Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance. FGFR tyrosine kinase plays an essential role in many biological processes including regulation of development and differentiation, tissue regeneration, angiogenesis, cell migration, cell death and metabolism3. Genetic studies have shown that signalling via specific FGF ligands to different members of the FGFR family controls a wide variety of developmental events in embryonic, foetal, and postnatal development4. The intracellular pathways activated by FGFR signalling include the well-characterised RAS/RAF/MAPK/ERK and PI3K/PDK1/AKT pathways both of which involve sequential activation of protein (and lipid) kinases initiated by activated FGFR kinase mediated by phosphorylation of intermediate adaptor proteins such as FRS2, Grb2 and Gab17. The biological functions of FGFs are, as a consequence, executed by the phosphorylation of the terminal targets of kinase-dependent pathways leading to specific changes in gene expression, cell motility, metabolism and survival.The Fibroblast Growth Factors (FGFs) are a family of evolutionarily conserved ligands that signal via binding to a cognate family of four structurally related transmembrane receptor tyrosine kinases, Fibroblast Growth Factor receptors 1–4 (FGFRs1–4)8. The activity of FGF ligands regulates tumour/stromal cell interactions such as angiogenesis, metastasis and immune evasion9. It has become increasingly clear, from genomic sequencing studies, that mutation of FGFRs is a prominent feature of diverse common cancer types10. FGFR mutations in tumours exhibit diverse manifestations. Glioblastoma and haematological malignancies exhibit gene fusions leading to FGFR kinase activation via forced dimerisation of the kinase domain with partners such as TACC11. Many tumour types that exhibit point mutations in FGFR genes lead to ligand-independent, or hypersensitive signalling12. Amplification of FGFR gene loci are the most common lesions observed in specific tumour types such as breast (18% of tumours), endometrium and lung (13%) and ovary (9%)14. It is generally assumed that these mutations result in activation of FGFR kinase-dependent signalling pathways15 but the exact structure of these pathways and their terminal targets in tumour cells is poorly understood. Analysis of FGFR signalling in tumour cells harbouring mutant FGFRs is therefore required.Many forms of cancer involve manifestations of aberrant FGFR signalling17. Different strategies have been employed to inhibit FGFR signalling the most common being the development of small molecule inhibitors of the FGFR kinase that interact with the ATP-binding cleft. These can exhibit multi-kinase specificity (e.g. Dovitinib18) or selective inhibition of the FGFR kinase (e.g. AZD454719). Despite exhibiting potent inhibition of FGFR kinase activity in preclinical models, clinical trials with these agents have displayed weak efficacy20. Clinical responses are heterogeneous and vary according to the type of genetic lesion21. There is, for example, increasing evidence that small molecule inhibition is most effective in the cases of tumours harbouring FGFR amplifications and fusions but less in the case of point mutations22. An additional finding is the acquisition of drug resistance via second-site gatekeeper mutations in FGFRs24 or by re-activation of signalling via alternative receptor tyrosine kinases such HER2 and PDGFR-alpha25. It is also possible that activating mutations in critical elements of the FGFR signalling pathway such as PI3K/PTEN could lead to the acquisition of drug resistance26. These observations show that to improve the clinical efficacy of FGFR inhibitors more information is needed on the impact of these compounds on FGFR kinase-dependent signalling in the context of genetic lesions in the FGF pathway.Given the prevalence of FGFR genetic lesions in human tumours, the pathway has become a target for drug development27. FGFR-amplified cell lines can exhibit FGFR addiction22 in which cell proliferation and viability are critically dependent on continuous FGFR activity as inhibition by small molecule inhibitors leads to cell death. We selected two well-curated triple-negative breast tumour cell lines for analysis; SUM5228 and MFM22329. These exhibit high-level amplification of FGFR2, high levels of catalytically active FGFR2 protein and are addicted to FGFR2 activity as FGFR kinase inhibitors or RNAi-mediated silencing of FGFR2 induce cell death31. The design of the experiment is to stimulate the two cell lines with FGF in the presence or absence of the FGFR kinase inhibitor SU540232 and analyse differences in phosphosite abundance within treatments and between the two cell lines using the differential labelling SILAC mass spectrometry technique33. This approach provides a comprehensive perspective on the structure of FGFR2 kinase-initiated signalling pathways; identifies phosphorylation events that are drug-dependent and reveals similarities and differences in responses between SUM52 and MFM223. The data also identifies potential biomarkers of drug response and mechanisms of intrinsic resistance to inform the design of future clinical trials35. The results reveal that, although sharing a common histopathological classification and FGFR2-addiction, the two cell lines differ significantly in their response to FGFR kinase inhibition.The biological consequences of genetic lesions in tumours are articulated through the protein networks in which the mutant gene products participate. These are far less well characterised, are often inferred rather than observed, and involve critical roles for non-mutant proteins which are not detected in mutational data. A central regulatory mechanism in these networks is post translational modification by protein phosphorylation of crucial regulatory sites that control protein activity, associations, localisation and abundance. This is the core mechanism that controls the biological behaviour of the tumour. Therefore, analysing patterns of protein abundance and phosphorylation gives direct access to the activity status of the pathway in the tumour; allows stratification based on pathway activity; and identifies key nodes for therapeutic targeting and novel nodes for drug development. In this study, we take a differential phosphoproteomics approach to analyse the impact of FGFR kinase inhibition on subsequent kinase-dependent phosphorylation events. Triple-negative breast tumours frequently harbour amplifications of FGFR1 or FGFR239 . The singly phosphorylated Y656 peptide is also highly enriched in SUM52. However, mutation of the equivalent residue in FGFR1, Y653 to Phe has no impact on kinase activity47. Mutation of FGFR1 Y654  inhibits kinase activity and is thought to boost intrinsic kinase activity ~10-fold after initial phosphorylation of residues earlier in the activation sequence, including Y653. FGFR2_Y657 singly phosphorylated peptide was not detected suggesting the occurrence of phosphorylated Y657 is low compared to Y656 singly, or Y656Y657 doubly phosphorylated peptides. This is consistent with sequential phosphorylation seen in FGFR1, which requires Y653 to be phosphorylated before Y654 for maximal activation45.The FGFR2 activation loop tyrosines, Y656 and Y657, are autophosphorylation sites required to be phosphorylated for full activation of FGFR2 kinase activityels Fig. , suggest48. Ubiquitination of JunB leads to proteasomal degradation and down-regulation of JunB-regulated transcriptional activity. S259 is the priming phosphorylation site that initiates phosphorylation of T255 (and S251) by GSK3β. Under growth factor stimulated conditions, JunB levels increase due to downregulation of the phosphorylation of this phospho-degron, mediated by the inhibitory effects of Akt on GSK3β. The low SUM52/MFM223 ratio suggests that JunB is more stable in SUM52 than MFM223 cells.One double phosphorylated peptide with a very low SUM52/MFM223 ratio is the JunB phosphopeptide T255S259. Both T255 and S259 on JunB are part of the phospho-degron motif recognised by the E3 ubiquitin ligase SCFFBXW749. Another phosphopeptide with significantly higher abundance in SUM52 than MFM223 is BAD_S99. Phosphorylation of serine 99 by AKT or p70S6K (RPS6KB1) inhibits apoptosis by preventing the pro-apoptotic interaction between BAD and anti-apoptotic BCL2 proteins50. This may indicate different mechanisms controlling apoptosis between the cell lines.A singly phosphorylated peptide with significantly higher abundance in SUM52 than MFM223 is PIN4_Y122. Signalling through PIN4_Y122 in glioblastoma cells with FGFR3-TACC gene fusion has been associated with tumour survival via regulation of mitochondrial metabolism32. Cells were either left untreated or pre-treated with SU5402 before stimulation with FGF1 for a further 30 min ; SUM52 . There were large numbers of serine/threonine phosphorylation events sensitive to SU5402 in both, indicating that serine/threonine-directed kinases or phosphatases propagate downstream events of the tyrosine-directed FGFR2 kinase.From a clinical perspective, patients harbouring triple-negative breast cancers that exhibit amplified FGFR2 are potential candidates for receiving treatment with an FGFR kinase inhibitor. However, the full extent of the downstream effects of this inhibition is unknown. In order to address this, we used SILAC-based quantitative phosphoproteomics to identify phosphorylation events that changed within the two FGFR2-overexpressing triple-negative breast cancer cell lines MFM223 and SUM52 upon treatment with the FGFR inhibitor SU5402min Fig. . The conmin Fig. , and resmin Fig. . In totamin Fig. . The dismin Fig. . If thisA total of 218 phosphosites were SU5402-sensitive in SUM52 and 178 in MFM223 with 55 (16%) exhibiting shared SU5402-sensitivity across both cell lines Fig. , highlig52 which tests a literature-curated database of kinase/substrate pairs against a submitted list of phosphosites to identify kinases that are predicted with high probability to be active in the test sample. The output of this analysis  terms to gain insights into processes impacted by inhibition of FGFR2 activity. This analysis revealed different patterns of enrichment in the two cell lines  leading to attenuation of signalling to the MAP Kinase pathway57. These data indicate that, in SUM52, the SOS1/Grb2 association is in an inhibited state which is released by the addition of SU5402, mediated by RSK signalling.Following activation of intrinsic FGFR kinase activity, part of the downstream signalling mechanism involves phosphorylation of the receptor-associated docking protein FRS2 and the recruitment of docking proteins such as SOS1, Grb2, SHP2 and Gab158. The additional negative charge on the adjacent residue S447 has also been shown to contribute to BRAF priming58. The autophosphorylation site BRAF_T37359 was also SU5402-insensitive, however, only detected in MFM223. Thus, BRAF exhibits basal kinase activity and priming that is not inhibited by SU5402.Active SOS1 regulates activity of the RAS-RAF-MAPK cascade by directly activating RAS which in turn recruits RAF leading to its subsequent activation. Phosphorylation of one RAF isoform, BRAF was detected in both cell lines. Phosphorylation of BRAF on S446 and S447 is insensitive to SU5402 treatment in both SUM52 and MFM223. S446 phosphorylation by an unidentified Ser/Thr kinase creates a negatively charged N-terminal region that elevates basal BRAF kinase activity and primes BRAF for binding RAS-GTP by regulating the interaction with RAS-GTP61 and inhibiting membrane association62. Phosphorylated RAF1_S259 is insensitive to growth factor stimulation63 and is de-phosphorylated by PP1–PP2A to activate MAPK64. Mutation of RAF1_S259 to inhibit its phosphorylation elevates basal kinase activity by enhancing membrane association and RAS-GTP binding leading to Noonan