Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction

Type I interferons (IFNs) increase the excitability of dorsal root ganglion (DRG) neurons via activation of MNK-eIF4E translation signaling to promote pain sensitization in mice. Activation of STING signaling is a key component of type I IFN induction. Manipulation of STING signaling is an active area of investigation in cancer and other therapeutic areas. Vinorelbine is a chemotherapeutic that activates STING and has been shown to cause pain and neuropathy in oncology clinical trials in patients. There are conflicting reports on whether STING signaling promotes or inhibits pain in mice. We hypothesized that vinorelbine would cause a neuropathic pain-like state in mice via STING and signaling pathways in DRG neurons associated with type I IFN induction. Vinorelbine (10 mg/kg, i.v.) induced tactile allodynia and grimacing in WT male and female mice and increased p-IRF3 and type I IFN protein in peripheral nerves. In support of our hypothesis, vinorelbine-mediated pain was absent in male and female StingGt/Gt mice. Vinorelbine also failed to induce IRF3 and type I IFN signaling in these mice. Since type I IFNs engage translational control via MNK1-eIF4E in DRG nociceptors, we assessed vinorelbine-mediated p-eIF4E changes. Vinorelbine increased p-eIF4E in DRG in WT animals but not in StingGt/Gt or Mknk1−/− (MNK1 KO) mice. Consistent with these biochemical findings, vinorelbine had an attenuated pro-nociceptive effect in male and female MNK1 KO mice. Our findings support the conclusion that activation of STING signaling in the peripheral nervous system causes a neuropathic pain-like state that is mediated by type I IFN signaling to DRG nociceptors.


Introduction
Vinorelbine is a chemotherapeutic agent that belongs to the semisynthetic vinca alkaloid family (1). This drug is FDA-approved for non-small cell lung cancer (NSCLC) treatment (2). Vinorelbine is also used to treat metastatic breast carcinoma, and has shown effectiveness in advanced forms of melanoma and renal cell carcinoma (1). NSCLC and breast cancer are estimated to be the top two cancer incidences in 2023 in the US (3).
Despite serving as a first-line treatment in advanced NSCLC (4), vinorelbine has been reported to cause peripheral neuropathy (5-10) and induce pain in cancer patients, mostly at the tumor site or in areas innervated by nerves compressed by the tumor (1).
Vinorelbine mainly acts as a destabilizing agent of microtubules (4, 11). Microtubule destabilizers have been shown to activate stimulator of interferon genes (STING)interferon regulatory factor 3 (IRF3) dependent production of IFN-β and downstream interferon-stimulated genes (ISGs) in both immune and triple negative breast cancer cells (12). Type I IFNs are powerful monomeric cytokines that induce an antiviral response upon infection through the upregulation of molecules that antagonize viral replication (13).
Regulation of type I IFNs by STING depends on the IRF family members IRF3/7 (14). The expression of IRF7 in most cells is low, while IRF3 is ubiquitously expressed triggering the phosphorylation of IRF3 upon viral infection in nearly all cell types (15). Loss-of-function mutation in the IRF3 gene impairs interferon expression in patients, suggesting a critical role of this transcription factor in the antiviral interferon responses (16,17). an antifade mountant (Cat. #P36390, Invitrogen). Images were captured on Olympus FV1200 Confocal Microscope System with 20X objective. Images were analyzed using Olympus CellSens Software. To determine the percentage of p-IRF3 and p-eIF4E immunoreactivity in neurons of each mouse, about 200 neurons were counted per DRG section. For the size-fluorescence data, measurements of area of peripherin, p-IRF3, and p-eIF4E in neurons were performed using a computerized image analysis system (Olympus CellSens) and plotted in 100µm 2 (cross sectional area) increments. In addition, neuronal population with high peripherin mean fluorescence intensity (<400 µm 2 ) considered as peripherin-positive, which are likely to contain nociceptors (33), were used to assess the changes in immunoreactivity of p-IRF3 and p-eIF4E. Results are reported as mean fluorescence intensity within each size population.

FLIR Imaging
Changes in temperature either on the site of administration (tail base) or abdomen wall were assessed using a FLIR T-Series Thermal Imaging Camera (FLIR Systems, Inc).
