Vancomycin intermediate-resistant Staphylococcus aureus (VISA) is a pathogen of concern. VraS, a histidine kinase, facilitates the VISA phenotype. Here, we reveal a benzoxazolyl urea (chemical 1) that directly inhibits VraS and enhances vancomycin to below the clinical breakpoint against an archetypal VISA strain, Mu50. 50 μM of 1 enhances vancomycin 16-fold to 0.25 μg/mL. The MIC of oxacillin is enhanced 32-fold to 8 μg/mL, only slightly above its clinical breakpoint. The chemical also showed promising enhancement of oxacillin against several MRSA strains. 1 shows ∼30 % inhibition of ATPase activity in VraS and reduces vra gene auto-upregulation typical upon vancomycin exposure. Therefore, 1 inhibits VraS to block normal vra operon function, leading to potent enhancement of cell wall-directed antibiotics. Interestingly, a molecular modeling approach suggests 1 does not displace ATP from the active site, but acts elsewhere. While VraS inhibitors have previously been reported to function against MRSA, to the best of our knowledge, this is the first direct VraS inhibitor ever reported that shows significant enhancement of vancomycin against VISA.

Neurofibromatosis Type 1 (NF1) is a common neurogenic condition characterized by heterozygous loss of function mutations in the neurofibromin gene. NF1 patients are susceptible to the development of neurofibromas, including plexiform neurofibromas (pNFs), which occurs in about half of all cases. Plexiform neurofibroma are benign peripheral nerve sheath tumors originating from Schwann cells after complete loss of neurofibromin; they can be debilitating and also transform into deadly malignant peripheral nerve sheath tumors (MPNSTs). Here, our data indicates that silver nanoparticles (AgNPs) may be useful in the treatment of pNFs. We assessed the cytotoxicity of AgNPs using pNF cells and Schwann cells derived from the same NF1 patient. We found that AgNPs are selectively cytotoxic to pNF cells relative to isogenic Schwann cells. We then examined the role of neurofibromin expression on AgNP-mediated cytotoxicity; restoration of neurofibromin expression in pNF cells decreased sensitivity to AgNP, and knockdown of neurofibromin in isogenic Schwann cells increased sensitivity to AgNP, outlining a correlation between neurofibromin expression and AgNP-mediated cytotoxicity. AgNP was able to selectively remove pNF cells from a co-culture with patient-matched Schwann cells. Therefore, AgNPs represent a new approach for clinical management of NF1-associated pNF to address significant clinical need.

Neurofibromatosis type 1 (NF1) is among the most common neurogenic disorders, characterized by loss of function mutations in the neurofibromin gene (NF1). NF1 patients are extremely susceptible to developing neurofibromas, which can transform into deadly malignant peripheral nerve sheath tumors (MPNSTs). At the center of these tumors are NF1-null Schwann cells. Here, we found that nanomedicine shows promise in the treatment of NF1-associated MPNSTs. We assessed the cytotoxicity of silver nanoparticles (AgNPs) in NF1-null NF1-associated MPNSTs, NF1-wildtype sporadic MPNST, and normal Schwann cells. Our data show that AgNP are selectivity cytotoxic to NF1-associated MPNSTs relative to sporadic MPNST and Schwann cells. Furthermore, we found that sensitivity to AgNPs is correlated with the expression levels of functional neurofibromin. The restoration of functional neurofibromin in NF1-associated MPNSTs reduces AgNP sensitivity, and the knockdown of neurofibromin in Schwann cells increases AgNP sensitivity. This finding is unique to AgNPs, as NF1 restoration does not alter sensitivity to standard of care chemotherapy doxorubicin in NF1-associated MPNSTs. Using an in vitro model system, we then found that AgNP can selectively eradicate NF1-associated MPNSTs in co-culture with Schwann cells at doses tolerable to normal cells. AgNP represents a novel therapy for the treatment of NF1-associated MPNSTs and addresses significant unmet clinical need.

Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.

Kratom (Mitragyna speciosa) is consumed by 10-15 million individuals in the US for its mood-elevating effects, as an alternative to traditional opiates for pain relief and to attenuate opiate withdrawal symptoms. Complex natural products, such as kratom, contain a variety of chemicals whose combined effects can only be understood by the meticulous identification and characterization of the individual chemicals and their effects in preclinical model systems. Twenty-five indole and oxindole kratom alkaloids were evaluated for mu (MOR), delta (DOR) and kappa (KOR) opiate receptors binding affinities, opiate receptor mediated G-protein activation, inhibition of forskolin stimulated cAMP accumulation and b-arrestin2 recruitment. Several indole and oxindole alkaloids exhibited partial agonism at the MOR (7-hydroxymitragynine, corynoxine A > epiallo-isopaynantheine, isospeciofoline, speciociliatine, corynoxine B > mitragynine, 3-epicorynoxine B > paynantheine and speciogynine) - all of which also exhibited strong G protein bias. Mitraciliatine and isopaynantheine were weak MOR antagonists. Speciophylline exhibited positive allosteric modulatory activity at the MOR as evidenced by increased affinity of DAMGO binding and potentiation of [met]-enkephalin-induced inhibition of forskolin stimulated cAMP activity in the absence of intrinsic activity. Corynoxine A induced displayed similar affinity at the mu opiate receptor as 7 hydroxymitragynine but was less potent in the nociceptive hot plate assay and the mechanical allodynia von Frey test. Corynoxine did not significantly alter respiration or heart rate or induce a conditioned place preference. Results demonstrate the pharmacological complexity of kratom alkaloids and have important implications for determining the combined effects of less abundant indole and oxindole alkaloids with regard to the therapeutic as well as potential harmful effects of kratom.