inhibition syndrome65. This suggests that RAF1 in both cell lines is in an, at least partially, inhibited cytosolic conformation. RAF1_S642 was also detected only in MFM223 cells and was found to be SU5402-sensitive. MAPK1 (ERK2) mediates phosphorylation of RAF1_S642, inhibits the RAS/RAF-1 interaction and desensitises RAF-1 to stimulation66. Thus, SU5402-sensitive ERK activation in MFM223 cells leads to relief of inhibition of RAF1 activity.Another RAF isoform, RAF1, is phosphorylated on S259 in both cell lines and not sensitive to SU5402 treatment. RAF1_S259 is phosphorylated by AKT, creating a 14-3-3 binding site, blocking association with RAS-GTP67 and induced phosphorylation via RAF1/BRAF and are essential for MAP2K1/MAP2K2 catalytic activity70. Thus, SU5402 mediates inhibition of MAP2K1/2 catalytic activity, despite the evidence that BRAF and RAF1 are in a partial or wholly inhibited activity state.Activation of RAF activates the dual-specificity protein kinases MAP2K1/MEK1 and MAP2K2/MEK2 to propagate signalling to MAP-Kinases. The homologous phosphopeptide MAP2K1_S222/MAP2K2_S226 was observed to be inhibited by SU5402 in both SUM52 and MFM223. These residues are subject to basal phosphorylation by PDK173. SU5402 treatment caused a decrease in the abundance of the doubly phosphorylated MAPK1_T185Y187 and the singly phosphorylated Y187 peptides in MFM223. Although the doubly phosphorylated peptide was not detected in SUM52, the abundance of the singly phosphorylated Y187 peptide was also decreased in the presence of SU5402. These residues are targets for phosphorylation by MAP2K1/2 and are required for full activation of MAPK kinase activity76. In the presence of SU5402, MAPK1 activity is therefore inhibited in both cell lines. Phosphorylation of the doubly phosphorylated MAPK3_T202Y204, both residues also targets for phosphorylation by MAP2K1/2 and required for full activation of MAPK kinase activity, was observed to be inhibited by SU5402 in MFM223. The singly phosphorylated MAPK3_Y204 was also detected and was SU5402-sensitive in MFM223 but not detected in SUM52 cells.The ERKs MAPK1 (ERK2) and MAPK3 (ERK1) are the targets for phosphorylation by MAP2K1/277 including negative feedback via phosphorylation of SOS. RPS6KA1_S221 was found to be constitutively phosphorylated in both SUM52 and MFM223 and unresponsive to SU5402. This site is constitutively phosphorylated by PDK1 at the plasma membrane and inducibly via activation of ERK activity79 and contributes to increased activity that also depends on phosphorylation of S363 and S380. RPS6KA1_S363 is an ERK substrate in the N-terminal kinase linker region80 and phosphorylation at this site is significantly reduced in SUM52 cells but not MFM223 in the presence of SU5402. RPS6KA1_S380 autophosphorylation is also inhibited by SU5402 in MFM223 cells but not detected in SUM52. Phosphorylation of S380 provides a docking site for phosphoinositide-dependent kinase 1 (PDK1)82 which can then phosphorylate S221 in the activation loop of the N-Terminal kinase domain. RPS6KA1_S380 autophosphorylation requires prior ERK-mediated phosphorylation of T573 which was not detected83. Thus, SU5402 supresses Rsk1 activity via inhibition of ERK activity in both cell lines.The ribosomal protein S6 kinases RPS6KA1(Rsk1) and RPS6KA3 (Rsk2) are downstream targets of the RAF/MEK/ERK pathway and mediate many cellular responses to ERK activation84 required for activation of RSK2 kinase activity. However since MAPK1 and 3 are inhibited by SU5402, this may indicate regulation of these sites by alternative kinases. RPS6KA3_S227 (homologous to RPS6KA1_S221) is constitutively phosphorylated in both cell lines and uninhibited by SU5402 treatment. Thus, RPS6KA3 activity is induced by SU5402 in MFM223 cells but not SUM52.Phosphorylation of RPS6KA3_S369 is elevated by SU5402 treatment in MFM223 but not SUM52 cells. RPS6KA3_T365 is also elevated in MFM223, but not detected in SUM52. RPS6KA3_S369 and RPS6KA3_T365 are reported ERK substrates85. mTOR activity is negatively regulated by association with AKT1S1(PRAS40). Constitutive SU5402-insensitive phosphorylation of mTOR phosphosite MTOR_S1261 was detected in MFM223 and MTOR_1166 in SUM52. Phosphorylation of MTOR_S1261 promotes phosphorylation of MTOR substrates and mTOR86. RPTOR is a mTOR adaptor protein that dictates mTORC1 complex substrate specificity. SU5402-insensitive phosphorylation of RPTOR_S722 and S877 was observed in both MFM223 and SUM52 and additionally RPTOR_S863 and RPTOR_T865 in SUM52 only. RPTOR_S863 and RPTOR_T865 are mTORC1-mediated rapamycin-sensitive phosphorylation sites and sensitive to MAPK and PI3K inhibition87. Phosphorylation of these residues primes RPTOR for subsequent phosphorylation of other sites including RPTOR_S87788. RPTOR_S722 is phosphorylated by AMPK89. Thus, RPTOR is in an SU5402-insensitive/mTORC1-activating configuration in both cell lines.An alternative pathway downstream of FGFR2 is the PI3K/AKT/mTOR/S6K pathway. Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes via association with RPTOR (mTORC1) or RICTOR (mTORC2) important for nutrient and growth factor signalling91. Thus, treatment with SU5402 relieves a negative feedback pathway to mTORC1 kinase activity in both cell types. Taken together the evidence is that mTORC1 complex kinase is constitutively active and potentially further amplified by inhibition of FGFR kinase activity in both cell lines.AKT1S1(PRAS40) binds the mTOR kinase domain and inhibits mTORC1 mediated signalling. Phosphorylation of AKT1S1_S183 and AKT1S1_T246 is inhibited by SU5402 treatment in both SUM52 and MFM223. AKT1S1_T246 is phosphorylated by AKT which leads to binding of 14-3-3 proteins and relief of AKTS1 inhibition92 that regulate cell responses such as apoptosis, metabolism and biosynthesis including regulation of ribosomal protein S6 phosphorylation93. RPS6KB1_S447 was observed to be uninhibited by SU5402 in both cell lines. Phosphorylation of this site is a rapamycin and wortmannin-sensitive priming step required for full activation of S6K1 kinase activity96. SU5402 inhibits RPS6KB2_S423 phosphorylation in both cell lines. Although no function has been described for this site, the sequence context is identical to RPS6KB1_S447 suggesting that S6K2 activity is inhibited in both cell lines. The S6K1 substrate residues in RPS6: RPS6_S235, S23698 are also inhibited by SU5402 in SUM52 but not MFM223.The S6 kinases RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are downstream targets of mTORA comprehensive comparison of SU5402-inhibited phosphorylation events in the two cell lines using the pathway reconstruction approach above is limited by instances where phosphosites are only found in one of the two datasets. As an alternative approach, we created a database of the 1,385 phosphosites that were identified in both cell lines and examined divergence in SILAC ratios between the two cell lines Fig. . 52 phos99. Phosphorylation of EIF4EBP1 at multiple sites induces release from EIF4E permitting cap-dependent protein translation. A singly phosphorylated peptide, EIF4EBP1_S65, and a doubly phosphorylated peptide containing phosphorylated T70 were inhibited by SU5402 in both cell lines whereas doubly phosphorylated peptides containing a phosphorylation at residues T37, T41, T46 or T77 were down-regulated in SUM52 but not in MFM223. EIF4EBP1_S65 and T70 are serum-sensitive and rapamycin-sensitive mTOR mediated phosphorylation sites101 but have also been reported to be sensitive to inhibition of GSK3β103 and MAPK104 in different settings. EIF4EBP1_T37 and T46 have also been identified as mTOR substrates106 and have been defined as priming sites whose phosphorylation is required for phosphorylation of EIF4EBP1_S65 and 70 and full inhibition of EIF4EBP1 binding to EIF4E106. We conclude that SU5402-mediated inhibition of EIF4EBP1_T37 and T46 occurs via inhibition of mTOR in SUM52 cells and EIF4EBP1_S65 and 70 via inhibition of MAPKinase (or an undefined kinase) in both cell lines. Downstream of mTOR, S6K1 substrate residues in RPS6: RPS6_S235 and S23698 are also inhibited by SU5402 in SUM52 but not MFM223.A striking example of a differential response to SU5402 inhibition is Eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) Fig. . EIF4EBPAs previously discussed, downstream of the RAS/MEK/MAPK pathway are the RSK kinases. RSK1 (RPS6KA1) activity is downregulated in SUM52 on addition of SU5402 and unchanged in MFM223, as evidenced by a decrease in S363 phosphorylation. In contrast, RSK2 (RPS6KA3) is upregulated on addition of SU5402 and unchanged in SUM52. These data show therefore that SU5402 differentially alters specific mTOR-mediated signaling and RAS/MEK/ERK events in SUM52 and MFM223. Thus, in the two cell lines, inhibition of FGFR kinase activity is propagated intracellularly to distinct functional outcomes.107 and is required to inhibit degradation of anti-apoptotic MCL1. Thus, inhibition of MCL1_T163 phosphorylation will lead to depletion of MCL1 and execution of apoptosis. Phosphorylation of BAD_S99 is inhibited in both cell lines. Phosphorylation of BAD_S99 by AKT or RAF109 leads to the binding of 14-3-3 proteins and inhibition of pro-apoptotic function111. Thus, SU5402-mediated apoptosis involves simultaneous activation of pro-apoptotic and inhibition of anti-apoptotic functions.We examined the common and divergent datasets for potential explanations for the FGFR2-addicted phenotype of SUM52 and MFM223 and induction of apoptosis in response to SU5402. MCL1_T163 is inhibited by SU5402 in SUM52. This is a substrate of MAPK7 and identifies many new targets for future investigation. Using a pathway reconstruction approach, we observe inhibition of the canonical RAF/MEK/ERK/RSK, PI3K/AKT/PDK/mTOR/S6K pathways. By focusing on phosphorylation sites identified in both cell lines, we also observe significant differences in response to FGFR2 inhibition in the two cell lines. These are not, however, mutually exclusive as elements of both pathways are inhibited in both cell lines. Our conclusion is that the FGF pathway is a dynamic system whose behaviour is defined by quantitative and qualitative features of pathway architecture.This study set out to define the proteomic landscape and phosphorylation-dependent intracellular signalling pathways impacted by pharmacological inhibition of the FGFR2 kinase. It focuses on two triple-negative breast tumour cell lines, MFM223 and SUM52 that exhibit the shared property of amplification of the FGFR2 gene and protein and ongoing dependance on FGFR2 signalling for cell survival. The motivation is to better comprehend the molecular responses to FGFR2 activation in cancer and identify new biomarkers and targets for more effective use of agents directed at inhibition of FGFR2 signalling. Using a differential phosphoproteomics approach, we identify 341 sites on 219 proteins that are sensitive to inhibition of FGFR2 kinase activity. This data significantly expands the known landscape of FGFR2 signalling112 as, by definition, prolonged activation of a particular pathway might involve desensitisation before drug inhibition. Identification of intrinsic and induced resistance mechanisms is therefore crucial for stratifying tumours and understanding the heterogeneity of drug responses observed in clinical trials20. A prominent mechanism of intrinsic resistance revealed in this dataset is the use of Ser/Thr directed phosphorylation of binding sites for 14-3-3 proteins114. This can have both inhibitory and activating functions. AKT phosphorylates both BRAF and RAF1 at critical 14-3-3 binding residues which, in the 14-3-3 bound form, acts to inhibit membrane binding and the interaction with RAS-GTP thereby attenuating BRAF/RAF1 signalling115. This connects prior activation of the AKT pathway and suppression of the RAS/RAF/MEK/ERK pathway. The adaptor protein