Tomivosertib reduces ectopic activity in dorsal root ganglion neurons from patients with radiculopathy

Abstract

Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in patients suffering from this largely untreated disease. While many intracellular signalling mechanisms have been examined in preclinical models that drive spontaneous activity, none have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we showed that inhibition of mitogen-activated protein kinase interacting kinase (MNK) with tomivosertib (eFT508, 25 nM) reversibly suppresses spontaneous activity in human sensory neurons that are likely nociceptors based on size and action potential characteristics associated with painful dermatomes within minutes of treatment. Tomivosertib treatment also decreased action potential amplitude and produced alterations in the magnitude of after hyperpolarizing currents, suggesting modification of Na+ and K+ channel activity as a consequence of drug treatment. Parallel to the effects on electrophysiology, eFT508 treatment led to a profound loss of eIF4E serine 209 phosphorylation in primary sensory neurons, a specific substrate of MNK, within 2 min of drug treatment. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain.

Keywords: MNK; eFT508; human DRG excitability; ion channels.

Figure 1

Treatment with eFT508 (tomivosertib) reverses spontaneous activity in human dorsal root ganglion neurons associated with painful dermatomes. A representative recording of a human dorsal root ganglion (DRG) neuron with spontaneous activity (SA) is shown in A that is unaffected by vehicle treatment, and a similar recording where SA is suppressed by bath application of eFT508 (25 nM) is shown in B. The summary graph (C) shows the combined results from all whole cell patch clamp experiments in human DRG neurons with SA treated with vehicle (Veh) or eFT508 analysed using two-way ANOVA with Tukey’s multiple comparison tests. In graphical representations, different symbols are used to denote various experimental groups defined by treatment conditions. Open symbols indicate the vehicle groups, while filled symbols represent the groups treated with eFT508. The specific shapes of the symbols provide further details: circles are used to signify baseline measurements taken before the administration of any treatment, squares indicate the effects post-treatment with either dimethyl sulphoxide (DMSO) or eFT508, and triangles are used to depict the outcomes observed with washout. *P < 0.05. ns = not significant.

Figure 2

Effect of eFT508 (tomivosertib) on action potential characteristics in neurons with and without spontaneous activity. Neurons were divided into two subgroups based on whether or not the cell showed spontaneous activity (SA) at baseline and compared using non-parametric Wilcoxon matched-pairs signed rank tests. Example traces of neurons without SA (A, red = baseline; blue = after eFT508 treatment) and with SA (B, red = baseline; black = after eFT508 treatment). The treatment effect of eFT508 for no SA and SA neurons is shown for resting membrane potential (RMP) (C), current thresholds (D), action potential (AP) threshold (E), AP amplitude (F), AP overshoot (G), AP afterhyperpolarization (H), AP rising time (I), AP falling time (J) and AP width at 0 mV (K). *P < 0.05, **P < 0.01.

Figure 3

MNK inhibition with eFT508 (tomivosertib) rapidly decreases phosphorylated eIF4E in human dorsal root ganglion neurons. (A) Representative enlarged images of p-eIF4E immunostaining (red) and neuronal marker, peripherin (blue). Enlarged image scale bar = 5 μm. (B) Normalized mean grey intensity values of p-eIF4E signal shown starting at 2 min through 20-min post eFT508 treatment. Data are presented as replicates of independently treated culture wells (n = 4) for vehicle (Veh) and 25 nM eFT508 at all time points. Two-way ANOVA with Bonferroni’s multiple comparisons (****P < 0.0001). MNK = mitogen-activated protein kinase interacting kinase.