Neurofibromatosis type 1 (NF1) is the most common neurogenic disorder affecting 1 in every 3,000 people worldwide. NF1 is defined by heterozygous loss-of-function of the neurofibromin 1 gene (NF1). NF1 patients develop neurofibromas with near complete penetrance. These neurofibromas include peripheral nerve sheath tumors (PNSTs) such as plexiform neurofibromas (pNFs) and malignant PNSTs (MPNSTs). Current standard of care shows incomplete penetrance limiting clinical utility. Therefore, treatments to selectively and safely remove PNST cells would be advantageous. Biallelic loss of functional NF1 is the major driving force in the development of PNSTs. NF1 is a tumor suppressor gene responsible for suppressing Ras activity. Loss of NF1 increases Ras activity and subsequently increases intracellular reactive oxygen species (ROS). We and other groups have demonstrated that systemically administrable silver nanoparticles (AgNPs) are cytotoxic to a variety of cancers. We further showed that AgNP-mediated cytotoxicity is dependent upon intracellular ROS which induces rapid ionization (activation) of Ag0 to cytotoxic Ag+. Therefore, AgNPs represent a rational cancer-selective therapy for NF1-deficient PNSTs as these cells are enriched for ROS relative to non-cancerous cells. In this study, we found that AgNPs are significantly (~5-fold) more cytotoxic to NF1-null MPNST cells relative to tumor cell of origin Schwann cells and ~7-fold more cytotoxic to pNF cells relative to patient matched Schwann cells. Sporadic NF1 wild-type MPNST cells were not sensitive to AgNP. We further explored the relationship between oxidative stress and AgNP-mediated cytotoxicity in PNSTs. We found that co-administration of oxidative stress inducing cumene hydroperoxide with AgNP augments the cytotoxicity of AgNP in otherwise AgNP-tolerant cells (Schwann cells, NF1-wildtype MPNSTs). Importantly, either agent alone only showed modest cytotoxicity. As NF1 gene expression levels correlated with AgNP-sensitivity, we further studied this possible interaction by reintroducing NF1 into NF1-null MPNST. Remarkably, NF1 restoration decreased sensitivity to AgNP. This change is unique to AgNP as there was no altered sensitivity to standard of care chemotherapy doxorubicin. Conversely, knockdown of NF1 expression in Schwann cells increased sensitivity to AgNP with no corresponding change in sensitivity to pNF standard of care MEK inhibitor selumetinib. In conclusion, our work provides evidence that a therapeutic window exists for the safe use of AgNPs as a precision medicine to treat NF1-null PNSTs. Citation Format: Garrett Alewine, Adithya Ghantae, Christina Mamrega, Jerrica L. Knight, Bashnona Attiah, Robert A. Coover, Cale D. Fahrenholtz. Silver nanoparticles are selectively cytotoxic to neurofibromin 1 deficient peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 373.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation. Editor's evaluation This study explores a role for the purinergic receptor P2RY14 and cAMP signaling in Schwann cell precursor self-renewal and neurofibroma development. Importantly, the authors show that genetic and chemical inhibition of P2RY14 inhibits Schwann cell precursor self-renewal in vitro and suppresses neurofibroma development in vivo. The authors also report that these effects are mediated by an increase in cAMP signaling. https://doi.org/10.7554/eLife.73511.sa0 Decision letter eLife's review process Introduction Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that affects up to 1:2000 individuals worldwide (Kallionpää et al., 2018). To date, there is no cure for NF1, which is characterized by multiple, variable, clinical manifestations (Friedman, 1998; Tabata et al., 2020). At least half of the children with NF1 develop plexiform neurofibromas (PNs), which are tumors within peripheral nerves. PN may be present at birth and show most rapid growth during the first decade of a child’s life (Nguyen et al., 2012). PNs can occur in any cranial or peripheral nerve and have the potential to transform into lethal malignant peripheral nerve sheath tumors (MPNST) (Prudner et al., 2020). Neurofibroma infiltration of normal nerves in NF1 patients results in a complicated risk profile because it can cause nerve damage and compress nearby vital organs (Kim et al., 2017). Therefore, understanding how neurofibromas form and how to treat them is under intense investigation. Peripheral nerve glial cells, Schwann cells (SCs), are the only cell type in neurofibromas that shows bi-allelic loss-of-function mutations in the NF1 tumor suppressor gene (Serra et al., 1997; Serra et al., 2001). Neurofibroma SCs also show aberrant properties ex vivo, consistent with it being the primary pathogenic cell type in neurofibromas (Sheela et al., 1990; Kim et al., 1995). In mice, neural crest cells develop into SC precursors (SCPs) between embryonic day 11 (E11) and E13 (Jessen and Mirsky, 2019). SCPs or related boundary cap cells can serve as cells-of-origin for neurofibromas, as loss of Nf1 in these cells causes PN formation (Zhu et al., 2002; Wu et al., 2008; Chen et al., 2014; Chen et al., 2019). In vitro, embryonic SCPs retain multi-lineage differentiation potential and self-renewal capabilities for several passages, indicating that they are progenitor-like cells (Jessen and Mirsky, 2019). Mouse SCPs also express epidermal growth factor receptor+ (EGFR+) and respond to EGF with limited self-renewal (Williams et al., 2008). EGFR+ cells that co-express the SC marker S100 account for about 1.8% human neurofibroma cells (DeClue et al., 2000). The idea that these cells may be tumor-initiating cells is consistent with the finding that human neurofibromas sorted for co-expression of the SC marker p75+ and EGFR show limited self-renewal in vitro. Also, EGFR-dependent Nf1-/- SCPs show increased self-renewal