is potentially associated with the onset of seizure. Saracatinib, a Fyn inhibitor, suppresses epileptogenesis and reduces epileptiform spikes. However, whether saracatinib inhibits cognitive deficits after SE is still unknown. METHODS: In the present study, a pilocarpine-induced SE mouse model was used to answer this question by using the Morris water maze and normal object recognition behavioral tests. RESULTS: We found that saracatinib inhibited the loss in cognitive function following SE. Furthermore, we found that the number of hippocampal neurons in the saracatinib treatment group was increased, when compared to the SE group. CONCLUSIONS: These results showed that saracatinib can improve cognitive functions by reducing the loss of hippocampal neurons after SE, suggesting that Fyn dysfunction is involved in cognitive deficits after SE, and that the inhibition of Fyn is a possible treatment to improve cognitive function in SE patients.

Combination treatment of Src inhibitor Saracatinib with GMI, a Ganoderma microsporum immunomodulatory protein, induce synthetic lethality via autophagy and apoptosis in lung cancer cells.

Saracatinib is an oral Src-kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed-resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase-7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B-II in A400 cells. ATG5 silencing abolished the caspase-7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib.

Biophysical and computational view on the in vitro combination between an anticancer drug, saracatinib and human serum albumin.

Interaction behaviour of an anticancer drug, saracatinib (SCB) with human serum albumin (HSA), the major carrier protein in human blood circulation was investigated using fluorescence and absorption spectroscopy as well as computational methods. Analysis of the fluorescence quenching data along with absorption results confirmed the complex formation between SCB and HSA, based on the inverse correlation of the Stern-Volmer constant (KSV) with temperature and hyperchromic effect in the absorption spectra. Moderate binding affinity between SCB and HSA was evident from the binding constant, Ka value (1.08-0.74 × 104 M-1), while the SCB-HSA complexation was anticipated to be stabilized by hydrophobic and van der Waals interactions along with hydrogen bonds, as revealed from the thermodynamic data (ΔS = + 29.40 J mol-1 K-1 and ΔH = - 13.90 kJ mol-1). Addition of SCB to HSA significantly defended the thermal denaturation of the protein, though it perturbed the surrounding medium around Tyr and Trp residues. Site marker displacement results elucidated Sudlow's site I, positioned in subdomain IIA of HSA as the preferred binding site of SCB, which was well supported by molecular docking. Molecular dynamics simulation results suggested the stability of the SCB-HSA complex.Communicated by Ramaswamy H. Sarma.

Pivotal Role of Fyn Kinase in Parkinson's Disease and Levodopa-Induced Dyskinesia: a Novel Therapeutic Target?

The exact etiology of Parkinson's disease (PD) remains obscure, although many cellular mechanisms including α-synuclein aggregation, oxidative damage, excessive neuroinflammation, and dopaminergic neuronal apoptosis are implicated in its pathogenesis. There is still no disease-modifying treatment for PD and the gold standard therapy, chronic use of levodopa is usually accompanied by severe side effects, mainly levodopa-induced dyskinesia (LID). Hence, the elucidation of the precise underlying molecular mechanisms is of paramount importance. Fyn is a tyrosine phospho-transferase of the Src family nonreceptor kinases that is highly implicated in immune regulation, cell proliferation and normal brain development. Accumulating preclinical evidence highlights the emerging role of Fyn in key aspects of PD and LID pathogenesis: it may regulate α-synuclein phosphorylation, oxidative stress-induced dopaminergic neuronal death, enhanced neuroinflammation and glutamate excitotoxicity by mediating key signaling pathways, such as BDNF/TrkB, PKCδ, MAPK, AMPK, NF-κB, Nrf2, and NMDAR axes. These findings suggest that therapeutic targeting of Fyn or Fyn-related pathways may represent a novel approach in PD treatment. Saracatinib, a nonselective Fyn inhibitor, has already been tested in clinical trials for Alzheimer's disease, and novel selective Fyn inhibitors are under investigation. In this comprehensive review, we discuss recent evidence on the role of Fyn in the pathogenesis of PD and LID and provide insights on additional Fyn-related molecular mechanisms to be explored in PD and LID pathology that could aid in the development of future Fyn-targeted therapeutic approaches.

IGF2BP1 is a targetable SRC/MAPK-dependent driver of invasive growth in ovarian cancer.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.

Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Disease Modifier in the Rat DFP Model: Sex Differences, Neurobehavior, Gliosis, Neurodegeneration, and Nitro-Oxidative Stress.

Diisopropylfluorophosphate (DFP), an organophosphate nerve agent (OPNA), exposure causes status epilepticus (SE) and epileptogenesis. In this study, we tested the protective effects of saracatinib (AZD0530), a Src kinase inhibitor, in mixed-sex or male-only Sprague Dawley rats exposed to 4-5 mg/kg DFP followed by 2 mg/kg atropine and 25 mg/kg 2-pralidoxime. Midazolam (3 mg/kg) was given to the mixed-sex cohort (1 h post-DFP) and male-only cohort (~30 min post-DFP). Saracatinib (20 mg/kg, oral, daily for 7 days) or vehicle was given two hours later and euthanized eight days or ten weeks post-DFP. Brain immunohistochemistry (IHC) showed increased microgliosis, astrogliosis, and neurodegeneration in DFP-treated animals. In the 10-week post-DFP male-only group, there were no significant differences between groups in the novel object recognition, Morris water maze, rotarod, or forced swim test. Brain IHC revealed significant mitigation by saracatinib in contrast to vehicle-treated DFP animals in microgliosis, astrogliosis, neurodegeneration, and nitro-oxidative stressors, such as inducible nitric oxide synthase, GP91phox, and 3-Nitrotyrosine. These findings suggest the protective effects of saracatinib on brain pathology seem to depend on the initial SE severity. Further studies on dose optimization, including extended treatment regimen depending on the SE severity, are required to determine its disease-modifying potential in OPNA models.

Src Inhibition Attenuates Liver Fibrosis by Preventing Hepatic Stellate Cell Activation and Decreasing Connetive Tissue Growth Factor.

The SRC kinase family comprises non-receptor tyrosine kinases that are ubiquitously expressed in all cell types. Although Src is reportedly activated in pulmonary and renal fibrosis, little is known regarding its role in liver fibrosis. This study investigated whether the inhibition of Src protects against liver fibrosis. The expression of Src was upregulated in thioacetamide (TAA)-induced fibrotic mouse liver and cirrhosis of patients, and phospho-Src was upregulated during activation of hepatic stellate cells (HSC). In addition, Src inhibition reduced the expression of α-smooth muscle actin (αSMA) in primary HSCs and suppressed transforming growth factor ß (TGF-ß)-induced expression of connective tissue growth factor (CTGF) in hepatocytes. Src inhibitor Saracatinib also attenuated TAA-induced expression of type I collagen, αSMA, and CTGF in mouse liver tissues. The antifibrotic effect of Src inhibitors was associated with the downregulation of smad3, but not of signal transducer and activator of transcription 3 (STAT3). In addition, Src inhibition increased autophagy flux and protected against liver fibrosis. These results suggest that Src plays an important role in liver fibrosis and that Src inhibitors could be treat liver fibrosis.