Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene.

Trk (NTRK) receptor and NTRK gene fusions are oncogenic drivers of a wide variety of tumors. Although Trk receptors are typically activated at the cell surface, signaling of constitutive active Trk and diverse intracellular NTRK fusion oncogenes is barely investigated. Here, we show that a high intracellular abundance is sufficient for neurotrophin-independent, constitutive activation of TrkB kinase domains. In HEK293 cells, constitutive active TrkB kinase and an intracellular NTRK2-fusion oncogene (SQSTM1-NTRK2) reduced actin filopodia dynamics, phosphorylated FAK, and altered the cell morphology. Atypical cellular responses could be mimicked with the intracellular kinase domain, which did not activate the Trk-associated MAPK/ERK pathway. In glioblastoma-like U87MG cells, expression of TrkB or SQSTM1-NTRK2 reduced cell motility and caused drastic changes in the transcriptome. Clinically approved Trk inhibitors or mutating Y705 in the kinase domain, blocked the cellular effects and transcriptome changes. Atypical signaling was also seen for TrkA and TrkC. Moreover, hallmarks of atypical pTrk kinase were found in biopsies of Nestin-positive glioblastoma. Therefore, we suggest Western blot-like immunoassay screening of NTRK-related (brain) tumor biopsies to identify patients with atypical panTrk or phosphoTrk signals. Such patients could be candidates for treatment with NTRK inhibitors such as Larotrectinhib or Entrectinhib.

Dabigatran Etexilate Reduces Thrombin-Induced Inflammation and Thrombus Formation in Experimental Ischemic Stroke.

Dabigatran etexilate (DE), a direct-acting, oral inhibitor of thrombin, significantly reduces the risk of stroke compared with traditional anticoagulants, without increasing the risk of major bleeding. However, studies on the fate of cerebral tissue after ischemic stroke in patients receiving DE are sparse and the role of dabigatran-mediated reduction of thrombin in this context has not yet been investigated. Here, we investigated whether pretreatment with DE reduces thrombin-mediated pro-inflammatory mechanisms and leakage of the blood-brain barrier (BBB) following ischemic stroke in rats. Male Wistar rats received DE (15 mg/kg) or a vehicle solution 1 hour before transient middle cerebral artery occlusion (tMCAO) for 90 minutes. Infarct volume, neurologic outcome and intracranial hemorrhage (ICH) were determined after tMCAO. Thrombin generation was indirectly assessed by measuring thrombin/antithrombin III complex. Microvascular patency was evaluated histologically. Cytokine expression and immunoreactivity of cluster of differentiation (CD) 68 were examined to characterize inflammatory processes after pretreatment with DE. BBB integrity was examined by quantifying brain edema. Rats given DE revealed a significant reduction in infarct size without an increase in ICH and significant recovery of neurologic deficits compared to controls. Administration of DE decreased thrombin generation and thrombus formation, dampened the CD68-immunoreactivity and attenuated pro-inflammatory cytokine expression in the cerebral parenchyma ipsilateral to the ischemic lesion. BBB permeability was unaltered following treatment with DE. In summary, prophylactic anticoagulation with DE improves stroke outcome by reducing thrombin-induced inflammation and thrombus formation without increasing the rate of ICH.

Pretreatment with rivaroxaban attenuates stroke severity in rats by a dual antithrombotic and anti-inflammatory mechanism.

Stroke outcome is more favourable in patients receiving oral anticoagulants compared with non-anticoagulated patients. The reasons for this "stroke-attenuating" property of oral anticoagulants are largely unknown. This study examined whether prestroke anticoagulation with rivaroxaban, a novel direct factor Xa inhibitor, influences stroke severity, thrombin-mediated intracerebral thrombus formation and pro-inflammatory processes in a rat model of brain ischaemia/reperfusion injury. Male Wistar rats were anticoagulated with rivaroxaban and subjected to 90 minutes of transient middle cerebral artery occlusion. Infarct size, functional outcome and the occurrence of intracranial haemorrhage (ICH) were assessed until day 7. Thrombin generation was determined by measuring the amount of thrombin/antithrombin complex. Intracerebral thrombus formation was evaluated by histology and Western blot. CD68-immunoreactivity and the expression of cytokines and adhesion molecules were investigated to assess postischaemic inflammation. The integrity of the blood-brain barrier was analysed using fluorescein isothiocyanate-dextran. Rats pretreated with rivaroxaban developed significantly smaller strokes and less severe functional deficits compared with controls. Although rivaroxaban strongly reduced thrombin-mediated thrombus formation, this was not accompanied by an increased risk of ICH. In addition, rivaroxaban dampened the inflammatory response in the ischaemic brain by downregulating ICAM-1 expression and the activation of CD68+-immune cells. In contrast, rivaroxaban had no effect on the integrity of the blood-brain barrier after stroke. Here, we identified reduced thrombo-inflammation as a major determinant of the stroke-protective property of rivaroxaban in rats. Further studies are needed to assess the therapeutic potential of novel oral anticoagulants in the acute phase after a stroke.