and form neurofibromas upon transplantation (Joseph et al., 2008; Williams et al., 2008). Together, these studies suggest the presence of progenitor-like cells in neurofibromas, which depend on EGFR for self-renewal. EGFR signaling may also play additional roles in transformed SCs, as MPNST cells re-acquire EGFR expression that is absent in mature SC (DeClue et al., 2000). SCPs differentiate into SCs. When associated with a single large-diameter axonal segment SCs form myelin. SCs associated with multiple smaller diameter axons become non-myelinating Remak cells (Mirsky et al., 2008). Neurofibroma SCs show a dramatic change in Remak SC morphology, bundling one or two few axons (Erlandson and Woodruff, 1982; Zheng et al., 2008), rather than up to 20 small diameter axons in WT nerve Remak bundles (Harty and Monk, 2017). Notably, while neurofibromas rarely form, elevation of EGFR in WT SCPs and SCs is sufficient to mimic this nerve disruption phenotype (Ling et al., 2005). The NF1 gene encodes neurofibromin, a GTPase activating protein that accelerates the hydrolysis of RAS-GTP to its inactive GDP-bound form downstream of EGFR (Simanshu et al., 2017). In SCs, loss of neurofibromin causes increases in GTP-bound RAS (Kim et al., 1995; Sherman et al., 2000), and RAS-GTP stimulates the mitogen-activated protein kinase (MAPK) pathway and other downstream pathways, including deregulation of the PI3K-AKT signaling (Dasgupta et al., 2005; Johannessen et al., 2008; Patmore et al., 2012; Keng et al., 2012). Loss of NF1 also causes reduced levels of cyclic AMP (cAMP) in Nf1 mutant mouse, fly and zebrafish (Hegedus et al., 2007; Tong et al., 2002; Wolman et al., 2014; Anastasaki and Gutmann, 2014). Whether cAMP deregulation occurs downstream of increased RAS-GTP is unclear. Neurofibromin shares homology with the yeast proteins Ira1 and Ira2, which are inhibitory regulators of the RAS-cAMP adenylyl cyclase pathway, but no evidence shows a direct role for NF1 or IRA proteins in direct regulation of cAMP in mammals (Ballester et al., 1990; Martin et al., 1990; Xu et al., 1990). It is unclear how, or if, regulation of cAMP is relevant to neurofibroma initiation or growth but reducing cAMP drove formation of brain tumors in cells lacking Nf1 (Warrington et al., 2010). We sought to identify molecules that might affect neurofibroma development in the SC lineage. We identified P2ry14 as a G-protein-coupled receptor (GPCR) expressed in neurofibroma SCPs. P2ry14 is activated by extracellular UDP and UDP-sugars and signal through Gi to inhibit adenylate cyclase (AC), decreasing cAMP (Abbracchio and Ceruti, 2006; Conroy et al., 2016). Intriguingly, P2ry14 regulates homeostasis of hematopoietic stem/progenitor cells (Cho et al., 2014). Also, satellite glial cells and SCs have been reported to express P2ry14 in vitro (Patritti-Cram et al., 2021). Here, we show that, in vitro, P2ry14 inhibition decreases mouse SCP self-renewal by modulation of cAMP. In vivo, P2ry14 knockout increased mouse survival, decreased SC proliferation, improved nerve Remak bundle morphology, and decreased tumor initiation. Pharmacological elevation of cAMP diminished NF1 deficient SCP self-renewal in vitro and reduced SC proliferation in neurofibroma bearing mice in vivo. We suggest that targeting the P2ry14 receptor pathway could be relevant for treatment of NF1. Results P2RY14 is expressed in human neurofibroma SCP and promotes SCP self-renewal in vitro To characterize SCPs we used flow cytometry. We dissociated cells from human PNs resected for therapeutic purposes from three neurofibroma patients. Cells were sorted into p75+/EGFR- SCs and p75+/EGFR+ SCP-like tumor-initiating cells. We performed gene expression analysis on these cells and found that P2ry14 mRNA is elevated in p75+/EGFR+ SCP-like tumor-initiating cells (Figure 1A). Western blot also showed expression in human SCs and neurofibroma SCPs, with 1.9-fold increase of P2ry14 protein in SCP-like cells (Figure 1B). Anti-P2ry14 staining also showed P2ry14 protein membranes of myelinating SCs in human neurofibroma tissue sections (Figure 1C). To test if P2ry14+ SCP-like cells derived from human neurofibromas have altered ability to self-renew, we performed fluorescence-activated cells sorting (FACS) and sorted SCP-like cells into p75+/EGFR+/P2ry14- and p75+/EGFR+/P2ry14+ cells and plated them at low density to generate unattached spheres in vitro (Figure 1D–F). Unsorted neurofibroma cells rarely form SCP-like spheres. FACS analysis (of cells from three additional neurofibroma tumors) showed that on average p75+/EGFR+/P2ry14- cells formed spheres at a frequency of 23.4%, while 64.8% of p75+/EGFR+/P2ry14+ cells formed spheres. The p75+/EGFR+/P2ry14 + cells maintained their significantly enhanced ability to form spheres in vitro for three passages (Figure 1G and H). Thus, P2ry14 is overexpressed in human neurofibroma SCPs in vitro, and marks SCP with the potential to self-renew in vitro. Figure 1 Download asset Open asset P2RY14 is expressed in human neurofibromas and promotes Schwann cell precursor (SCP) self-renewal in vitro. (A) Microarray heatmap shows P2ry14 receptor expression in p75+/EGFR+ SCP-like tumor-initiating cells derived from human plexiform neurofibroma tumor cells compared to p75+/EGFR- SCP-like cells. (B) Western blot of human Schwann cells and neurofibroma SCP shows the latter has a 1.9-fold increase in P2ry14 protein expression. (C) Immunohistochemistry of human neurofibroma shows P2ry14 expression (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]). (D) Representative fluorescence-activated cells sorting (FACS) plot shows live sorted human plexiform neurofibroma tumor cells. (E) Representative FACS plot shows human plexiform neurofibroma tumor cells sorted into p75+/EGFR+ SCP-like tumor-initiating cells (pink square). (F) Representative FACS plot