Animals were allowed to acclimate in the testing room for 1 h (ambient temperature of 21°C ± 2°C) in Plexiglas chambers (11.4 × 7.6 × 7.6 cm) before testing. We captured colorized infrared thermogram images containing the abdomen wall and tail of mice before experimental treatment and at 1, 3, 5 and 8 days after the first administration of vinorelbine. The temperature was obtained drawing a straight line at the base of the tail or a square on the abdomen of mice. The mean temperature was recorded from the average of each pixel along the drawn line or in the square. Three thermograms were averaged to obtain the mean temperature of the tail and the abdomen per animal.

Mouse behavior
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 We performed behavioral experiments in wild type (WT) or transgenic male and female mice. Animals that were cohabiting were arbitrarily assigned to groups (control or treatment). All behavioral measurements were performed in age-matched animals.
Animals were habituated to the experimental setup (Plexiglas chambers (11.4 × 7.6 × 7.6 cm)) for the von Frey test 1 h before each experiment. Behavioral testers were blinded to treatment and genotype in all experiments. Mechanical paw withdrawal thresholds were measured using the up-down method with calibrated von Frey filaments (Stoelting Company, Wood Dale, IL). We calculated this threshold by using the formula: 50% g stimuli. Increasing or decreasing forces with different von Frey filaments were applied during three seconds to the mouse paw in order to calculate the 50% withdrawal threshold.
Mouse grimace scoring was performed as a behavioral test for examination of spontaneous pain response (35). Mice were placed individually in a Plexiglas chamber and allowed to acclimate for 1 h, and then scored by blinded scorers at 1, 3, 5, 8, and 10 days after the first vinorelbine administration. The scores of each animal were averaged at each time point.

Statistics
Data were analyzed with Graphpad Prism V9 (Graphpad, San Diego, CA). All data are shown as mean ± standard error of the mean (SEM). Repeated measures two-way ANOVA was used to analyze behavioral data plotted as group by time. Students t-test was used to assess effect sizes and Western blot data. Other statistical tests are described in figure legends. We used at least 5 mice per group of each sex in behavioral experiments based on power analysis predicated on our previous work with type I IFNs. Because no . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint 1 1

Vinorelbine-induced STING-type I interferon signaling in peripheral nerve and DRG.
Type I interferon induction is predominantly controlled at the transcriptional level, wherein the main molecular mechanism controlling its production is the canonical STING-TBK1-IRF3-IFN pathway (36). We determined changes in the activation of these proteins in DRG and sciatic nerve in WT male and female mice at day 10 after the first administration of vinorelbine. We did not observe changes in the phosphorylation of STING (Ser366) (Fig.   1E, 1K) or the phosphorylation of TBK1 (Ser172) in WT mice at this time point (Fig. 1F,   1L). However, we found an increase in the phosphorylation of IRF3 (Ser396) in DRG and a trend toward increased phosphorylation in the sciatic nerve (Fig. 1G, 1M). We also observed an increase in the total levels of IFN-β in DRG (Fig. 1I), and an increase in IFN-α in sciatic nerve (Fig. 1H). This suggests that vinorelbine administration results in the activation of IRF3 stimulating type I IFN production in the periphery. These findings are in line with the observed vinorelbine-induced hypersensitivity in mice and with our previous work showing that type I interferons cause mechanical hypersensitivity in mice (18).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made showing STING-IRF3-IFN pathway in vinorelbine treated WT sciatic nerve compared to vehicle. P) Representative western blot images showing STING-IRF3-IFN pathway in vinorelbine treated WT sciatic nerve compared to vehicle. Data are presented as the mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001 (n = 13 per group, with 7 male and 6 female mice) as determined by two-way ANOVA followed by Bonferroni's test in A, C. *p<0.05, ***p<0.001 as determined by t test in B, D, E-I and K-O. Vino: vinorelbine, veh: vehicle.