SOS1 is phosphorylated by MAPK and Rsk eliciting 14-3-3 binding which inhibits the association of Grb2 and signalling to RAS/RAF116. Both these pathways were observed to be insensitive to SU5402 indicating prior adaptation to amplified FGFR2. An example of induced resistance mediated by 14-3-3 proteins is the SU5402-senstive inhibition of phosphorylation of the mTOR complex inhibitor AKT1S191. In this case the inhibtion of phosphorylation leads to release of 14-3-3 proteins, inducing binding of AKT1S1 to mTOR and inhibition of function.An essential issue in the clinical application of kinase-directed inhibitors is intrinsic resistance: the propensity of downstream responses to deploy compensatory changes to inhibit the pathway. These might be particularly important in the case of oncogene addiction mechanisms66. We also observed SU5402-sensitive RAF mediated phosphorylation of MAP2K1 which acts to inhibit MAP2K1 activity69. The evidence from this data is that there exist multiple pathways regulating resistance to drug inhibition operating over different timescales. Both mechanisms provide potential explanations for heterogeneity of tumour responses to drug inhibition22.Induced resistance can occur by the phosphorylation of upstream pathway elements by downstream kinases to dynamically mediate feedback inhibition. We observed SU5402-sensitive phosphorylation of RAF1 by MAPKinase which inhibits the RAS-GTP/RAF1 interaction118. These data reveal differences in sensitivity to inhibition of specific pathways. SUM52 cells exhibit particular sensitivity to inhibitors of the mTOR pathway: Rapamycin, GSK2119563, GSK2126458, Temsirolimus26. MFM223 is insensitive to mTOR inhibitors, such as rapamycin, but exhibits sensitivity to inhibitors of the PI3K/AKT pathway such as Wortmannin, Akt Inhibitor IV and MK-2206118. Functional profiling of 714 kinases in siRNA screens120 showed that MFM223 and SUM52 are both sensitive to depletion of FGFR2 and MAPK3. MFM223 are also sensitive to depletion of PI3K, AKT3 and MAPK1. Both cell lines are resistant to depletion of PDK, AKT1/2 MAP2K1, MAP2K2, BRAF, RAF1, RPS6KA1/3, and RPS6KB1/2120. MFM223 has an activating mutation H1047R in PI3KCA122 whereas SUM52 are WT PI3KCA and PTEN deleted27. This evidence, taken with the phosphoproteomics data presented here, shows that the divergence in response to FGFR2 inhibition initiates in processes proximal to FGFR2 activation where signalling into the RAS/RAF/MEK/ERK/RSK and AKT/PDK/mTOR/S6K systems are regulated. Our results show that, in MFM223, FGFR2 kinase dependent signalling into RAS/RAF/MEK/ERK predominates whilst signalling into PI3K/AKT/PDK/mTOR/S6K is more prominent in SUM52 as previously suggested22. This suggests that deletion of PTEN and activating mutations of PI3K, and feedback processes controlling their activity, may have different quantitative effects on downstream pathways123.SUM52 and MFM223 have previously been screened with panels of candidate therapeutic compounds to establish drug sensitivity profiles13 but quantitative differences in the abundance of pathway components124. This points to a pressing need to categorise tumours not only by their repertoire of somatic mutations but by analysis of pathway status before and after drug treatment. The data in this study, therefore, provides a catalogue of functional phosphoprotein biomarkers for further analysis and tumour stratification35.Two aspects of this study have implications for improving the application of current therapies targeting FGFRs. High-level amplification and FGFR-addiction may lead to long-term desensitisation of downstream pathways and consequential blunting of the effects of drug inhibition. It is also conceivable that inhibiting FGFR signalling as a therapeutic strategy could lead to an increase in basal signalling by relief of inhibition. Second, not all FGFR-addicted cells exhibit the same response to pathway inhibition. This may involve, not only the consequences of additional mutations that impact pathway behaviour2 in RPMI-1640 or DMEM containing 2 mM L-Glutamine (Lonza), respectively, supplemented with 0.1 mg/ml streptomycin, 100 U/ml penicillin (Sigma-Aldrich), and 10% v/v fetal calf serum (Biosera). For SILAC labelling, SUM52 cells were prepared as previously described125 and MFM223 cells were cultured in SILAC DMEM (Thermo Fisher Scientific) supplemented with 0.798 mM L-lysine and 0.398 mM L-arginine , “medium” 4,4,5,5-D4 L-lysine and 13C6 L-arginine, or “heavy” 13C6 L-lysine and 13C615N4 L-arginine (CK Isotopes)), supplemented with 10% dialyzed FBS (Biosera), 0.1 mg/ml streptomycin, and 100 U/ml penicillin at 37 °C with 5% CO2.SUM52 and MFM223 cells were cultured at 37 °C, 5% CO125.Following 4 hrs serum starvation in serum-free media, cells were pre-treated with 20 μM SU5402 for 30 min, followed by addition of 20 ng/ml FGF1 and 20 mg/ml heparin for 30 mins, or treated as above in the absence of chemical inhibitor. Cell lysis and protein concentration determination were carried out as previously described36. Primary antibodies used in this study were obtained from Santa Cruz Biotechnology , Cell Signaling Technology , AKT, and AKT pT308) and Sigma-Aldrich (tubulin).Western blotting was carried out as previously described3 cells per well into opaque-walled, flat-bottom 96-well plates, and incubated overnight at 37 °C, 5% CO2. The medium was changed to the experimental conditions after 24 hours and incubated for 72 hours at 37 °C, 5% CO2. 100 μl of CellTiter-Glo Reagent was added into each well, incubated for 10 minutes at room temperature before measuring luminescence in a Tecan F200pro luminometer.Cell viability was determined using the CellTiter-Glo assay (Promega). Cells were seeded at 7 ×103PO4, 20% acetonitrile, pH 3) and loaded onto a 100 × 4.6 mm polysulfoethyl A column . Separation used a gradient elution profile that started with 100% mobile phase A, increased from 0 to 50% mobile phase B  over 30 min, increased to 100% B over 5 min, and then returned to 100% A. The peptides eluted from the column were split into 20 fractions. Each fraction was desalted using a C8 macrotrap cartridge (Michrom Bioresources) according to manufacturer’s instructions. Phosphopeptides were enriched using TiO2 tips  as previously described38. Each phospho-enriched sample was split into two in order to run technical repeats.10 mg of each labelled cell population were pooled prior to trypsin digestion. Proteins were reduced with 8 mM DTT, alkylated with 20 mM iodoacetamide in 50 mM ammonium bicarbonate and digested with Trypsin Gold  at 37 °C overnight. Digested samples were acidified by addition of 0.5% TFA. Peptides were desalted using Sep-Pak C18 Cartridges (Waters) according to manufacturer’s instructions. Desalted and dried peptides were resuspended in 100 μL mobile phase A (10 mM KH38. Peptides eluted directly (350 nL/min) via a Triversa nanospray source (Advion Biosciences) into a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). The mass spectrometer alternated between a full FT-MS scan (m/z 380–1600) and subsequent CID MS/MS scans of the seven most abundant ions. Survey scans were acquired in the Orbitrap cell with a resolution of 60,000 at m/z 400. Precursor ions were isolated and subjected to CID in the linear ion trap. Isolation width was 2 Th. Only multiply-charged precursor ions were selected for MS/MS. CID was performed with helium gas at a normalized collision energy of 35%. Precursor ions were activated for 10 ms. Data acquisition was controlled by Xcalibur 2.1 software.On-line liquid chromatography was performed as previously described127. Analysis of the comparative proteome and phosphoproteome between MFM223 and SUM52 cells included data from two biological replicates, each split into two technical replicates. Analysis of the SU5402-sensitive phosphoproteome included data from two biological replicates, each split into two technical replicates, plus data from a further biological replicate for SUM52 acquired as previously described125. Data were searched, using the Andromeda search engine within MaxQuant128 against the human Swiss-Prot database (downloaded 16.6.17). The human database contained 20,205 reviewed protein entries. The search parameters were: minimum peptide length 7, peptide tolerance 20 ppm (first search) and 4.5 ppm (main search), mass tolerance 0.5 Da, cleavage enzyme trypsin/P, and 2 missed cleavages were allowed. Carbamidomethyl (C) was set as a fixed modification. Oxidation (M), acetylation (Protein N-term), and phospho (STY) were set as variable modifications. The appropriate SILAC labels were selected and the maximum labelled amino acids was set to 3. All experiments were filtered to have a peptide and protein false-discovery rate (FDR) below 1% and the match between runs featured was enabled. Within the MaxQuant output, phosphorylation sites were considered to be localised correctly if the localisation score (PTM score) was at least 0.75. Bioinformatics analysis was performed using Perseus129. Significance testing was carried out using a Student’s t-test on log2 transformed ratios and controlled with a Benjamini-Hochberg FDR threshold of 0.05. Peptides quantified in three or more experimental repeats, including at least two biological repeats, were deemed significantly changed if they had a p-value of <0.05 and a ratio of <0.667 or >1.5 (at least a 1.5-fold change in abundance). Also deemed significantly changed were peptides identified in at least two biological repeats with a median ratio <0.5 or >2.0 with each individual ratio showing >1.5-fold change in the same direction.Mass spectra were processed using the MaxQuant software (version 1.5.3.8)130 was used to identify over-represented GO terms within the datasets. Phosphopeptides containing well localised phosphosites were analysed for predicted kinase motifs using KEA52 with a high stringency setting.DAVID Supplementary FiguresSupplementary Table S1Supplementary Table S2Supplementary .Table S3Supplementary Table S4 Supplementary Table S5 Supplementary Table S6 Supplementary Table S7


Magnetic monopoles have been proposed as emergent quasiparticles in pyrochlore spin ice compounds. However, unlike semiconductors and two-dimensional electron gases where the charge degree of freedom can be actively controlled by chemical doping, interface modulation, and electrostatic gating, there is as of yet no analogue of these effects for emergent magnetic monopoles. To date, all experimental investigations have been limited to large ensembles comprised of equal numbers of monopoles and antimonopoles in bulk crystals. To address these issues, we propose the formation of a two-dimensional magnetic monopole gas (2DMG) with a net magnetic charge, confined at the interface between a spin ice and an isostructural antiferromagnetic pyrochlore iridate and whose monopole density can be controlled by an external field. Our proposal is based on Monte Carlo simulations of the thermodynamic and transport properties. This proposed 2DMG should enable experiments and devices which can be performed on magnetic monopoles, akin to two-dimensional electron gases in semiconductor heterostructures. Heterostructure interfaces have physical properties distinct from bulk materials, providing the basis for many electronic devices. Miao et al. propose a spin ice heterostructure that can host a two-dimensional gas of emergent magnetic monopoles with a net magnetic charge. While exotic phases of matter such as fractional quantum Hall states5 or Wigner crystals6 can be realized in electronic systems, a natural question is whether magnetic monopoles can also exhibit rich behaviors if their charge and dimensionality can be controlled in a likewise fashion to those of electrons. However, to the best of our knowledge, all the current experiments have been restricted to bulk spin ice systems with equally populated