shows p75+/EGFR+ SCP-like tumor-initiating cells further sorted into p75+/EGFR+/P2ry14- (left, purple square) and P75+/EGFR+/P2ry14+ (right, blue square). (G) Photomicrographs of human neurofibromas dissociated using FACS to yield: unsorted, p75+/EGFR+/P2ry14- and P75+/EGFR+/P2ry14+ cells. (H) Quantification of unsorted, p75+/EGFR+/P2ry14- and P75+/EGFR+/P2ry14+ cells plated in sphere medium. (n = 3; two-way ANOVA; primary: **p = 0.0057, ****p < 0.0001; secondary: *p = 0.0487; **p < 0.0024, ****p < 0.0001; tertiary: *p = 0.0321, ***p = 0.0006). Mouse Nf1 mutant SCPs P2RY14-cAMP signaling regulates self-renewal To test if P2ry14 signaling is relevant in cultured SCP spheres from wild-type (WT) and Nf1-/- mouse embryos, we treated these cells with the highly selective P2ry14 inhibitor PPTN (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid) hydrochloride (Robichaud et al., 2011). We found that treatment with PPTN reduced the percentage of Nf1-/- mouse SCPs that formed spheres, but had little effect on WT SCPs, consistent with a role in Nf1 mutant SCP self-renewal (Figure 2A). Photographs of spheres are shown in Figure 2—figure supplement 1A; this experiment was repeated in three biological replicates with similar results. Dose-response analysis confirmed that the optimal concentration of the P2ry14 inhibitor (PPTN) is 300 nM; as 500 nM PPTN was toxic (Figure 2—figure supplement 2A–F). Figure 2 with 3 supplements see all Download asset Open asset Mouse Nf1 mutant Schwann cell precursors (SCPs) use P2RY14 signaling to regulate self-renewal. (A) Quantification of percent of sphere forming cells in mouse wild-type (WT) and Nf1-/- SCPs treated with the selective P2ry14 inhibitor (300 nM 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) (primary, secondary, tertiary passage) (n = 3; two-way ANOVA; primary: *p = 0.0375, ***p = 0.0001, ****p < 0.0001; secondary: *p = 0.0101, **p = 0.0015, ***p = 0.0009; tertiary: *p = 0.0101, **p = 0.0050, ***p = 0.0005). (B) P2ry14 mRNA expression in WT and Nf1-/- E12.5 mouse SCP treated with sh non-target (shNT) control and shP2ry14 (****p < 0.0001). (C) Western blot of WT and Nf1-/- SCPs treated with shNT and shP2ry14 showing P2ry14 knockdown. WT shP2ry14 show a 0.4-fold decrease of P2ry14 protein compared to WT shNT. Nf1-/- shP2ry14 show a 0.5-fold decrease compared to Nf1-/-. (D) Quantification of percent of sphere forming cells in mouse WT and Nf1-/- SCPs treated with shNT and shP2ry14 (n = 3; two-way ANOVA; primary: *p = 0.0288, ****p < 0.0001; secondary: **p = 0.0029,***p = 0.0005, ****p < 0.0001; tertiary: *p = 0.0154, ***p = 0.0007). (E) Western blot of WT and Nf1-/- Schwann cell (SC) spheres shows changes in pPKA substrate phosphorylation after shP2ry14 knockdown. WT shP2ry14 shows a 1.43-fold increase in pPKA after P2ry14 knockdown. Nf1-/- cells have a 1.31-fold increase in pPKA expression after P2ry14 knockdown. (F) Quantification of percent of sphere forming cells in Nf1-/- mouse SCPs treated with 1 µM rolipram or 300 nM PPTN (n = 3; two-way ANOVA; primary: ***p = 0.0002, ****p < 0.0001; secondary **p = 0.0030, ****p < 0.0001; tertiary: *p = 0.0476, ***p = 0.0004). To confirm these results, we silenced P2ry14 gene expression using short-hairpin RNAs (shRNAs) targeting P2ry14. WT and Nf1-/- mouse SCPs were treated with non-target control (shNT) or shRNA P2ry14 (shP2ry14). shP2ry14 treated cells showed reduced P2ry14 mRNA (Figure 2B) and P2ry14 protein (Figure 2C). Sphere formation was significantly decreased in Nf1-/- SCPs but not WT treated with P2ry14 shRNA, and this phenotype also persisted for three passages (Figure 2D). Photomicrographs are shown for one shRNA (sh09) (Figure 2—figure supplement 1B); but the experiment was repeated with two additional P2ry14 shRNAs in three biological replicates each, with similar results (Figure 2—figure supplement 3A-D). We also analyzed cAMP-dependent protein kinase (PKA) substrate phosphorylation (using anti-p-PKA substrate antibody) as an indirect read out of cAMP levels in cells. p-PKA substrate phosphorylation increased in WT and Nf1-/- SCPs shP2ry14 treated cells. Importantly, in Nf1-/- SCPs knockdown of P2ry14 increased levels of pPKA to those in WT shNT levels (Figure 2E). Thus, PPTN or shRNA targeting P2ry14 affects Nf1 mutant SCP spheres more than WT spheres, a differential effect consistent with deregulated cell signaling in Nf1 mutant cells. To understand if P2ry14-mediated changes in cAMP signaling affect SCP self-renewal, we treated WT and Nf1-/- SCPs with the specific phosphodiesterase-4 (PDE4) inhibitor, rolipram. Rolipram blocks degradation of cAMP by PDE4, increasing intracellular levels of cAMP (Mackenzie and Houslay, 2000). Treatment with 1 µM rolipram or 300 nM of the P2ry14 inhibitor (PPTN) decreased Nf1-/- SCP self-renewal; the combination showed additional effect, largely at early passage (Figure 2F). Photographs of these experiments are shown in (Figure 2—figure supplement 1C). These results support the idea that the elevated self-renewal in Nf1-/- SCP is due, at least in part, to P2ry14 Gi-mediated changes in cAMP. P2RY14 is expressed in vivo in mouse SCPs and SCs In the Nf1fl/fl;DhhCre neurofibroma mouse model, Cre recombinase is expressed from Desert Hedgehog (Dhh) regulatory sequences to effect recombination of the Nf1fl/fl allele, resulting in loss of both Nf1 alleles in developing SCPs at E12.5 (Wu et al., 2008). Nf1fl/fl;DhhCre mice develop paraspinal neurofibromas that have loss of axon-SC interaction in Remak bundles, mast cell and macrophage accumulation, and nerve fibrosis, all characteristics of human PNs (Wu et al., 2008; Liao et al., 2018; Fletcher et al., 2019a; Fletcher et al., 2019b). In this setting, PNs are present by 4 months of age, and neurofibromas enlarge as the mice age, ultimately compressing the spinal cord causing paralysis and