shp2 inhibitor protects achrs from effects of …serum dilutions with agrin but without the shp2...

ARTICLE OPEN ACCESS

SHP2 inhibitor protects AChRs from effects ofmyasthenia gravis MuSK antibodySaif Huda MD DPhil Michelangelo Cao MD PhD Anna De Rosa MD Mark Woodhall PhD

Pedro M Rodriguez Cruz MD DPhil Judith Cossins DPhil Michelangelo Maestri MD Roberta Ricciardi MD

Amelia Evoli MD David Beeson PhD and Angela Vincent MD FRCPath FRS

Neurol Neuroimmunol Neuroinflamm 20207e645 doi101212NXI0000000000000645

Correspondence

Dr Vincent

angelavincentndcnoxacuk

AbstractObjectiveTo determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2(SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and overridethe inhibitory effect of MuSK-antibodies (Abs)

MethodsThe effect of the SHP2 inhibitor NSC-87877 onMuSK phosphorylation and AChR clustering wastested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MGIgG4 preparations

ResultsIn the absence of MuSK-MG Abs NSC-87877 increased MuSK phosphorylation and thenumber of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12myotubes that form spontaneous AChR clusters In the presence of MuSK-MG sera the AChRclusters were reduced as expected but NSC-87877 was able to protect or restore the clustersTwo purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChRcluster formation and in both clusters were restored with NSC-87877

ConclusionsStimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877 orother drugs could represent a novel therapeutic approach for MuSK-MG and could potentiallyimprove other NMJ disorders with reduced AChR numbers or disrupted NMJs

RELATED ARTICLE

EditorialTreating muscle-specifickinase myasthenia gravisfrom the inside out

Page e646

Joint first authorsmdashthese authors contributed equally to the manuscript

From the Department of Clinical Neurosciences (SH MC MW PMRC JC DB AV) Weatherall Institute of Molecular Medicine and Nuffield University of Oxford UKDepartment of Clinical and Experimental Medicine (ADR MM RR) Neurology Unit Pisa and Department of Neuroscience (AE) Catholic University Rome Italy

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by Medical Research CouncilOxford University

This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Myasthenia gravis (MG) is an autoimmune disease of theneuromuscular junction (NMJ) In a variable percentage ofpatients muscle-specific kinase antibodies (MuSK-Abs) arepresent1 In humans symptoms occur particularly in cranialbulbar and respiratory muscles with frequent respiratorycrises2 Although immunomodulatory treatments can bebeneficial in the longer term there is an unmet need for cheapfast and effective treatments

MuSK is normally activated following binding of agrin secretedfrom the motor nerve terminal to low-density lipoproteinreceptor-related protein 4 (LRP4) LRP4 then binds to MuSKleading to its dimerization and to auto- and trans-phosphorylation in the MuSK juxtamembrane region3 Thesubsequent recruitment of intracellular downstream of kinase 7(DOK7) further stimulates MuSK phosphorylation causingactivation of a phosphorylation cascade that ultimately leads toclusters of acetylcholine receptor (AChR) anchored by in-tracellular rapsyn on the postsynaptic membrane of the NMJ4

MuSK-Abs are predominantly of the IgG4 subclass and in-hibit the interaction between LRP4 and MuSK preventingMuSK phosphorylation and AChR clustering56 Considering

this mechanism increasing MuSK phosphorylation couldrepresent a potential therapeutic strategy for the developmentof specific treatments

Among several regulators of the AChR clustering pathway theSRC (Rous sarcoma gene) homology 2 domain-containingphosphotyrosine phosphatase 2 (SHP2) is a phosphatase thatreduces MuSK phosphorylation Importantly for the purposesof this study NSC-87877 an SHP2 inhibitor has been shownto enhance agrin-induced and agrin-independent AChR clus-tering in vitro7 We tested NSC-87877 for its ability to increaseMuSK phosphorylation and to reverse or prevent the effects ofMuSK-Abs in 2 in vitro models

MethodsMaterialsSerum samples were collected with informed consent from31 patients with typical MuSK-MG symptoms and immu-notherapy responses MuSK-Ab titers of patientsrsquo sera andpurified immunoglobulin G (IgG) fractions were determinedby radioimmunoassay and cell-based assay (CBA)89 Sera

GlossaryAChR = acetylcholine receptor CBA = cell-based assay DOK7 = downstream of kinase 7 FACS = fluorescence-activated cellsortingKO = knockout LRP4 = low-density lipoprotein receptor-related protein 4MG =myasthenia gravisMuSK =muscle-specific kinase NMJ = neuromuscular junction SHP2 = Src homology 2 domain-containing phosphotyrosine phosphatase 2SK = specific kinase