monopoles and antimonopoles13. Here we propose such a platform, a heterointerface between a spin ice (R2Ti2O7)  and an antiferromagnet (R2Ir2O7) in the pyrochlore family. At the boundary, the exchange field between the iridate and the spin ice has a non-zero flux flowing toward/away from the spin ice, resulting in a charged 2DMG in the adjacent layers of the spin ice. Such boundary conditions are reminiscent of the polar discontinuity at the interface between LaAlO3 and SrTiO3 which hosts a two-dimensional electron gas15, and inspired by recent theoretical proposals suggesting the surface crystallization of magnetic monopole and antimonopoles at the material/vacuum interface of spin ice thin films17. Such a heterostructure represents an example of a magnetically charged monopole system, providing a unique opportunity to investigate their properties. In the following, we begin by describing the proposed heterostructure where such a 2DMG can be formed. We then investigate its transport and thermodynamics properties, and demonstrate that its properties can be modified by an external magnetic field. Finally, we demonstrate that the 2DMG remains robust even when including longer-ranged dipolar interactions.Similar to electrons in a metal, monopoles in spin ice compounds2Ti2O7 and antiferromagnetic R2Ir2O7. In the spin ice, R3+ has Ising moments on the order of  ~10 μB, and obey the “2-in-2-out” ice rules below  ~1K. Spin-flip excitations generate tetrahedral sites with “3-in-1-out/3-out-1-in”, which have been described as quasiparticles akin to magnetic monopoles. In the iridate, the Ir4+ forms another tetrahedral network, with an antiferromagnetic “all-in-all-out” (AIAO) order at TN ~ 130K. The R3+ moments in the iridate experience an additional d–f exchange interaction between R3+ and Ir4+ moments. Depending on their relative strength to the nearest neighbor interaction Jdf /Jeff, R3+ moments exhibit an AIAO ordering for Jdf /Jeff > 1, a 3-in-1-out fragmentation configuration for 1/3 < Jdf /Jeff < 1, and remain 2-in-2-out for Jdf /Jeff < 1/318. For simplicity, we first consider the case with AIAO ordering of R3+ moments. The typical ordering temperature of R3+ moments is a few Kelvins, significantly smaller than ordering temperature of Ir4+ moments at  ~130K19. As a result, the only moments of interest are the R3+, while Ir4+ moments are treated as a static antiferromagnetic background. In Fig. 2Ir2O7/R2Ti2O7 (001) interface. Due to the incomplete coordination of Ir4+ nearest neighbors, the exchange fields experienced by the interfacial R3+ moments in the R2Ir2O7 and R2Ti2O7 layers are 2/3 and 1/3, respectively, of the strength of those in a bulk R2Ir2O7. When the exchange field is sufficiently strong , the interface tetrahedra (the topmost red layer) will adopt an AI/AO configuration, forcing the spin ice to adopt a fully polarized state to avoid the formation of monopoles.Figure −2 must necessarily exist inside the spin ice.On the other hand, when the spin ice layer is sandwiched in between two iridate slabs, and polarization directions from both interfaces are opposing, as illustrated in Fig. 20. Also, unlike monopoles in bulk spin ice, which are associated with a spin-flip excitation, the emergence of these monopoles is not accompanied by the creation of antimonopoles in the spin ice, nor defects in R2Ir2O7. In practice, a macroscopic sample will possess random domains where AO (AI) terminations on both interfaces lead to positively (negatively) charged monopoles, whereas AO/AI combinations should not lead to monopoles -oriented heterostructures, or even artificial spin ice systems23  orientation for Hloc /Jeff = 14. In the R2Ti2O7 slab, a gas of singly charged monopoles is evident. Averaging over snapshots gives a cross-sectional monopole density profile as shown in Fig. 2Ir2O7/R2Ti2O7 interface. Investigations of the thickness dependence of the spin ice slab shows that the 2DMG is confined within 3–4 unit cells of the interface due to entropic considerations  = 20 Monte Carlo steps) and calculate the sheet conductivity σ2D = (BS)−1dM /dt ~ Jm /B of the sandwich structure, as well as a pure spin ice and a pure iridate slab for baseline comparisons, where M is the total magnetization of the system, S is the in-plane area of the heterostructure, and Jm is the magnetic charge current, as shown in Fig. To investigate the transport characteristics of the 2DMG, we apply an oscillating longitudinal magnetic field n+ and antimonopoles n− as a phase diagram of temperature and Hloc /Jeff. Above the spin ice freezing temperature of a few Kelvins, the system is paramagnetic and dominated by thermal fluctuations. For 0 < Hloc /Jeff < 12, a singly charged 2DMG always exists within a finite temperature window, due to the non-zero flux of the exchange field that flows toward/away from the spin ice. As the temperature is lowered toward the ground state, the system exhibits different phases for various Hloc /Jeff values. For Hloc /Jeff > 12, a metallic 2DMG persists to the lowest temperatures, which is the case as shown in Fig. Hloc /Jeff < 12 and 2 < Hloc /Jeff < 6 regions, where the iridate adopts AIAO and fragmented phases respectively, the boundary tetrahedral layers have pinned, fragmented charges due to the insufficient interfacial exchange fields. As a result, a 2DMG exists at finite temperatures for these Hloc /Jeff values, but is not the ground state. At Hloc /Jeff = 6, the iridate is on the phase boundary between the AIAO and fragmentation phases, and the 2DMG state arises from the resulting spin fluctuations. Nevertheless, a charged 2DMG exists over all values of Hloc /Jeff in the phase diagram, and thus should be experimentally accessible in real material systems.In Fig. 2Ir2O7/R2Ti2O7/R2Ir2O7 (111) sandwiches with a Kagomé R3+ termination on the iridate layer, as shown in Fig. Hloc /Jeff-T phase diagram of the sheet density of monopoles and antimonopoles. This phase diagram is qualitatively similar to that of a (001)-oriented heterostructure, but here the 2DMG ground state is even more robust. In the region with 3 < Hloc /Jeff < 6, a 2DMG (which we label as 2DMG-K1) exists with a sheet density of a−2 is a sheet density when 3-in-1out monopoles fully occupy a single tetrahedra layer along the (111) direction), and covers the experimentally determined value of Hloc /Jeff = 4.5 for Ho2Ir2O718. For Hloc /Jeff > 6, the total sheet density increases to 3+  termination, and found that its behavior is qualitatively similar to the Kagomé termination.We have also investigated the 2DMG in RBG (similar to a gate voltage in a field effect transistor), with positive direction defined as pointing from the iridate to the spin ice. With a negative field, the monopoles are driven away from the interface and pushed into the bulk of the iridate, and the 2DMG can then be fully depleted at a BG of 10 mT, while with a positive field, the monopoles are driven toward the interface, and saturate at a density of 2qmu.c.−2, since the maximum sheet density is determined by the boundary conditions. Figure σ2D as a function of BG. In negative BG, σ2D decreases toward zero as the 2DMG is depleted, whereas at positive bias σ2D also decreases since the the monopoles are pushed toward the boundary, thereby reducing the number of empty sites into which a monopole can hop.In Fig. 3+ − R3+, which should act as an effective Coulomb interaction between monopoles. We find that the existence of the charged 2DMG is robust against dipolar interactions. We now add these additional non-nearest neighboring terms into the Hamiltonian:ei is a unit vector along the positive direction of an Ising pseudo-spin σi.Having demonstrated the existence and properties of a 2DMG in the simplest nearest neighbor-only model, we now explore the effects of the long-range dipolar interaction between RHloc /Jeff = 10, taking into account dipolar interactions. Due to this effective Coulomb repulsion, monopoles are pushed to the boundary layer and form a two-dimensional stripe-like ordering with q = , reminiscent of the charge-stripe ordering observed in complex oxides such as cuprates with strong electron correlations25. Upon warming, this ordered state melts and some monopoles overcome their Coulomb repulsion and enter the interior of the spin ice layer, forming a 2DMG similar to the simpler nearest-neighbor-only case illustrated in Fig.  Figure 29. While the lattice mismatch between bulk spin ice and iridate compounds is comparatively small (1%), even a modest epitaxial strain can lift frustration in the spin ice layer26. To avoid this complication, the heterostructure can be synthesized on a substrate which is directly lattice matched to the spin ice material , since the antiferromagnetism in the iridate layer is likely more robust against small anisotropies. Finally, the magnetization signal from the motion of the 2DMG should, in principle, be detectable by conventional superconducting quantum interference device magnetometers (10−8 emu sensitivity), since the sheet density of monopoles is estimated to be  ~4 × 1014 cm−2 or  ~7 × 1012 cm−2 for the nearest neighbor only and dipolar calculations, respectively. Assuming a detection area on the order of a few square millimeters, the hopping of the dipolar 2DMG should yield a changing magnetization signal of ~10−7 emu.These proposed heterostructures can be realized using modern thin film growth techniques such as molecular beam epitaxy or pulsed laser deposition, owing to the stable +4 valence of Ir and Ti, as demonstrated by the recent successful growth of the spin ice and the iridate thin films30, while the lowered dimensionality of the system should facilitate the fabrication of transistor-like geometries where the density of monopoles can be actively controlled. This will allow one to address outstanding questions including whether emergent monopoles carry an electric dipole akin to the spin of an electron30, whether they experience Lorentz force in electric fields and exhibit an analogous Hall effect, and their energy-momentum dispersion relationship.The proposed 2DMG should enable possibilities in the study of emergent magnetic monopoles not possible in bulk materials. Its active magnetic charge degree of freedom should allow for the isolation of monopoles or antimonopolesσi = ±1 being the Ising pseudo-spin representation of a R3+ moment pointing toward or away from a given tetrahedron. The value of σi is then simulated according to the spin model and the Monte Carlo method with the standard Metropolis algorithm24. For the nearest neighbor-only model, a single spin-flip algorithm was combined with a worm-loop algorithm to prevent the system from being trapped in a local minimum at low temperatures. For the long-ranged interactions, a parallel algorithm was also used. The worm-loop algorithm developed for the pyrochlore heterostructures was based on the loop algorithm used in previous works32 (see Supplementary Note 2Ir2O7/R2Ti2O7/R2Ir2O7 heterostructures, the simulations were performed on 16 × I2 × K lattice sites with periodic boundary conditions, where I = 4 and K = 12. For R2Ir2O7/R2Ti2O7 heterostructures, the simulations were performed on 16 × I2 × K lattice sites with periodic boundary conditions along in-plane directions, where I = 4 and K = 30. We have increased the in-plane model size from I = 4 to I = 10 and did not see difference in 2DMG properties. We have also varied the spin ice slab thickness, and discovered the natural confinement depth of the 2DMG. In total, 106 hybrid Monte Carlo steps were used for the thermalization, including at least 107 single spin-flip steps.The Monte Carlo simulations have been performed based on the Ising model of the pyrochlores, with Supplementary InformationPeer Review File