thus, necessitating sacrifice (Wu et al., 2008). We bred P2ry14-/- mice (Meister et al., 2014) and generated P2ry14-/-; Nf1fl/fl;DhhCre, P2ry14+/-; Nf1fl/fl;DhhCre littermates, and Nf1fl/fl;DhhCre controls (Figure 3A). Genotyping and western blotting verified reduced P2ry14 in P2ry14-/-; Nf1fl/fl;DhhCre sciatic nerve and neurofibroma tumors compared to Nf1fl/fl;DhhCre controls (Figure 3B and C). In the P2ry14-/- mice, most of the coding region of P2RY14 is replaced by a β-galactosidase cassette, so that where the P2ry14 gene is expressed, β-galactosidase is detectable (Meister et al., 2014). We visualized P2ry14 in SOX-10 positive SCPs in the spinal cord dorsal and ventral roots (VR) and in the dorsal root ganglion (DRG) of E12.5 mice (Figure 3D and E). P2ry14-/-; Nf1fl/fl;DhhCre SCs in 7-month-old mice were also positive for β-galactosidase staining (Figure 3F). CNPase (2’,3’ cyclic nucleotide 3’ phosphodiesterase), a known SC marker, co-localized with β-galactosidase staining in the sciatic nerve (Figure 3F, inset). P2ry14 antibody staining confirmed P2ry14 expression in myelinating SC in mouse WT nerve and in Nf1fl/fl;DhhCre neurofibromas (Figure 3G). Figure 3 Download asset Open asset P2RY14 is expressed in Schwann cell precursors (SCPs) and Schwann cells (SCs) in vivo in a mouse model of neurofibromatosis. (A) Schematic of generation of neurofibroma mice breeding of P2ry14-/- mice with Nffl/fl mice to obtain P2ry14+/-; Nf1fl/fl;DhhCre and P2ry14-/-; Nf1fl/fl;DhhCre littermates after several crosses. (B) Genotyping confirmation of wild-type (WT) and P2ry14 knockout (KO) alleles. (C) Western blot of sciatic nerve and neurofibroma tumors of Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice show decrease in P2ry14 expression upon P2ry14 knockdown (1- to 0.1-fold decrease in sciatic nerve and 1- to 0.2-fold decrease in neurofibroma tissue). (D) Spinal cord (SC) immunofluorescent staining of mouse embryos at embryonic day 12.5 (E12.5) shows P2ry14 expression (β-galactosidase) co-localization with SOX-10 SCs at the dorsal root ganglion (DRG) and ventral roots (VR). (E) Spinal cord (SC) immunofluorescent staining of mouse embryos at E12.5 shows P2ry14 expression (β-galactosidase) co-localization with SOX-10 SCs at DRG. E1 and E2 insets show enlarged sections of the DRG. (F) Immunofluorescent staining of 7-month-old mouse sciatic nerve shows β-galactosidase positive staining as a confirmation of P2ry14 knock-in; co-labeling of β-galactosidase and CNPase shows that P2ry14 co-localizes with SCs (inset). (G) DAB staining of 7-month-old WT nerve and Nf1fl/fl;DhhCre mouse neurofibromas (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]). P2RY14 deletion in mouse model of neurofibroma increases survival, delays neurofibroma initiation, and improves SC Remak bundle disruption Kaplan-Meier survival analysis showed that P2ry14-/-; Nf1fl/fl;DhhCre have a significant survival advantage compared to neurofibroma-bearing mice (Nf1fl/fl;DhhCre; p = 0.0256). In fact, P2ry14-/-;Nf1fl/fl;DhhCre did not differ significantly from non-neurofibroma bearing Nf1fl/fl;DhhCre control littermates (p = 0.1367) (Figure 4A). To test if P2RY14 deletion plays a role in neurofibroma initiation, we analyzed Nf1fl/fl;DhhCre and P2ry14 -/-;Nf1fl/fl;DhhCre mice to 4 months of age, when tumor is first detectable. Gross dissection of spinal cords from these mice showed that P2ry14-/-;Nf1fl/fl;DhhCre have decreased neurofibroma number, consistent with a role in tumor initiation, and only slightly reduced neurofibroma diameter (Figure 4—figure supplement 1A–C). Ki67 and H&E staining of neurofibromas in these 4-month-old mice showed no evident changes between genotypes in cell proliferation or cell morphology (Figure 4—figure supplement 1D–F). Figure 4 with 1 supplement see all Download asset Open asset P2RY14 deletion in a mouse model of neurofibroma increases survival and delays neurofibroma initiation. (A) Kaplan-Meier survival plot of Nf1fl/fl;DhhCre (red line; n = 8); P2ry14+/-; Nf1fl/fl;DhhCre (black line, n = 14); P2ry14-/-; Nf1fl/fl;DhhCre (blue line; n = 13); Nf1fl/+ control (green line, n = 11) (*p = 0.0256) shows P2ry14-/-;Nf1fl/fl;DhhCre have increased survival. (B) Representative image of gross dissection of Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice at 7 months of age. (C) Neurofibroma tumor number quantification at 7 months of age (unpaired t-test ****p < 0.0001). (D) Neurofibroma diameter quantification at 7 months (unpaired t-test ***p = 0.0004) (for C and D: Nf1fl/fl;Dhh+ n = 8 mice, 48 neurofibroma tumors; P2ry14-/-;Nf1fl/fl;Dhh+ n = 8 mice, 11 neurofibroma tumors). (E) Ki67 staining of mouse dorsal root ganglion (DRG) and neurofibroma tumor tissue at 7 months of age. (F) Quantification of Ki67+ cells in mouse DRG and neurofibroma tumor tissue at 7 months of age (one-way ANOVA; multiple comparisons ***p = 0.0008; ****p < 0.0001). (G) Anti-p-PKA substrate staining in wild-type (WT), Nf1fl/fl;DhhCre and P2ry14-/-; Nf1fl/fl;DhhCre mice. p-PKA substrate phosphorylation labeling co-localized with CNPase Schwann cell (SC) marker (insets). To determine if the effects of P2ry14 differ over time, we aged Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice. At 7 months P2ry14-/-;Nf1fl/fl;DhhCre mice also showed significantly fewer neurofibromas compressing the spinal cord on gross dissection (Figure 4B and C); tumor diameter was reduced to a lesser degree (Figure 4D). At this age, P2ry14 loss decreased Ki67+ cells in neurofibroma tissue sections (Figure 4E–F); many of these proliferative cells expressed the SC marker CNPase (Figure 4—figure supplement 1G). However, H&E staining showed characteristic neurofibroma morphology (Figure 4—figure supplement 1H). To determine if cAMP is affected by loss of P2ry14 in tumors, we stained with anti-p-PKA substrate antibody. Nf1fl/fl;DhhCre nerves showed