Vinorelbine-induces p-IRF3 in DRG neurons in mice.
p-IRF3 is the main downstream effector of STING inducing the transcription of Ifna1 and 1 4

Figure 2. Vinorelbine increases p-IRF3 immunoreactivity in DRG neurons. A)
Representative confocal micrographs showing p-IRF3 (red) and peripherin (green) immunofluorescence in WT ICR mouse DRG neurons day 3 post-second administration of vinorelbine (10 mg/kg i.v.) or vehicle (3% DMSO i.v.). DAPI (cyan) stains nuclei in the . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint 1 6 used as part of the same cohort in these experiments. As expected, vinorelbine induced an increase in grimace score in WT mice from day 5 after first administration (day 0) and lasting until day 10. However, vinorelbine had no effect in Sting Gt/Gt mice (Fig. 3A). This genotype difference was also observed in the effect size (Fig. 3B). Additionally, a reduction in the paw withdrawal threshold was observed in WT mice that was not observed with vinorelbine in Sting Gt/Gt mice ( Fig. 3C-D). These results indicate that STING activation is needed for the pronociceptive effects of vinorelbine in mice.
We performed Western blots to further elucidate the changes of the canonical STING-IFN pathway underlying the blunted hypersensitivity of vinorelbine in Sting Gt/Gt mice. In the absence of a functional STING pathway, the administration of vinorelbine was not able to result in the increased production of IFN or p-IRF3 in DRG and sciatic nerve ( Fig. 3E-P), which aligns with the lack of hypersensitivity induced by the chemotherapeutic in Sting Gt/Gt mice. These results support the conclusion that vinorelbine induces type I IFN production via STING activation, consistent with the loss of nociceptive sensitization in Sting Gt/Gt mice.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made (n = 10-12 per group, 6 males and 4-6 females per group) as determined by two-way ANOVA followed by the Bonferroni's test in A,C. ***p<0.001, ****p<0.0001 as determined by two-way ANOVA followed by Bonferroni's test in B,D. t test in E-I and K-O (n=3-6 mice per group). Vino: vinorelbine, veh: vehicle.

MNK1 KO mice.
MNK1 is a serine/threonine kinase that phosphorylates eIF4E, the 5' mRNA Cap-binding protein, and is a key contributing factor in the development of chronic pain (41-44).
Previously, we showed that type I IFNs stimulate ERK/MAP kinase-MNK-eIF4E signaling in mouse DRG neurons (18). Since vinorelbine promotes the production of type I IFNs in DRG and sciatic nerve, we tested the hypothesis that vinorelbine-evoked nociceptive behaviors require MNK1 signaling. Vinorelbine was administered to MNK1 KO mice and their wild type controls (C57BL/6J) and compared its effects with vehicle in behavioral assays. As expected, we found an increase in the grimace score in WT mice from day 1 after administration to day 10 (Fig. 4A). A reduction in the vinorelbine-induced . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint pronociceptive effect was observed in MNK1 KO mice with a significantly lower grimace score the day after the second administration compared to WT animals (Fig. 4A). The statistical difference in the genotype effect can also be observed in the effect sizes in vinorelbine treated animals (Fig. 4B). Similarly, we found a reduction of paw withdrawal threshold in WT animals that reached almost 0.2 g at its peak. While a significant decrease in von Frey threshold was observed in the MNK1 KO mice 1 day after treatment, this effect was short lived and not seen on subsequent days (Fig. 4C). In line with this, the paw withdrawal threshold effect sizes of vinorelbine were significantly greater in WT animals compared to MNK1 KO mice (Fig. 4D).