Little is known about the signaling pathways involved in the differentiation of human osteoclasts. The present study evaluated the roles of the Ras/PI3K/Akt/mTOR, Ras/Raf/MEK1/2/ERK1/2, calcium-PKC, and p38 signaling pathways in human osteoclast differentiation. Mononuclear cells were isolated from the peripheral blood of control persons and patients with neurofibromatosis 1 (NF1), and the cells were differentiated into osteoclasts in the presence of signaling pathway inhibitors. Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase 5B. Inhibition of most signaling pathways with chemical inhibitors decreased the number of human osteoclasts and disrupted F-actin ring formation, while the inhibition of p38 resulted in an increased number of osteoclasts, which is a finding contradictory to previous murine studies. However, the p38 inhibition did not increase the bone resorption capacity of the cells. Ras-inhibitor FTS increased osteoclastogenesis in samples from control persons, but an inhibitory effect was observed in NF1 samples. Inhibition of MEK, PI3K, and mTOR reduced markedly the number of NF1-deficient osteoclasts, but no effect was observed in control samples. Western blot analyses showed that the changes in the phosphorylation of ERK1/2 correlated with the number of osteoclasts. Our results highlight the fact that osteoclastogenesis is regulated by multiple interacting signaling pathways and emphasize that murine and human findings related to osteoclastogenesis are not necessarily equivalent. Osteoclast differentiation from the cells of the monocyte-macrophage lineage is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) , 2. SeveThe p38 can be blocked by BIRB796, a potent inhibitor of all p38 isoforms. Another inhibitor of p38, SB203580, is selective for the α and β isoforms of p38 , 5. InhiThe inhibition of the Raf/MEK-pathway is largely reflected by the phosphorylation of ERK1/2 . U0126 aM-CSF and RANKL enhance the proliferation of osteoclast precursors, survival, and activity of osteoclasts through the phosphoinositide 3-kinase (PI3K) / protein kinase B (Akt) pathway –18. The Downstream pathways influenced by Ras may activate or suppress osteoclast resorption . FarnesyNF1 gene encodes the tumor suppressor protein neurofibromin, which is a negative regulator of Ras. Germline NF1 mutations cause the autosomal dominant neuro-cutaneous-skeletal syndrome neurofibromatosis type 1 (NF1), which has an incidence of 1/2000 [NF1 gene may cause skeletal abnormalities. Bone lesions in NF1 are variable and include scoliosis, short stature, osteopenia, and osteoporosis [The f 1/2000 –27. Neurf 1/2000  and mutaoporosis , 30. A goporosis , 32. NF1oporosis .This study focuses on the roles of different signaling pathways in the differentiation of human osteoclasts. Osteoclasts from control persons and patients with NF1 were compared to model genetically up-regulated Ras-signaling in osteoclast differentiation.The study was carried out at the Turku University Hospital and the University of Turku, Finland. It complied with the Declaration of Helsinki, was approved by the Ethics Committee of Southwest Finland Hospital District, and had research permission granted by the Turku University Hospital. The participants gave their written informed consent for establishing osteoclast cultures from their peripheral blood samples. The participants did not use any medication known to affect bone, and they were aged 18–62 years (median age was 31 years). NF1 patients fulfilled the NIH diagnostic criteria .Mononuclear cells including osteoclast precursors were isolated with Ficoll-Paque PLUS  centrifugation from fresh blood samples of 7 healthy controls  and 8 NF1 patients , aged 18–59 years (median age was 27 years), using the protocol described by Husheem et al.  and modiThe differentiation of osteoclasts was assessed by analyzing tartrate-resistant acid phosphatase form 5b (TRACP), which is a commonly approved cytochemical marker of osteoclast activity . The difIn a separate experiment, osteoclast progenitors of three control persons and three NF1 patients matched by age and gender were differentiated into osteoclasts essentially as described above, but on bovine bone slices . Half a million cells were seeded per bone slice on a 96-well-plate and cultured in the presence of the inhibitors listed in Table The carboxy-terminal cross-linking telopeptide of type I collagen (CTX) in cell culture media was used as an indicator of the bone resorption activity of the osteoclasts . CultureThe effects of the different inhibitor treatments on osteoclast differentiation on bovine bone slices were studied by fluorescent staining of nuclei and F-actin rings. First, osteoclasts were fixed with 4% paraformaldehyde, permeabilized with 0.01% Tween-20 in PBS on ice for 5 min, and non-specific binding was blocked with 1% BSA in PBS for 30 min. Cells were stained with STAR635 phalloidin  in 1:100 dilution to visualize F-actin and with Hoechst 33342 at a 1:10000 dilution in 1% BSA in PBS to visualize the nuclei, both for 1 h at room temperature. Glass coverslips were embedded with 30% glycerol in PBS and imaged with Carl Zeiss Axioimager microscope and Carl Zeiss Zen 2012 (blue edition) software.Osteoclasts from three healthy donors and three NF1 patients matched by age and gender were cultured in 24-well plates for 5 days for western blot analysis. The cell lysates were collected by scraping in Laemmli sample buffer supplemented with Phosphatase Inhibitor Cocktail II  and Complete Mini Protease Inhibitor Cocktail , and lysates from three parallel wells were pooled. The lysates were denatured for 5 min at 95 °C, run in 8% SDS-PAGE gels, and western blotted onto polyvinyl difluoride membranes . The membranes were blocked with 5% milk. The membranes were incubated with primary antibodies for phospho-p38 MAPK (Thr180/Tyr182)  and total p38 MAPK  in 5% BSA / TBS-Tween-20, and for phospho-Akt (Thr308) , and pan-Akt  in 5% milk / TBS-Tween-20 overnight at 4 °C. The incubations with primary antibodies for phospho-p44/42 MAPK   and total p44/42 MAPK (ERK1/2)  were in 1% BSA/PBS-Tween-20 at room temperature for 1 h. The primary antibodies were followed by washing and incubation with horseradish peroxidase-linked goat anti-rabbit antibody  at room temperature for 30 min. The antibodies were detected by chemiluminescence and imaged with a LAS-4000 device . For each protein, the phosphorylated and total protein were always assayed from the same membrane. Detection of the phosphorylated form was followed by stripping of the membrane in a solution containing 2% SDS, 0.8% β-mercaptoethanol, and 65 mM Tris-HCl pH 6.8 at 55 °C for 30 min.imagej.nih.gov/ij/). The ratios of phosphorylated-to-total protein were computed for each band. Thus, the amounts of total p38, Akt and ERK1/2 served as loading controls for the respective phosphorylated proteins. Data from each donor’s cells were normalized relative to the vehicle control.The resulting blots were analysed by subtracting background, and each band was quantified using the built-in tools of the ImageJ software  and packages lmerTest (2.0–32) and lme4 (version 1.1–12). P-values <0.05 were considered as significant.Comparisons of cell counts between inhibitors and between healthy and NF1 were performed using generalized linear mixed effects models with Poisson distribution and nested random intercepts for matched pairs and each person. The diameter of osteoclasts was compared between treatments using square root transformed values and linear mixed effects models with a random intercept for each person. In the CTX assay, the wells with the highest and lowest readings from each donor were excluded to avoid any erroneous values, leaving data from three parallel assays. Statistical analyses and visualization were performed using the R software version 3.3.0 , MEK (PD98059 or U0126), PI3K (LY294002), and mTOR (Rapamycin) revealed viable, normal looking cells on the entire substratum . Adding an inhibitor of mTOR enhanced the effect of PKC inhibition only in NF1 cells (P < 0.001)  Fig. b.Western blotting showed that the inhibition of PKC, MEK, PI3K, and mTOR had no major effect on the phosphorylation of ERK1/2 or p38 in healthy cells. However, in NF1 cells, decreased phosphorylation of ERK1/2 was observed with all of the used inhibitors . The respective numbers were 1.81 and 1.57 in NF1 cells (P < 0.001). The osteoclasts displayed undisturbed F-actin ring formation. However, the p38 inhibition did not increase the level of CTX in the culture media indicating stable resorption activity (Fig. Inhibition of p38 resulted in marked changes in the morphology of osteoclasts (Fig. Increased osteoclastogenesis induced by p38 inhibition was counteracted by the inhibitors of PKC, MEK, and mTOR (Fig. The number of osteoclasts increased, when cells from control persons were treated with the Ras inhibitor FTS Fig. . InteresMost previous studies related to osteoclast signaling pathways have been performed using rodent cells. Thus, the current data extend our understanding on the signaling involved in human osteoclast differentiation and function, even though further mechanistic studies are required to fully understand the present observations. In all conditions, viable osteoclasts were observed. Inhibition of PKC, MEK, PI3K, and mTOR decreased the number of human osteoclasts, which corroborates the earlier studies carried out with mouse osteoclasts –18, 20.One of our major findings was that the inhibition of p38 promoted differentiation of human peripheral blood monocytes into osteoclasts in samples derived from control persons and NF1 patients. This finding is contradictory to the observations in rodent studies where p38 inhibition suppressed osteoclast differentiation , 7. In oThe inhibition of p38 increased the phosphorylation of ERK1/2 in healthy cells but not in NF1 cells. It appears that there is a strong interaction between the p38 and Ras/Raf/MEK/ERK1/2 pathways. Combined inhibition of p38 and MEK counteracted the osteoclastogenesis-promoting effect of p38 inhibition alone. This further corroborates the conclusion that p38 interacts with the Ras/Raf/MEK/ERK1/2 pathway in osteoclast differentiation. Since dose-response analysis of p38 inhibition was outside the scope of the present study, we cannot fully exclude off-target effects of the p38 inhibitors. However, both SB203580 and BIRB796 are relatively specific to p38 –41.Another major finding of our study was that the number of osteoclasts appeared to correlate with the phosphorylation of ERK1/2. Different responses to Ras inhibition with FTS in cells from NF1 and control samples were observed; FTS increased osteoclastogenesis and ERK1/2 phosphorylation of control samples and decreased osteoclastogenesis and ERK1/2 phosphorylation in NF1 samples. A similar association was observed also in our other experiments showing that p38 inhibition increases ERK1/2 phosphorylation, a correlation previously observed in another cell type , and ostThe inhibition of PI3K in healthy cells did not have a significant effect on the number of osteoclasts, but the formation of F-actin rings was disrupted. This is in accordance with earlier results in mice, which have shown that the inhibition of PI3K interferes with the formation of F-actin structure and osteoclast attachment on the bone . In the In conclusion, the previously suggested effects of PKC, MEK, PI3K, and mTOR inhibition were confirmed in human osteoclasts, but not in the case of p38, where inhibition of p38 resulted in increased osteoclastogenesis. The increase in osteoclastogenesis induced by p38 inhibition seemed to associate with increased ERK1/2 phosphorylation. Multiple observations in the current study support the speculation that ERK1/2 phosphorylation may act as a key driver of human osteoclast differentiation. These results highlight the need for a deeper understanding of molecular mechanisms and cellular events that affect human osteoclastogenesis.