a decrease in p-PKA substrate phosphorylation versus WT mice. Remarkably, this decrease was reversed in P2ry14-/-;Nf1fl/fl;DhhCre nerves (Figure 4G). pPKA labeling co-localized with CNPase, suggesting that the changes in p-PKA substrate phosphorylation expression are at least in part, in nerve SCs (Figure 4G, insets). Based on these results, we conclude that P2ry14 deletion in vivo in neurofibroma mice increases mouse survival and delays neurofibroma initiation and has lesser effects on SC proliferation. We also examined the effects of P2ry14 loss on nerve disruption phenotype using electron microscopy. At 4 months, Nf1fl/fl;DhhCre nerves already show disrupted Remak bundles (reduced numbers of axons ensheathed by individual SCs); this phenotype was rescued in P2ry14-/-;Nf1fl/fl;DhhCre mice (Figure 5A and C). By 7 months, Nf1fl/fl;DhhCre nerves were even more severely disrupted, and rescued to near WT levels by P2ry14 loss (Figure 5B and D). We conclude that genetic knockout of P2ry14 in neurofibroma-bearing mice improves the Remak bundle defects characteristic of neurofibroma-bearing mice, and that are present in both nerve and neurofibroma. Figure 5 Download asset Open asset P2RY14 deletion improves nerve ultrastructure. (A) Electron micrograph of saphenous nerve of 4-month-old wild-type (WT), Nf1fl/fl;DhhCre, P2ry14-/-; Nf1fl/fl;DhhCre and P2ry14-/- mice. (B) Electron micrograph of saphenous nerve of 7-month-old WT, Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice. (C) Remak bundle quantification at 4 months of age (n = 3; two-way ANOVA: ****p < 0.0001). (D) Remak bundle quantification at 7 months of age (n = 3; two-way ANOVA: **p = 0.0027, ****p < 0.0001). Increasing cAMP in neurofibroma-bearing mice by rolipram or a P2RY14 inhibitor decreases SC proliferation in neurofibromas Results presented above indicate that loss of P2ry14 during embryonic development delays phenotypes caused by Nf1 loss in SCP and SCs. To test if P2ry14 signaling might also play roles in tumor maintenance, we took pharmacological approaches. We treated Nf1fl/fl;DhhCre mice with vehicle or 5 mg/kg rolipram for 60 days to test if cell proliferation in established Nf1fl/fl;DhhCre neurofibromas is affected by increasing cAMP levels (Figure 6A). All mice survived rolipram treatment without significant weight loss. Neurofibroma lysates from rolipram treated mice confirmed increases in p-PKA substrate phosphorylation (Figure 6B). Numbers of mice assessed did not allow for tumor volume analysis. However, cell proliferation (Ki67 staining) in tissue sections revealed that rolipram treated neurofibromas show significantly decreased cell proliferation in vivo (Figure 6C and D). Figure 6 Download asset Open asset Cyclic AMP (cAMP) increase in a mouse model of neurofibromatosis either by rolipram or P2RY14 inhibitor treatment decreases Schwann cell (SC) proliferation. (A) Rolipram drug treatment experimental design. (B) Tumor lysates of vehicle and rolipram treated Nf1fl/fl;DhhCre mice show changes in p-PKA substrate. (C) Ki67 staining at 9 months of age in vehicle and rolipram treated mice. (D) Quantification of Ki67+ cells in vehicle treated versus rolipram treated mice (unpaired t-test: ****p < 0.0001; n = 3). (E) P2ry14 inhibitor (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) drug treatment experimental design. (F) Immunofluorescent staining of sciatic nerve of 4-month-old wild-type (WT), Nf1fl/fl;DhhCre (vehicle) and Nf1fl/fl;DhhCre (PPTN treated) shows increased p-PKA expression after PPTN treatment. (G) Ki67+ staining of Nf1fl/fl;DhhCre (vehicle) and Nf1fl/fl;DhhCre (PPTN treated) neurofibroma tissue. (H) Quantification of Ki67+ cells after PPTN treatment in neurofibroma tissue. To test if short-term inhibition of P2ry14 in Nf1fl/fl;DhhCre neurofibroma mice similarly affects pPKA levels and cell proliferation, we treated 4-month-old Nf1fl/fl;DhhCre mice with a P2ry14 inhibitor (PPTN) (Robichaud et al., 2011; Battistone et al., 2020). Oral bioavailability for this inhibitor is poor, therefore, osmotic minipumps were implanted subcutaneosuly to release a daily dose of 4.55 mg/kg for 14 days as described (Battistone et al., 2020). At day 14, mice were sacrificed and tissue harvested (Figure 6E). Immunostaining showed the expected increase in pPKA-substrate staining in the sciatic nerve of Nf1fl/fl;DhhCre mice after PPTN treatment (Figure 6F). Short-term treatment with the P2ry14 inhibitor also decreased cell proliferation (Ki67+ cells) in neurofibroma tissue sections PPTN treated mice (Figure 6G and H). These results suggest that P2ry14 and cAMP play at least a partial role in tumor maintenance. Altogether, these in vitro and in vivo studies show that in a mouse model of neurofibromatosis, P2ry14 is a key regulator of SCP self-renewal, SC proliferation, neurofibroma initiation, and neurofibroma maintenance. Discussion GPCR-mediated regulation of cAMP occurs upon NF1 loss in mammals (Dasgupta et al., 2003; Deraredj Nadim et al., 2016), fish, and dropsophila (Tong et al., 2002; Wolman et al., 2014). In these systems, PACAP receptors and serotonin receptors have been identified as GPCRs that act upstream of NF1 (Anastasaki and Gutmann, 2014; Deraredj Nadim et al., 2016). However, the potential role of cAMP in neurofibroma remains unclear. Targeting GPCR signaling has been suggested as a potential therapeutic option to treat NF1, so exploring the relevance of this pathway to peripheral nerve tumors is important. We found that human neurofibroma-derived SCP-like cells sorted for GPCR P2ry14 expression have increased self-renewal potential. Adult peripheral nerves do not contain a stem cell population (Stierli et al., 2018). Therefore, neurofibroma SCP-like cells may result from persistence of immature cells and/or from de-differentiation of mutant SCs. In either case, neurofibroma also