Considering the reduction of the pronociceptive effects of vinorelbine in MNK1KO, in an independent set of experiments we measured the effects of vinorelbine on STING-TBK1-IRF3-IFN signaling. Vinorelbine treatment was associated with a trend toward decreased levels of p-TBK1 and p-IRF3 in MNK1 KO mice ( Fig. 4F-G) in DRG, and it did not modify the levels of p-STING ( Fig. 4E) or type I IFN production ( Fig. 4H-I). Vinorelbine provoked similar changes in the STING-TBK1-IRF3 pathway in sciatic nerve decreasing p-TBK1 and p-IRF3 (Fig. 4M), but in addition the drug also downregulated IFN-α levels in MNK1 KO mice (Fig. 4N). Together, these results show an interplay between the STING-IRF3-IFN pathway and MNK1 signaling, highlighting that vinorelbine fails to induce increases in IFN production in peripheral nerves in the absence of MNK1.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint  Because we observed a reduction in the pro-nociceptive effect of vinorelbine in MNK1 KO animals as well as abrogated associated changes in the STING pathway, we investigated the effect of vinorelbine on p-eIF4E in WT, Sting Gt/Gt , and MNK1 KO animals. Moreover, translation regulation through p-eIF4E has been shown to be associated with antiviral response and translation of mRNAs encoding proteins such as type I IFNs (45). An increase in p-eIF4E in DRG neurons was observed across all neuronal sizes ( Fig. 5A-B).
Specifically, peripherin-positive neurons from vinorelbine administered animals had an increase in p-eIF4E of 0.4 times the mean intensity value compared to control (Fig. 5C).
To give more insight about p-eIF4E changes induced by vinorelbine, western blot experiments in animals of the different genotypes were performed in DRG and sciatic nerve. In line with our immunohistochemistry experiments we found a clear trend toward . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint an increase of p-eIF4E in DRG from wild type animals (Fig. 6A). On the other hand, no changes were found in DRGs of Sting Gt/Gt and MNK1 KO mice ( Fig. 6B-C). An increase in p-eIF4E was observed in sciatic nerve in wild type mice (Fig. 6D). Administration of vinorelbine resulted in no change in p-eIF4E levels in Sting Gt/Gt in sciatic nerve (Fig. 6E).
Further, a trend toward reduction in p-eIF4E with vinorelbine administration was observed in MNK1 KO sciatic nerve (Fig. 6F). Both MNK1 and MNK2 are expressed in mouse DRG neurons (46) so the remaining p-eIF4E in peripheral nerves in MNK1 KO mice is likely support by the MNK2 isoform, which is known to have constitutive activity (26).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint  Altogether, our results reinforce the idea that IFNs cause pain by acting on nociceptors and explain, mechanistically, how chemotherapeutics that are STING activators and microtubule destabilizers such as vinorelbine, have the potential to cause pain and peripheral neuropathy. Furthermore, this study gives insight into the complexity of IFN induction within peripheral nerves and provides additional evidence that translational regulatory mechanisms play a critical role in chemotherapy induced pain (41).

Discussion
We reach several primary conclusions from the experiments described here. First, we have discovered a plausible mechanism through which vinorelbine induces pain hypersensitivity in male and female mice by activating the STING signaling pathway, including IRF3 phosphorylation (S396), and subsequent type I IFN induction in the peripheral nervous system. This conclusion is supported by the finding that vinorelbineinduced pain is abrogated in Sting Gt/Gt mice and peripheral nerve induction of type I IFN by vinorelbine was absent in these mice. Second, our work shows that a downstream effector involved in vinorelbine-induced pain is MNK1-eIF4E signaling, a finding consistent with our previous work on mechanisms through which type I IFNs cause pain in mice (18).
Therefore, our work also provides a clear mechanistic understanding of vinorelbineinduced peripheral neuropathy and leads us to the third conclusion, that MNK signaling is . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint a therapeutic avenue for reducing the neuropathy caused by this chemotherapeutic. Future work will be aimed at understanding how MNK inhibition can be used as a therapeutic approach to reduce chemotherapy neuropathies and improve cancer chemotherapeutic efficacy.
Our work was primarily motivated by the consistent clinical finding that vinorelbine causes painful peripheral neuropathy in humans treated with this drug. Our work fills an important gap in knowledge by providing a mechanistic explanation for vinorelbine-induced pain.
Neuropathy has been observed in oncological patients receiving vinorelbine in phase II/III trials for metastatic breast cancer, with neurotoxicity ranging from 33 to 43.6% (47, 48).