The BONEBRIDGE  is a bone-conduction implant used in the treatment of conductive and mixed hearing loss. The BONEBRIDGE consists of an external audio processor and a bone-conduction floating mass transducer that is surgically implanted into the skull in either the transmastoid, retrosigmoid or middle fossa regions. The manufacturer includes self-tapping screws to secure the transducer; however, self-drilling screws have also been used with success. In cases where the skull is not thick enough to house the transducer, lifts are available in a variety of sizes to elevate the transducer away from the skull. The objective of the present study was to investigate the effects of screw type, lift thickness, and implant location on the sound transmission of the BONEBRIDGE.Six cadaveric temporal bones were embalmed and dried for use in this study. In each sample, a hole was drilled in each of the three implant locations to house the implant transducer. At the middle fossa, six pairs of screw holes were pre-drilled; four pairs to be used with self-tapping screws and lifts , one pair with self-tapping screws and no lifts, and one pair with self-drilling screws and no lifts. At the transmastoid and retrosigmoid locations, one pair of screw holes were pre-drilled in each for the use of the self-tapping screws. The vibration of transmitted sound to the cochlea was measured using a laser Doppler vibrometry technique. The measurements were performed on the cochlear promontory at eight discrete frequencies . Vibration velocity of the cochlear wall was measured in all samples. Measurements were analyzed using a single-factor ANOVA to investigate the effect of each modification.No significant differences were found related to either screw type, lift thickness, or implant location.This is the first known study to evaluate the effect of screw type, lift thickness, and implant location on the sound transmission produced by the BONEBRIDGE bone-conduction implant. Further studies may benefit from analysis using fresh cadaveric samples or in-vivo measurements. Bone conduction implants (BCIs) stimulate the cochlea through vibratory excitation of the temporal bone using an actuator . Bone coPercutaneous BCIs were first introduced by Tjellström and his team in Sweden in the late 1970s . The mosIn comparison, transcutaneous BCIs benefit from the skin overlying the implanted device remaining intact. In passive transcutaneous BCIs such as the Baha Attract  and Sophono  , the actActive transcutaneous BCIs, such as the BONEBRIDGE bone conduction implant  or Osia  are semi-implantable such that the vibratory energy does not need to be transmitted through the skin , 8. The The transmitted vibration to the cochlea using BCIs has been widely studied in the literature using accelerometers –10 and lThe BB-BCI is commonly implanted at the transmastoid (TM), retrosigmoid (RS) or recently-introduced middle fossa (MF) locations , 10, 11.Six cadaveric temporal bones (two right ears and four left ears) were used in this study. All cadaveric specimens were obtained with permission from the body bequeathal program at Western University, London, Ontario, Canada in accordance with the Anatomy Act of Ontario and Western’s Committee for Cadaveric Use in Research . Samples were received previously fixed in a 10% formaldehyde solution and dried.On each sample, appropriate craniotomies were made in the TM, RS and MF locations to house the implant transducer. To expose the cochlear promontory, the external auditory canal was widened by removing cartilage and drilling the anterior and inferior walls. To expose the cochlear promontory for LDV measurements, the tympanic membrane and the malleus were removed as previously described . To maxiCochlear velocity was compared in eight different conditions with varying combinations of implant location , lift thickness  and screw type (self-drilling or self-tapping). Six conditions (C1-C6) were studied at the MF location: four with self-tapping screws and lifts , one with self-tapping screws and no lifts, and one with self-drilling screws and no lifts. One condition (C7) was studied in the TM location with self-tapping screws and no lifts, and one condition (C8) in the RF location with self-tapping screws and no lifts. A summary of the test conditions is given in Table For each condition, the vibration of the transmitted sound to the cochlea was measured using the LDV  pointed directly on the cochlear promontory. A two degree-of-motion goniometer  was used to accurately position the sample under the LDV device. The measurements were performed on the cochlear promontory at eight discrete frequencies . A 45 dB HL signal was directly transmitted to the transducer via a wired connection using the BB-BCI programming software . The noise vibration was also measured with no stimulation and compared with the BCI stimulated vibration. The cochlear promontory velocity was measured with an accuracy of 2 mm/s/V and is presented as frequency responses expressed in mm/s. Measurements were averaged over five repetitions.The effect of location, lift and screw type was calculated in relative terms. Since condition C1 had two out of three common effects with the other 7 conditions (C2 to C8), it was chosen as the reference condition for analysis in relative terms, meaning the velocity of all conditions was compared to the reference and reported in dB unit.p < 0.006 was used to control the familywise error rate at p ≤ 0.05.A pilot study was performed that showed that ±5 dB HL input would change the output velocity from 0.1 mm/s to 0.13 mm/s with a standard deviation of 0.02 mm/s. Based on that study, a sample size calculation was performed to confirm that 6 samples were needed to provide a statistical power of 80% to find the equivalent of a ± 5 dB HL difference. A one-way analysis of variance (ANOVA) was used to compare the effects of variations between location, lift thickness and screw type at each frequency. Bonferroni correction was used to account for multiple testing ; therefore, the The effect of location on transmitted sound to the cochlea was assessed by comparing the vibration velocity when screw type and lift presence were controlled for by comparing C7 and C8 against C1 Fig. .Fig. 2EThe effect of lift thickness on transmitted sound to cochlea was assessed by comparing the vibration velocity when the same type of screw and implant location were combined with various lift thicknesses  as shown in Fig. The effect of screw type on transmitted sound to cochlea was assessed by comparing the vibration velocity when location and lift presence was controlled for, but the screw type varied, i.e., comparing C2 against C1 Fig. .Fig. 4Ep-value found was 0.2.The one-way ANOVA did not find any statistically significant differences between any of the conditions at any of the test frequencies. The minimum The BB-BCI is the first active, transcutaneous BCI and multiple studies have shown it to be an effective device to treat hearing loss in patients with mixed or conductive hearing loss –15. The The current study is the first to investigate the effects of common surgical variables on the performance of the BB-BCI; however, similar investigations have been published for other direct-drive systems. The effects of stimulation distance to the cochlea and squamosal suture on percutaneous BCI performance were investigated by Eeg-Olofsson et al. . It was In studies focusing on the effect of surgical approach, it is important to only change one source of variability at a time. By using cadaveric temporal bones in the present study instead of intact cadaveric heads, we were able to fully control our experimental setup and testing. A total of eight distinct conditions (C1 – C8) were tested in each of six specimens. Since C1 had two out of three sources of variability in common with the remaining conditions, it was used as the reference condition. The mass of each temporal bone was constant at all conditions, since all three implant locations were drilled before measurements. Also, absence of brain matter precluded any additional source of variability when comparing implantations at the TM location compared to the MF and RS locations. In addition, pre-drilling of screw holes allowed for changing the location, lift and screw type with minimal movement, so the angle of the laser beam on the cochlear promontory could be kept constant in each sample. Lastly, the wired connection between the processor and the signal generator bypassed performance variabilities such as battery life and microphone-speaker angle.One limitation of the present study was that only one combination of lift and screw type was used in each of the TM and RS locations. This was because the air cells in the mastoid limited the possibility of finding multiple suitable screw hole locations. Also, in the RS approach, the area behind the sigmoid sinus was too small to allow multiple screw holes.The present findings did not reveal any significant differences associated with location, lifts or screw type. This allows surgeons the flexibility to choose the appropriate location and fixation for their particular clinical scenario without possible effects on sound transmission. We did not evaluate the potential effects of the amount of soft tissue removed on sound transmission, however since the BB-BCI is an active implant mounted directly to the bone via two screws, there should be minimal damping of sound caused by the overlying skin.Although no significant differences were found between locations, placement of the BB-BCI in the MF location may be preferred as it causes less disruption of the air cells and nuchal musculature, requires a shorter operating time, and offers improved post-operative scar cosmesis , 11. HowThe effects of implant location, lift thickness and screw type on BB-BCI were investigated on six cadaveric temporal bones using a LDV technique. This is the first known study to evaluate the effect of these variables on the sound transmission produced by the BB-BCI. No significant difference was found related to any of these modifications.