contain cells that also express P2ry14. Our findings are entirely consistent with findings that hematopoietic stem/progenitor cells marked by P2ry14 stimulate self-renewal (Lee et al., 2003; Cho et al., 2014; Holmfeldt et al., 2016). Purinergic receptor P2ry14 is activated by UDP and by the nucleotide sugars UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine (Chambers et al., 2000; Moore et al., 2003; Abbracchio et al., 2003; Carter et al., 2009; Conroy et al., 2016). NF1 mutant cells may release one or more of these ligands, because SCP self-renewal in serum-free medium was reduced by pharmacological P2ry14 inhibition, and by shRNA targeting P2ry14, in the absence of added UDP or UDP-sugars. The small increase in P2ry14 protein in mutant cells may contribute to increasing signaling downstream of the GPCR, and/or Nf1 deficient cells may release more UDP-sugars than WT cells. While it is difficult to measure UDP levels in the extracellular milieu without causing cell damage and concomitant release of UDP-sugars (Lazarowki & Harden, 2015), it will be of interest to measure UDP and UDP-sugars both in the neurofibroma extracellular milieu, and in SCP and SC culture medium. UDP, UTP, and other nucleotide sugars including UDP-glucose are present at high levels in tumor cells, and released from cells in tumors and injury sites, where they act as danger signals that trigger inflammatory responses (Eigenbrodt et al., 1992; Skelton et al., 2003). We focused on the role of P2ry14 in SCs and SCPs, because most of the P2ry14 expression in peripheral nerve neurofibromas is in CNPase+ myelinating SCs, based on our use of a β-galactosidase reporter and antibody staining. However, P2ry14 is also expressed in immune cells and in small sensory neurons and larger diameter sensory neurons (Skelton et al., 2003; Müller et al., 2005; Scrivens and Dickenson, 2005a; Scrivens and Dicken

We have ascertained that phenylindolylmethyldiaminopyrimidines (PIDAPs), stop the growth of USA300 MRSA at low micromolar concentrations. The controls, penicillin G and vancomycin, are able to stop the growth of MRSA at ~765 μM (256 μg/mL) and ~1.38 μM (2 μg/mL) respectively. We have also found out that PIDAPs are bactericidal at or close to the MIC. No activity was observed against Gram-negative pathogens. Other Gram-positive pathogens have not yet been tested. Based on a search through the ChEMBL database, PIDAPs are a novel class of chemicals with antimicrobial properties. A limited structure-function study suggests that the diaminopyrimidine is part of the pharmacophore. Unfortunately, we also detected potential dose-limiting toxicity on human cell lines. Further, detailed studies are needed.

Background and objective Neurofibromatosis 1 (NF1) is a genetic disorder that is accompanied by psychiatric comorbidities such as depression, anxiety, and attention-deficit hyperactivity disorder (ADHD) in more than half of the patients. However, there are limited data describing optimal treatment strategies for these conditions. This study aimed to address that gap in understanding and explore the neurobiological basis of psychiatric comorbidities in NF1. Materials and methods A retrospective cohort study was conducted among NF1 patients with a comorbid diagnosis of depression, anxiety, and/or ADHD. These disease states were chosen based on their relatively high reported prevalence in NF1 and shared pathophysiological mechanisms via monoaminergic dysfunction. Information regarding demographics, psychotherapeutic medication use, and clinical outcomes was gathered from electronic medical records. Relationships between patient- and medication-related factors and outcome measures were assessed using statistical analysis. Results The study population (n = 82) consisted of NF1 patients with a comorbid diagnosis of depression (76.8%), anxiety (53.7%), and/or ADHD (23.2%). The use of second-generation antipsychotic agent augmentation therapy or hydroxyzine monotherapy was associated with significantly more behavioral health (BH)-related emergency department (ED) visits, admissions, and inpatient days in the study population. Conversely, the use of bupropion augmentation therapy, buspirone augmentation therapy, and stimulants was associated with improved clinical outcomes, though these results were not statistically significant. Conclusions Based on our findings in this real-world study setting, patients with NF1 and psychiatric comorbidities appear to experience significant benefits from medications that enhance dopaminergic neurotransmission (e.g., bupropion, stimulants) when compared to drugs that oppose it (e.g., second-generation antipsychotics).

To facilitate analyses of purinergic signaling in peripheral nerve glia, we review recent literature and catalog purinergic receptor mRNA expression in cultured mouse Schwann cells (SCs). Purinergic signaling can decrease developmental SC proliferation, and promote SC differentiation. The purinergic receptors P2RY2 and P2RX7 are implicated in nerve development and in the ratio of Remak SCs to myelinating SCs in differentiated peripheral nerve. P2RY2, P2RX7, and other receptors are also implicated in peripheral neuropathies and SC tumors. In SC tumors lacking the tumor suppressor NF1, the SC pathway that suppresses SC growth through P2RY2-driven β-arrestin-mediated AKT signaling is aberrant. SC-released purinergic agonists acting through SC and/or neuronal purinergic receptors activate pain responses. In all these settings, purinergic receptor activation can result in calcium-independent and calcium-dependent release of SC ATP and UDP, growth factors, and cytokines that may contribute to disease and nerve repair. Thus, current research suggests that purinergic agonists and/or antagonists might have the potential to modulate peripheral glia function in development and in disease.