Notably, a heightened severity of neurotoxicity (87%) after 4 cycles of vinorelbine has also been reported (6). Patients who undergo treatment with vinorelbine report pain, primarily localized at the tumor site or in regions affected by nerve compression caused by the tumor (1, 47, 49). Moreover, some patients also present with paresthesia and myalgia consisting of acute pain in lower back muscles and extremities (47). There is also evidence that vinorelbine causes a peripheral neuropathy because the drug decreases the amplitude of evoked action potentials in sensory nerve bundles in patients (6), one of the main neurophysiological abnormalities observed in neuropathy (50). Finally, vinorelbine treatment is associated with the worsening of peripheral neuropathy in patients with preexisting diabetes or CIPN (51). Despite the induction of neuropathy, vinorelbine is approved for advanced NSCLC treatment (4) and is used in clinical trials for a wide variety of cancer types (1,5,47,48). Based on the rationale that vinorelbine causes neuropathic pain and is used alone or in combination with other chemotherapeutic drugs (52), we studied the signaling pathways associated with vinorelbine-related pain to find new strategies to mitigate or prevent the concomitant pain associated with its use. Our results . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint provide a detailed mechanistic rationale for blocking MNK signaling as a therapeutic strategy to mitigate the pain-promoting effects of vinorelbine. A second motivation for our work was to better understand the role of STING and type I IFN signaling in painful peripheral neuropathy. Recently, it was demonstrated that microtubule-targeted agents (MTAs), including vinorelbine and eribulin, activate the STING pathway leading to type I IFN production in human breast cancer and leukemia monocytic cell lines (12, 28). The STING pathway is an intracellular sensor system of external or selforigin DNA that is critical for recognizing pathogen infection and/or cellular damage to establish an effective host defense (53, 54). Our results clearly demonstrate that vinorelbine induces STING activation in mouse DRG neurons and peripheral nerve as reflected in increased IRF3 phosphorylation and type I IFN induction (55). Recently published work shows that STING induces peripheral nociceptive sensitization via NFKB activation in DRG neurons in a model of bone cancer pain in rats (25). Furthermore, the STING inhibitor C-176 has been shown to reduce mechanical hypersensitivity induced by nerve injury in rats suggesting a pronociceptive role for STING (24). Mounting evidence has shown that STING is anomalously activated in models of peripheral neuropathic pain (56), central neuropathic pain (57, 58), and inflammatory pain (25, 59) in rodents. These works have concluded that the pharmacological blockade of STING may be a promising target for pain (25,(56)(57)(58)(59). Collectively, our work is consistent with the emerging view that activation of STING signaling in the peripheral nervous system is pro-nociceptive and may play an important role in multiple types of painful peripheral neuropathies.
Evidence of an antinociceptive role of STING has also been reported (22, 23, 60).
Activators of STING, including ADU-S100, DMXAA and 3'3'-cGAMP, have been shown to reverse CIPN-, nerve injury-(22) and bone cancer-induced pain in mice (23) via suppression of nociceptor excitability. Under physiological conditions, the role of STING . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint has been shown to regulate steady-state nociception, since intrathecal administration of the STING agonists ADU-S100 or DMXAA produced antinociception in naïve mice. Moreover, naïve Sting Gt/Gt mice showed increased sensitivity to mechanical and cold stimuli, although we did not find this in our experiments for von Frey threshold, and DRG nociceptors from these mice displayed increased action potential firing and reduced rheobase suggesting nociceptor hyperexcitability (22). Thus, the aforementioned studies suggest an antinociceptive role for STING (23). A recent review concluded that STING signaling causes bidirectional effects on nociception but did not reach firm conclusions on why STING activation may be associated with increased pain in some contexts and antinociception in others (54). One hypothesis to explain this controversy is that whether STING is activated in the peripheral or central nervous system (CNS) is critical for the direction of the effects of the pathway on nociceptive signaling. From this perspective, it is important to note that the chemical properties of vinorelbine, like other vinca alkaloids such as vincristine, render the drug unable to cross the blood-brain barrier (61). Accordingly, we only observed pro-nociceptive effects of vinorelbine in mice.