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 1 | January 2020 NeurologyorgNN

were heated dialyzed and sterile filtered before use IgGfractions were purified from plasmapheresis of 2 additionalpatients with MuSK-MG using protein G sepharose and anIgG4 affinity matrix58 Effectiveness of IgG subclass purifica-tion was tested with CBA Briefly MuSK-transfected humanembryonic kidney cells were incubated with the different IgGsubclasses (120) and probed with anti-human-IgG1 -IgG2-IgG3 and -IgG4monoclonal mouse antibodies (150) (I2513I5635 I7260 and I7385 Sigma) After fixing with 3 formal-dehyde cells were stained with Alexa Fluor 488 goat anti-mouse IgG (1200) (A32723 Invitrogen) and images capturedusing the Olympus IX71 fluorescence microscope with SimplePCI (Digital Pixel) The SHP2 inhibitor NSC-87877 (2613)was obtained fromTocris Bioscience Bristol UnitedKingdom

C2C12 myotube cultures and AChRcluster analysisC2C12 mouse myoblasts (91031101 ATCC) were main-tained and differentiated into myotubes after 5ndash6 days in dif-ferentiation medium as previously reported (Dulbeccorsquosmodified Eagle medium [DMEM] with 2 fetal calf serumhorse serum)9 MuSK-MG sera with a broad range of MuSK-Abs (nM) were chosen according to availability MuSK-MGsera (110 130 and 190) or purified MuSK-Ab subclasses(05 nM) were applied to myotubes for 30 minutes and thenincubated overnight with agrin 1800 (short rat form pro-ducing approximately 50 of maximum AChR clusters) in thepresence and absence of NSC-87877 100 μM AChR clusterswere then labeled with Alexa Fluor 594 α-bungarotoxin (11000) (B13422 Invitrogen) and fixed in 3 formaldehydeTwenty fields selected with bright field were analyzed fornumber and cluster length (gt3 μm) using ImageJ software8ndash11

TheMusk ldquoknockoutrdquo (KO) C2C12s were generated with theclustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system using standard methods12 BrieflyMusk guide oligonucleotides (Integrated DNATechnologies)were designed and cloned into a modified plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n-D10A (42335 Addgene)Guide A oligonucleotide sequences were A-forward 59-CACCGCATTCTCCCGGATGCTGTAG-39 and A-reverse59-AAACCTACAGCATCCGGGAGAATGC-39 GuideB oligonucleotide sequences were B-forward 59-CACCGCTCCTCACCATTCTGAGCG-39 and B-reverse59-AAACCGCTCAGAATGGTGAGGAGC-39 Myoblastswere electroporated with 10 μg of each plasmid using theNeon Transfection System (Life Technologies) selected withGeneticin Antibiotic (10131035 Life Technologies) andcloned in 96-well plates using fluorescence-activated cellsorting Clones were screened by Sanger sequencingafter PCR of genomic DNA using the primers 59-TGGTGCTTTGGTTATGGAGCC-39 and 59-GAG-GAGGGGTCTAAGGCTTG-39 Musk KO generation wasconfirmed by Western blot

DOK7-overexpressing myoblasts were prepared as previouslydescribed1011 The myotubes were exposed to MuSK-Ab or

control sera or 05 nMMuSK-Ab of each purified preparation(IgG or IgG4) as illustrated in the figures

MuSK phosphorylationMuSK phosphorylation was assessed by Western blotting onimmunoprecipitates from the C2C12 myotubes They werestarved of fetal calf or horse serum for 3 hours incubated inmedium alone or medium plus NSC-87877 (1ndash1000 μMusually 100 μM) for 40 minutes at 37degC lysed in cold lysisbuffer (10 mM Tris-HCl 1 mM EDTA 100 mM NaCl 1Triton X-100 1times protease [P8340 Sigma] and phosphatase[78420 Thermo Fisher] inhibitor cocktails) and centrifugedThe supernatant was immunoprecipitated with anti-MuSK-Ab (AF562 Bio-Techne Minneapolis MN) using Protein GDynabeads (10004D Thermo Fisher) or directly withpatientsrsquo MuSK-Abs for experiments with purified IgG andIgG4 Immunoprecipitated proteins were eluted into sodiumdodecyl sulphate sample buffer and theWestern blots probedwith anti-phosphotyrosine antibody 11000 (4G10 UpstateBiotechnology MA) The nitrocellulose was then strippedand reprobed with AF562 Band densitometry was performedusing ImageJ software MuSK phosphorylation levels werenormalized to levels of immunoprecipitated MuSK

Statistical analysisStatistical tests were performed with GraphPad Prism 6 asindicated in the figure legends Error bars represent thestandard error of the mean unless otherwise stated Differ-ences with p values lt005 were considered statisticallysignificant

Standard protocol approvals registrationsand patient consentsThe study of archived patient samples was approved by theOxfordshire Research Ethics Committee A (07 Q160X28)

Data availabilityThe authors confirm that all the data supporting the findingsof this study will be made available on reasonable request tothe corresponding author