All the samples showed the pseudoplastic behavior and the power‐law model had a high efficiency (R2 ≥ .99) in describing their rheological behavior. The lowest span value, the lowest mean particle diameter, and the highest mean particle surface area, and thus, the most stable and homogenous samples were associated with the conventional ice cream followed by the optimized plant one. The number of air bubbles in the structure of the conventional and optimized ice creams was significantly (p < .05) higher than in the other samples. The soy ice cream had the lowest Tg (−58.04°C), whereas the conventional one had the highest Tg (−55.05°C). Unlike the plant‐based samples, especially the soy ice cream, the conventional ice cream had the lowest ice content (IC), the highest unfreezable water (UFW), and the lowest frozen water (FW). Overall, this ice cream was more acceptable in terms of sensory attributes in comparison with the control sample and could be supplied to consumers as a novel, high‐quality, and marketable ice cream.The aim of this study was to investigate the structural, physicochemical, and rheological properties of soy and sesame milk‐based ice cream in order to optimize its formula. The overrun percentage of the optimized ice cream was acceptable compared to the conventional ice cream (produced with cow milk). The hardness and consistency of the optimized ice cream were significantly ( The optimized ice cream based on soy and sesame milks have more acceptable in terms of sensory evaluation and other physical properties in comparison with the control sample and could be supplied to consumers as a novel, high quality, and marketable ice cream. The optimized plant‐based ice cream was produced by the most appropriate ratio of soy and sesame milk on textural parametersThe textural and sensory properties of optimized ice cream were acceptableThe high content of plant proteins in the plant‐based ice cream produced causes functional propertiesThe optimized plant ice cream could be supplied to consumers as a novel, high‐quality, and marketable ice cream1Ice cream is a frozen foam comprised of partially aggregated lipid particles, air cells, ice crystals, and a continuous aqueous phase (serum) in which components such as polysaccharides, proteins, lactose, and mineral salts are dispersed ) was purchased from Tus Suyan Co. . Sterilized and homogenized milk (1.5% fat), skimmed milk powder, and homogenized and sterilized cream (30% fat) were supplied from Pegah Dairy Co . Panisol ex  was provided form Danisco, Demark. Sugar and vanilla were all purchased from local market. High‐quality peeled sesame was obtained from Simorgh Tahini Halva Co. . All other chemicals used were of analytical grade unless otherwise specified.2.22.2.1Soy flour and distilled water (1:5) were mixed in a blender at a rate of 700 rpm for 15 min at 85°C. The resulting mixture was then cooled down to 40–50°C and filtered using a two‐layered filter cloth . Finally, it was cooled off and pasteurized at 80°C for 15 s and kept in sealed bags at 4–5°C until use  were blended in a blender at a rate of 700 rpm for 20 min. After keeping at room temperature for 1 hr, the resulting mixture was filtered using a filter cloth so that the final dry weight of the sesame milk was adjusted to 11%–12% . Eventually, the prepared milk was pasteurized in suitable containers at 80°C for 15 s and stored in sealed bags at 4–5°C for further experiments . The models were applied to terms with p values smaller than .10 to obtain a more accurate prediction with a higher R2 value.The D‐optimal mixture design was used to obtain the optimal ratio of soy milk: sesame milk in the formula of the plant ice cream by using three textural parameters, including hardness, consistency, and apparent elasticity modulus. In the mixture experiment design, the total amount of milk is held constant and the measured property of the samples changes when the proportions of the milk types are changed (0%–100%). All predicted models have been produced starting with full cubic models. Some of the terms have been omitted in order to achieve the most optimal model. In the cubic models, the higher‐order interaction terms with the highest 2.2.4The conventional ice cream was composed of 10% fat, 15% sugar, 11% skimmed milk powder, 0.5% stabilizer, and 0.1% vanilla. A three‐unknown equation was developed in Excel 2010 according to the formula above in addition to the dry matter and fat contents of each of the plant milk and the cow`s milk individually. The mass of all ingredients was determined based on this equation. After weighting all of the required ingredients for the three different formulas of soy, sesame, and conventional ice creams, the liquid substances, including each of the plant milk, cow`s milk, and cream, were stirred continuously upon heating moderately up to 45–50°C. Afterward, the mixture of the solid matter, including sugar, skimmed milk powder, emulsifier, and vanilla, was added to the heated liquid. After being dissolved, they were completely blended using a stirrer  for 3 min and pasteurized at 80°C for 25 s and homogenized using a homogenizer  at 15,000 rpm for 3 min. After that, the mix was immediately cooled down to 5°C using a bath of water, ice, and salt. Then, aging was performed at 5 ± 0.5°C for 24 hr. The prepared ice cream mix was placed in a batch ice cream maker  during freezing for 20 min. Eventually, the samples were poured into plastic containers, coded, and stored at −18°C for at least 24 hr. Cow's milk was used with the same material and methods for the production of the conventional ice cream.2.32.3.1The pH, total solid, protein, and fat contents were analyzed by the reference AOAC  method aOverrun was measured by the following equation:2.3.2−1 at 5 ± 0.5°C. The empirical data were fitted to the power–law model  equipped with a thermal circulator  at a shear rat of 51.8 s2.3.3Mean diameter, particle size distribution, and specific surface area (SSA) were measured by using a particle size analyzer  through the dynamic light scattering (DLS) method. This device measures the mean geometric diameter of the particles in addition to the width of the particle size distribution curve, called Span. For this purpose, the ice cream samples were kept at 4°C for 4 hr after the hardening step during 24 hr at −18°C. They were then diluted and homogenized with distilled water at a ratio of 1:1,000. The samples were stored in a refrigerator at a temperature of 4°C until the test was completed. The samples were completely homogenized before the analysis.2.3.4After the 50 g samples of the plant‐based and conventional ice creams were kept at ambient temperature for 5 min, a texture analyzer  was employed to analyze their texture. To that end, a cylinder probe, 6 mm in diameter, was chosen to penetrate 15 mm into the sample at a rate of 2 mm/s  was used after the aging process . The analysis was performed according to Soukoulis et al. . The the2.3.6An optical microscope  equipped with a camera mounted on it  was used to investigate the samples structure. For image acquisition, a certain amount of each sample (0.2–0.5 mg) was compressed between two glass slides after 24h of slow hardening. Then, 10 images were captured using the 40 μm lens to acquire the highest quality image. Subsequently, ImageJ 1.44P software was used to analyze the images , and the graphs were drawn using Microsoft Office Excel 2010. Different letters were used to indicate values which were significantly different.Design Expert software version 10, D‐optimal mixture design were applied to optimize the formula, and the best ratio of soy milk to sesame milk was selected. Then, this study was also conducted based on a completely randomized design using SPSS software version 20. All analyses were performed in triplicate from the same bulk of sample. Duncan's multiple range test was exploited for mean comparison at 95% confidence interval  to sesame milk (45%) with a low 3.2p < .05), because the percentages of the nonfat solids of the milk and the fat content of the formula were constant. There have been no significant differences among the pH values of different ice cream samples in similar studies  compared to the conventional ice cream sample, as its overrun value was almost equal to that of the conventional ice cream  from each other. The percentage of resistance to melting of the optimized ice cream was significantly (p < .05) less than that of the soy ice cream, and more than those of the conventional and sesame ice creams. Given that the type and amount of all effective non‐protein compounds, including stabilizers, dry matter, and fat, as well as the method of ice cream production, were similar in all samples; this result seems to be justified considering the difference in the amount and type of soy protein and casein in samples . Shahrabi et al.  to describe the rheological behavior of the plant ice cream mixes (Table p < .05) higher than those of the conventional and sesame ones . On the other hand, the hardness and consistency of the optimized ice cream were significantly higher than those of the conventional one (p < .05). It can be stated that the amount of the optimized ice cream hardness was closer to that of the conventional ice cream. Definitely, the change in the type and amount of protein and fat in the plant and cow's milk for the preparation of various samples had a significant effect on the functional and important properties of ice cream, which influenced the size and distribution of air bubbles and the formation of ice cream crystals, and could ultimately change the texture hardness and consistency of the ice cream. Hardness may be considered a reflection of the mixture components  and the process conditions  of the final product  higher than that of the other samples, and the adhesiveness of the optimized sample was very similar to that of the conventional ice cream. This parameter depends on the combined effect of adhesiveness and bonding forces in addition to other factors such as viscosity and viscoelasticity  more than that of the other ice cream samples.Since the size of the ice crystals and the ice‐free phase volume are involved in creating hard texture in ice cream, this factor can be considered to be a criterion for the growth of ice crystals  and the conventional one had the highest Tg (−55.5°C). These results seem to be directly related to the amount and type of protein present in the system so that the proteins of the system can maintain water molecules and therefore reduce the movement of these molecules, resulting in less water in the system and finally has a direct positive effect on phase change and Tg. By increasing the amount of casein proteins present in the system , Tg increased  higher than in the other samples  because of the use of soy milk which is justifiable. Nevertheless, in the case of the other sensory attributes, the scores showed that there was not a significant difference (p > .05) between among the samples in terms of texture, so the points were very close to each other.The results of sensory evaluation showed that Figure  the plan4p < .05). Investigation of the PSA curves show that the most stable samples were the conventional and optimized ice creams, respectively, . Also, the soy and conventional ice cream had the lowest Tg (−58.04°C) and the highest Tg (−55.05°C), respectively. The results of sensory evaluation also confirmed the quality and general acceptance of the plant‐based ice cream. According to these results, new products with unique characteristics can be produced which eliminate the healthy problems involved in many animal products, including the presence of cholesterol, and replace animal proteins and fats with the plant‐based ones.The results of this study showed that although there were significant differences in the physicochemical, textural, rheological, and thermal properties of the ice cream samples, the optimized plant‐based ice cream was close to the conventional one and, in some cases, even had a better quality. High quality and content of plant proteins were due to formation of functional properties in optimized ice cream sample. The overrun percentage of the optimized and conventional ice cream were close together .The hardness  and consistency  of the optimized ice cream were significantly higher than those of the conventional one (The authors confirm that there is no known conflict of interest associated with this publication. This article does not contain any studies with human participants or animals performed by any of the authors. Written informed consent was obtained from all study participants.


Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [ NEM was added together with the labelled substrate at time zero in the control samples. After terminating the transport reaction, the external substrate was removed by chromatography on Sephadex G-75 columns, and the intraliposomal radioactivity was measured [Transport was started by adding 0.1 mM [measured . Transpo3) that was filled with water molecules, within 12 Å from the surface of the protein, and 13 Cl-counter-ions. The entire systems were minimized and successively undertaken to MDs adopting AMBER16 software package, [The starting structure of CAC protein was obtained from homology modeling simulation. Two additional mutated structures were obtained by the in silico mutation C136S and C155S, respectively. The protonation state for each titrable residue was assigned using the H++ web server. The three different proteins were respectively surrounded by a rectangular box a(Ser)b(H2O)2)n+Cun = 1, 2 a, b = 0, 1, 2The amount of reconstituted protein was estimated from Coomassie blue stained SDS–PAGE gels by using the Chemidoc imaging system equipped with Quantity One software (Bio-Rad) as previously described .We demonstrate that copper interacts with the CAC and strongly inhibits its function. The molecular mechanism of the interaction was revealed by combining the transport assay of site-directed mutants with computational chemistry. All the data concur in demonstrating the occurrence of a cross-link among copper and two Cys residues of the CAC that are crucial for the conformational changes underlying the transport process. From a pathophysiological point of view, on the one hand, the functional inhibition may modulate the CAC and hence the β-oxidation contribution in protecting the cell from ROS that are produced during temporary alterations of the copper homeostasis . On the


Neurofibromatosis Type 1 (NF1)-related Malignant Peripheral Nerve Sheath Tumors (MPNST) are highly resistant sarcomas that account for significant mortality. The mechanisms of therapy resistance are not well-understood in MPNSTs, particularly with respect to kinase inhibition strategies. In this study, we aimed to quantify the impact of both the genomic context and targeted therapy on MPNST resistance using reverse phase phosphoproteome array (RPPA) analysis. We treated tumorgrafts from three genetically engineered mouse models using MET (capmatinib) and MEK (trametinib) inhibitors and doxorubicin, and assessed phosphosignaling at 4 h, 2 days, and 21 days. Baseline kinase signaling in our mouse models recapitulated an MET-addicted state (NF1-MET), P53 mutation (NF1-P53), and HGF overexpression (NF1). Following perturbation with the drug, we observed broad and redundant kinome adaptations that extended well beyond canonical RAS/ERK or PI3K/AKT/mTOR signaling. MET and MEK inhibition were both associated with an initial inflammatory response mediated by kinases in the JAK/STAT pathway and NFkB. Growth signaling predominated at the 2-day and 21-day time points as a result of broad RTK and intracellular kinase activation. Interestingly, AXL and NFkB were strongly activated at the 2-day and 21-day time points, and tightly correlated, regardless of the treatment type or genomic context. The degree of kinome adaptation observed in innately resistant tumors was significantly less than the surviving fractions of responsive tumors that exhibited a latency period before reinitiating growth. Lastly, doxorubicin resistance was associated with kinome adaptations that strongly favored growth and survival signaling. These observations confirm that MPNSTs are capable of profound signaling plasticity in the face of kinase inhibition or DNA damaging agent administration. It is possible that by targeting AXL or NFkB, therapy resistance can be mitigated. Targeting the RAS/ERK signaling pathway is an effective treatment for numerous cancers with hyper-activation of the RAS pathway. The most striking clinical responses to inhibitors of BRAF, MEK, and EGFR have been observed in melanoma and lung cancers where RAS pathway activation is intrinsic. Even though these targeted therapies have resulted in an extension of the overall survival in these inherently aggressive cancers, the clinical response is often transient and complete remission is rare. Resistance to kinase inhibition is a significant clinical challenge and numerous studies have identified multifactorial and heterogeneous mechanisms of resistance to kinase inhibition . NF1 gene and is the most common single-gene disorder, affecting 1 in 3000 live births. The NF1 gene encodes neurofibromin, a GTPase-activating protein that negatively regulates RAS , where the loss of NF1 leads to deregulated RAS signaling. Deregulated RAS signaling caused by the loss of neurofibromin is both permissive and instructive for MPNST progression (3–5). Recent clinical trials have focused on targeting members of the RAS signaling pathway or the PI3K/mTOR pathway. To date, these trials have failed to identify consistent therapeutic vulnerabilities in MPNSTs; however, few studies have examined why these therapies failed. These clinical results highlight our limited knowledge of the mechanisms that drive resistance to kinase inhibition in MPNSTs.Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive, highly chemoresistant sarcomas arising from Schwann cells that are a leading cause of death in patients with Neurofibromatosis Type 1 (NF1) ,3. NeuroNF1 gene, NF1-related MPNSTs exhibit highly complex genomic alterations that result in substantial tumor suppressor gene loss and oncogene copy number variations . . 6]. EveAt 21 days, significant tumor inhibition was observed in NF1-MET tumors and in the surviving cells, the kinome adaptations observed with single MET inhibition were intensified . SpecifiDoxorubicin is a topoisomerase II inhibitor that prevents cellular replication by indirectly stabilizing double-stranded DNA breaks . It has p < 0.0005); however, doxorubicin treatment was inferior to capmatinib or trametinib . The heterogeneity of response and growth patterns observed correlated with the diversity and intensity of the innate and acquired kinome responses delineated in these genomically distinct MPNST tumors.For the MPNST tumorgraft line, the NF1-P53 tumors had the most aggressive growth, highest response heterogeneity, and least impressive response to single-agent treatment. In NF1-P53 tumors, doxorubicin treatment did not result in tumor regression D–F. CombRPPA revealed the consistent de-repression or reactivation of ERK (T202/Y204) in surviving cells throughout 21 days of kinase inhibitor treatment. To determine if ERK reactivation was specific to resistant subpopulations or the entire tumor, we stained the tumors for phospho-ERK (T202/Y204) after 21 days of single or combination therapy. In vehicle-treated tumors, we observed moderate to strong pERK staining; however, distinct ERK activation patterns were observed in each tumorgraft line A. In thep value = 3.91 × 10−21) and grouped strongly by time point . This novel therapy mechanism has been underreported to date. We determined that pAXL expression was tightly correlated to pNFkB (Spearmen’s rank correlation rho = 0.729, me point A,B. Exprme point B, regardme point A.NF1 deficiency appears to be more difficult to target than constitutive RAS activation. One possible reason for this difference is that fewer discrete signaling dependencies exist with NF1 tumor suppressor loss than cancers that are critically dependent on RAS signaling as a result of activating RAS or EGFR mutations. That is to say, NF1-related MPNSTs are less susceptible to the perturbation of oncogenic signaling, unless a genomic event such as MET or HGF amplification exhausts the negative feedback loops that drive kinome adaptation. Our data confirms substantially broad and redundant kinome adaptation in NF1-related MPNSTs in response to MET or MEK inhibition. What is even more impressive is the diversity of early and late response mediators which sit atop prominent signaling cascades that regulate growth, proliferation, inflammation, and apoptosis. These data strongly point to an evolutionary advantage in clonal selection for cell populations that maintain this degree of signaling plasticity. Based on our findings, any successful treatment strategy that relies solely on kinase inhibition will be difficult to sustain. Even in our most ideal treatment scenario where the potent MET inhibitor capmatinib suppressed growth in MET-addicted MPNSTs, kinome adaptation occurred within 21 days, leading to a slight resumption in growth by the end of the study time frame.Currently, there is no effective chemotherapy for NF1-related MPNSTs. Despite ongoing clinical trialing efforts, neither RTK nor downstream kinase inhibition has resulted in meaningful improvements in survival, despite well-founded attempts to target both the RAS/ERK and PI3K/AKT/mTOR pathways. Fundamentally, RAS deregulation as a result of Proteomic profiling analysis provided significant insight into how the genomic context influenced the therapy response, particularly in the case of oncogene addiction. The “MET-addicted” NF1-MET tumor model is highly responsive to MET inhibition. We confirmed this finding with proteomic profiling by showing a sharp decrease in MET activation and downstream signaling immediately after capmatinib treatment. Even though MET inhibition was sustained at 2 days, strong AXL and AKT activation indicated the initiation of pathway reactivation and pathway bypass signaling. After 21 days of capmatinib treatment, resistant populations reprogrammed the kinome via AXL and EGFR. Besides activation of the AKT/mTOR pathway, ERK reactivation was consistently present in NF1-MET tumors, even in the minor cell populations that survived combined MET-MEK inhibition. In contrast to the MET-addicted tumors, few, if any, signaling dependencies were present in the capmatinib-treated NF1-P53 tumorgrafts. Interestingly, the pace and strength of kinome adaptations in NF1-P53 tumors were considerably reduced compared to NF1-MET and NF1 tumors. One possible reason for this observation is that MET inhibition failed to cause sufficient cellular stress to necessitate broad kinome adaptation in innately-resistant NF1-P53 tumors. MEK inhibition was confounded by a higher degree of innate resistance than MET inhibition and resulted in greater response heterogeneity and variability in kinome activation within genotype groups. As observed with MET inhibition in NF1-P53 tumors, less robust kinome activation was observed in response to trametinib in NF1-P53 tumors.In both capmatinib- and trametinib-treated tumors, inflammatory signaling was present at the 4-h time point, whereas kinase signaling associated with proliferation and invasion dominated at 2 days and 21 days. Inflammation has not been widely studied in MPNSTs; however, it is a key determinant of neurofibroma progression and Schwann cell homeostasis ,41,42. BAXL receptor activation was a consistent kinome adaptation observed in all of the MPNST models in response to kinase inhibition and doxorubicin treatment. Recently, AXL has been implicated in therapy resistance to multiple targeted therapies and cancer types, including MPNST. Resistance is often mediated through AXL dimerization with other RTKs, leading to the bypass of the RTK inhibitor effect ,44. For In summary, the phosphoproteomic profiling of MET and MEK inhibition revealed distinct pathways of drug resistance involving AXL activation, ERK reactivation, and inflammatory kinase signaling. As expected from our previous studies, P53-null MPNSTs were innately resistant to kinase inhibition and demonstrated the most heterogeneous kinome responses; however, combined MET-MEK inhibition exposed potential vulnerabilities in these tumors. As with other RAS-activated tumors, pathway reactivation and bypass signaling are common mechanisms of therapeutic resistance. The results in this study point toward specific vulnerable signaling nodes in MPNSTs that may be exploited through novel combination therapeutic approaches.