There is controversy over the effects of type I IFNs on nociception, which may also be explained by peripheral nervous system (PNS) versus CNS sites of action (62). We have previously shown that type I IFNs activate their receptors (IFNR) localized on nociceptors driving neuron hyperexcitability and pro-nociceptive actions in mice (18) was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint intrathecal administration of , again consistent with different actions of this signaling pathway depending on the site of administration. Other studies finding an antinociceptive role for type I IFNs have administered the IFNs intracerebroventricularly or intrathecally (63-65). Our work adds to the growing body of evidence that peripheral type I IFN signaling is pro-nociceptive. We did not test CNS-mediated effects of vinorelbine because this is not relevant for the clinical effects of this peripherally-restricted drug.
Findings in Sting Gt/Gt mice unequivocally implicate STING signaling in the pro-nociceptive effects of vinorelbine. Accordingly, we did not observe changes in STING downstream effectors in either the peripheral nerve or DRG with vinorelbine administration. In contrast, in WT mice, we consistently observed increased IRF3 phosphorylation using Western blotting and immunofluorescence. We also observed increases in type I IFNs in DRG and sciatic nerve in WT mice, which likely occurred due to the presence of IRF3-driven transcription of Ifna and Ifnb genes (37, 38). On the other hand, we did not find changes in the phosphorylation of STING (Ser366), which is essential to trigger STING pathway signaling (66). A possible explanation is that after trafficking to the Golgi apparatus, STING is phosphorylated by serine/threonine UNC-51-like kinase (ULK1/ATG1) to facilitate its degradation and prevent the deleterious effect of a long-lasting inflammatory state induced by persistent STING activation (67). Thus, our failure to observe changes in phosphorylation of STING could be due to degradation of the activated protein at the timepoint at which we harvested tissues for analysis. This could also explain the lack of changes in p-TBK1 in WT mice.
The exact mechanism by which the MTAs such as vinorelbine or eribulin enhance STING signaling has not been fully uncovered. Some studies have suggested that the STING pathway is activated by mitochondrial or nuclear DNA release induced by cellular damage resulting from the treatment with MTAs but it has also been proposed that STING . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint 3 0 activation occurs downstream of the microtubule disruption triggered by these chemotherapeutics (12,28,68). Whatever the precise mechanism turns out to be, the discovery that vinorelbine-induced pain depends on STING-type I IFN signaling makes it possible to implement strategies to alleviate the neuropathic pain state caused by the chemotherapeutic. We demonstrate that this can be achieved by targeting MNK1-eIF4E signaling. Vinorelbine caused an increase in eIF4E phosphorylation at a site that is specifically regulated by MNK in both the peripheral nerve and in DRG cell bodies.
Moreover, grimacing and mechanical hypersensitivity were profoundly reduced in MNK1 KO mice. Translation regulation via MNK-eIF4E signaling has been shown to participate in the development and maintenance of pain hypersensitivity in many different preclinical models (18,41,42,44). MNK1 has been shown to be expressed at the mRNA level in nearly all human nociceptors (46) indicating that this mechanism is likely to translate to humans. Our findings support the idea that MNK represents a promising mechanistic target for pharmacological relief or prevention of chemotherapy-induced pain. It will be important to understand whether MNK inhibition will interfere with the anti-cancer activity of the chemotherapeutic, but this is unlikely since increased MNK activity and increased eIF4E phosphorylation is associated with many cancers (69, 70) and MNK inhibitors are currently in clinical trials for cancer treatment (71).
There are several limitations of this study. We did not directly interfere with type I IFNs, which would have allowed us to dissect STING activation versus type I-IFN downstream effects. While this can be addressed in future studies, our main goal was to understand whether STING activation was linked to vinorelbine-induced pain and our use of Sting Gt/Gt mice provides a clear link between vinorelbine and STING signaling. In some cases, we found trends for signaling changes in sciatic nerve or DRG (p values between 0.06 and 0.09). These trends were consistent with findings using transgenic and knockout mice for . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint 1 signaling pathways and were observed in a complex tissue where there are a mix of cell types that contribute to the overall changes in protein levels. We do not think that these observed trends weaken the overall conclusions of the study. Unanswered questions remain to be uncovered regarding other STING activated pathways, which we did not examine. For instance, it is known that STING signaling can activate inhibitor of κ B kinase (IKKε)/nuclear factor κ B (NF-κB), which synergizes with IRF3 to induce higher levels of type I IFNs (72). Future studies can address nuances of these signaling effects now that our work has unequivocally demonstrated that vinorelbine-induced pain requires STING signaling. Finally, while we provide genetic evidence for MNK1-eIF4E signaling as a causative factor in vinorelbine-evoked pain, it will be interesting to assess the extent to which MNK inhibitors like eFT508 can reproduce this effect. Given that pharmacological inhibition of MNK has thus-far matched genetic manipulation in behavioral endpoints in neuropathic and other pain models (18,41,42,44,73,74) it is likely that these MNK inhibitors will be effective in reducing pain promoting effects of vinorelbine.