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas. Expression profiling of human Schwann cells (SCs) and neurofibroma SC precursors (SCPs) identified enriched P2RY14 expression in neurofibroma SCPs. We show that genetic and pharmacological inhibition of P2RY14 in human and murine NF1 mutant SCPs reduces SCP self-renewal in vitro. We identified the mechanism of action being, P2RY14 forms a complex with the epidermal growth factor receptor (EGFR) to regulate cAMP and RAS signaling. In vivo, genetic deletion of P2RY14 in NF1 murine model delayed neurofibroma initiation, reduced cAMP and increased Caveolin 1 ( Cav1 ) expression. In a murine model where EGFR is overexpressed in SCs, genetic deletion of Cav1 increased neurofibroma initiation. Thus, P2RY14 regulates EGFR signaling, cAMP and Cav1 expression, facilitating SCP self-renewal and neurofibroma initiation. These gain and loss of function experiments suggest critical roles for P2RY14-EGFR crosstalk perturbed by NF1 tumor suppressor loss.

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) lack NF1 and show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. Gene expression profiling and protein expression identified P2RY14 in SCs and SC precursors (SCPs) implicating P2RY14 as a candidate upstream regulator of cAMP in EGF-dependent SCP. We found that SCP self-renewal was reduced by genetic or pharmacological inhibition of P2RY14. In NF1 deficient SCs and malignant peripheral nerve sheath tumor (MPNST) cells, P2RY14 inhibition decreased EGFR-driven phospho-Akt and increased cAMP signaling. In a neurofibroma mouse model, genetic deletion of P2RY14 increased mouse survival, delayed neurofibroma initiation and rescued cAMP signaling. Conversely, elevation of cAMP diminished SCP number in vitro and diminished SC proliferation in neurofibroma bearing mice in vivo. These studies identify the purinergic receptor P2RY14 as a critical G-protein-coupled receptor (GPCR) in NF1 mutant SCPs and SCs and suggest roles for EGFR-GPCR crosstalk in facilitating SCP self-renewal and neurofibroma initiation via cAMP and EGFR-driven phospho-Akt.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft-tissue sarcomas that cause significant mortality in adults with neurofibromatosis type 1. We compared gene expression of growth factors in normal human nerves to MPNST and normal human Schwann cells to MPNST cell lines. We identified WNT5A as the most significantly upregulated ligand-coding gene and verified its protein expression in MPNST cell lines and tumors. In many contexts WNT5A acts as an oncogene. However, inhibiting WNT5A expression using shRNA did not alter MPNST cell proliferation, invasion, migration, or survival in vitro. Rather, shWNT5A-treated MPNST cells upregulated mRNAs associated with the remodeling of extracellular matrix and with immune cell communication. In addition, these cells secreted increased amounts of the proinflammatory cytokines CXCL1, CCL2, IL6, CXCL8, and ICAM1. Versus controls, shWNT5A-expressing MPNST cells formed larger tumors in vivo. Grafted tumors contained elevated macrophage/stromal cells, larger and more numerous blood vessels, and increased levels of Mmp9, Cxcl13, Lipocalin-1, and Ccl12. In some MPNST settings, these effects were mimicked by targeting the WNT5A receptor ROR2. These data suggest that the non-canonical Wnt ligand WNT5A inhibits MPNST tumor formation by modulating the MPNST microenvironment, so that blocking WNT5A accelerates tumor growth in vivo.

MicroRNAs (miRs) are small non-coding RNAs that can have large impacts on oncogenic pathways. Possible functions of dysregulated miRs have not been studied in neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNFs). In PNFs, Schwann cells (SCs) have biallelic NF1 mutations necessary for tumorigenesis. We analyzed a miR microarray comparing with normal and PNF SCs and identified differences in miR expression, and we validated in mouse PNFs versus normal mouse SCs by qRT-PCR. Among these, miR-155 was a top overexpressed miR, and its expression was regulated by RAS/MAPK signaling. Overexpression of miR-155 increased mature Nf1−/− mouse SC proliferation. In SC precursors, which model tumor-initiating cells, pharmacological and genetic inhibition of miR-155 decreased PNF-derived sphere numbers in vitro, and we identified Maf as a miR-155 target. In vivo, global deletion of miR-155 significantly decreased tumor number and volume, increasing mouse survival. Fluorescent nanoparticles entered PNFs, suggesting that an anti-miR might have therapeutic potential. However, treatment of established PNFs using anti-miR-155 peptide nucleic acid-loaded nanoparticles marginally decreased tumor numbers and did not reduce tumor growth. These results suggest that miR-155 plays a functional role in PNF growth and/or SC proliferation, and that targeting neurofibroma miRs is feasible, and might provide novel therapeutic opportunities.

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor β (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, β-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by ATP, and are resistant to the effects of β-arrestin-mediated signaling, including PP2-mediated de-phosphorylation of AKT. In patients with the disorder Neurofibromatosis type 1, NF1 mutant SCs proliferate and form SC tumors called neurofibromas. Elevating ATP levels in vivo reduced neurofibroma cell proliferation. Thus, the low proliferation characteristic of differentiated adult peripheral nerve may require ongoing, nerve activity-dependent, ATP. Additionally, we identify a mechanism through which NF1 SCs may evade growth suppression in nerve tumors.

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD+ as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure–activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

The design and development of irreversible kinase inhibitors is an expanding frontier of kinase drug discovery. The current approach to develop these inhibitors utilizes ATP-competitive inhibitor scaffolds to target non-catalytic cysteines in the kinase ATP-binding site. However, this approach is limited as not all kinases have a cysteine in the ATP-binding site that can be targeted. In this work, we report a complementary approach to developing irreversible kinase inhibitors that utilizes the substrate-binding site. Using the catalytic subunit of cAMP-dependent protein kinase (PKAC alpha) as a model system, we have designed and synthesized an irreversible inhibitor based on the substrate-competitive inhibitor scaffold PKI(14-22) that covalently modifies non-catalytic Cys199 in the PKAC alpha substrate-binding site. The new compound inhibits PKAC alpha (IC50 = 11.8 +/- 1.1 nM), is similar to 100-fold selective for PKAC alpha in a kinase panel, and covalently labels the kinase as demonstrated by fluorescence, mass spectrometry, and kinetics experiments. This study demonstrates the feasibility of utilizing this new approach to develop irreversible inhibitors for any of the eighty-nine kinases that possess a similar non-catalytic cysteine in their substrate-binding sites.