Acknowledgements
We acknowledge Zawge Johannes Daniel and Abdulfetah Abdo for the technical support to this project. This work was supported by NIH grant NS065926 to TJP.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint  i  m  m  u  n  o  r  e  a  c  t  i  v  i  t  i  e  s  ,  a  n  d  i  s  o  l  e  c  t  i  n  B  4  b  i  n  d  i  n  g  i  n  p  r  i  m  a  r  y  a  f  f  e  r  e  n  t  n  e  u  r  o  n  s  o  f  t  h  e  r  a  t  a  n  d   m  o  u  s  e  .   J  P  a  i  n  .   2  0  0  7  ;  8  (  3  )  :  2  6  3  -7  2  .   3  4  .  C  h  a  p  l  a  n  S  R  ,  B  a  c  h  F  W  ,  P  o  g  r  e  l  J  W  ,  C  h  u  n  g  J  M  ,  a  n  d  Y  a  k  s  h  T  L  .  Q  u  a  n  t  i  t  a  t  i  v  e  a  s  s  e  s  s  m  e  n  t  o  f   t  a  c  t  i  l  e  a  l  l  o  d  y  n  i  a  i  n  t  h  e  r  a  t  p  a  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint   3  5   5  0  .  Z  h  a  n  g  H  ,  a  n  d  D  o  u  g  h  e  r  t  y  P  M  .  E  n  h  a  n  c  e  d  e  x  c  i  t  a  b  i  l  i  t  y  o  f  p  r  i  m  a  r  y  s  e  n  s  o  r  y  n  e  u  r  o  n  s  a  n  d  a  l  t  e  r  e  d   g  e  n  e  e  x  p  r  e  s  s  i  o  n  o  f  n  e  u  r  o  n  a  l  i  o  n  c  h  a  n  n  e  l  s  i  n  d  o  r  s  a  l  r  o  o  t  g  a  n  g  l  i  o  n  i  n  p  a  c  l  i  t  a  x  e  l  -i  n  d  u  c  e  d   p  e  r  i  p  h  e  r  a  l  n  e  u  r  o  p  a  t  h  y  .   A  n  e  s  t  h  e  s  i  o  l  o  g  y  .   2  0  1  4  ;  1  2  0  (  6  )  :  1  4  6  3  -7  5  .   5  1  .  K  e  l  l  e  r  S  ,  S  e  i  p  e  l  K  ,  N  o  v  a  k  U  ,  M  u  e  l  l  e  r  B  U  ,  T  a  l  e  g  h  a  n  i  B  M  ,  L  e  i  b  u  n  d  g  u  t  K  ,  e  t  a  l  .  N  e  u  r  o  t  o  x  i  c  i  t  y  o  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint 6 7 . K  o  n  n  o  H  ,  K  o  n  n  o  K  ,  a  n  d  B  a  r  b  e  r  G  N  .  C  y  c  l  i  c  d  i  n  u  c  l  e  o  t  i  d  e  s  t  r  i  g  g  e  r  U  L  K  1  (  A  T  G  1  )   p  h  o  s  p  h  o  r  y  l  a  t  i  o  n  o  f  S  T  I  N  G  t  o  p  r  e  v  e  n  t  s  u  s  t  a  i  n  e  d  i  n  n  a  t  e  i  m  m  u  n  e  s  i  g  n  a  l  i  n  g  . C e l l . . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 5, 2023. ; https://doi.org/10.1101/2023.06.03.543579 doi: bioRxiv preprint