ResultsSHP2 inhibition increased MuSKphosphorylation and induced agrin-independent AChR clustersFirst we studied the effect of SHP2 inhibition on MuSKphosphorylation and on AChR clustering in the absence ofagrin We exposed C2C12 myotubes to increasing concen-trations of NSC-87877 (1ndash400 μM) for 40 minutes MuSKwas immunoprecipitated by commercial anti-MuSK AF562Phosphorylation was identified in the immunoprecipitatesby Western blotting with an anti-tyrosine phosphorylationantibody and MuSK expression by incubating the strippedmembranes with anti-MuSK AF562 AChR clusters weremeasured on parallel myotube cultures by labeling withfluorescent alpha-bungarotoxin after 12 hours The ratio of

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 1 | January 2020 3

phosphorylatedMuSK to total MuSK increased substantially(each normalized to that in DMEM alone) with maximal effectat 100 μM at higher concentrations phosphorylation de-creased (figure 1A) C2C12 myotubes in the absence of agrinexpress few spontaneous clusters (lt10 per field figure 1BDMEM) but after incubation with agrin the numbers in-creased up to 40field As previously shown7 NSC-87877 at100 μM without agrin increased the numbers of AChR clus-ters similar to those achieved with agrin alone (figure 1B upperpanel) Again the optimal concentration was 100 μM whichwas used for all experiments thereafter As expected neitherMuSK nor phosphorylated MuSK was detectable in the MuskKO myotubes (figure 1B lower panel) Mean results of dif-ferent experiments are shown in figures 1 C and D

To ensure that 40 minutes was appropriate for the experi-ments a time course was performed This showed thatMuSK phosphorylation increased over time and was clearlydetectable at 40 and 90 minutes for agrin NSC-87877 orboth NSC-87877 and agrin MuSK phosphorylation wasmodestly increased when both were given together com-pared with NSC-87877 alone (p lt 005 at 409 p lt 001 at909) (figure 1E) and the submaximal 40 minutes was chosenfor future experiments looking at changes in MuSKphosphorylation

C2C12 myotubes overexpressing DOK7 develop stableAChR clusters in the absence of agrin11 MuSK phosphory-lation was clearly present in the DOK7-overexpressing cellswithout added agrin and was further increased by NSC-87877(figure 2A) Moreover the addition of NSC-87877 to theDOK7-overexpressing myotubes increased the number ofAChR clusters that mirrored the increased phosphorylation(figure 2B)

Patients with MuSK-MGTo investigate whether SHP2 inhibition was able to protectagainst the effects of MuSK-Abs 31 MuSK-MG sera (femalemale 91 median age at onset 42 years [range 18ndash70 years])were studied All patients except 3 were on immunotherapiesmainly prednisolone andor azathioprine but all had pre-dominantly bulbar disease (as indicated by b grades) and 8still had severe involvement (Myasthenia Gravis Foundationof America 3b or 4b see table) MuSK-Ab levels measured byradioimmunoprecipitation were typically variable(025ndash2426 nM) CBAs showed that as expected IgG4MuSK-Abs were present and predominated over IgG1 2 or 3(data not shown)

SHP2 inhibition alleviates the effect ofMuSK-Abs on agrin-induced andagrin-independent AChR clustersThree dilutions (110 130 and 190) of 21 MuSK-Abs and2 control sera were incubated with the myotubes for 30minutes before adding agrin with or without NSC-87877 12hours later the numbers of AChR clusters were measured(see time line in figure 3A) In each case the results were

expressed as of those in the presence of healthy controlserum dilutions with agrin but without the SHP2 inhibitorMuSK-MG sera reduced the number and size of AChRclusters with most effect at 110 dilution as expected and ateach serum concentration NSC-87877 (100 μM) increasedthe number and size of AChR clusters (figure 3 Aa and Ab)There was a modest correlation between the number ofclusters at 130 serum dilution and MuSK-Ab radio-immunoprecipitation titers with and without NSC-87877(figure 3B) with similar results or trends at 110 and 190(data not shown)

When SHP2 inhibitor and agrin were added 6 hours after theMuSK-Abs to see whether the inhibitor could recluster theAChRs following dispersal the numbers of clusters were againincreased at each serum dilution (figure 3C)

On the DOK7-overexpressing myotubes where the clustersare well established MuSK-MG sera reduced the number ofAChR clusters but to a lesser extent than the same sera onagrin-induced AChR clusters At 130 serum dilutions forinstance the number of agrin-induced clusters was 279 plusmn93 (n = 10) of control whereas on DOK7-overexpressingmyotubes the number of clusters was 696 plusmn 109 n = 10 p= 001) of control-treated myotubes Nevertheless NSC-87877 increased the number of clusters (figure 3D) at 110and 130 dilutions At 190 dilution the sera did not signifi-cantly reduce the number of clusters and NSC-87877 had noeffect

SHP2 inhibition increases AChR clusters inIgG4-treated C2C12 myotubesFinally to demonstrate that the effects were due (at leastmainly) to IgG4 MuSK-Abs purified MuSK-Ab IgGs andIgG4 subfractions were prepared from plasmapheresismaterial of 2 additional patients with typical MuSK-MGFor myotubes not exposed to patient antibodies MuSK wasimmunoprecipitated by the anti-MuSK AF562 antibodyfrom the cell lysates (to avoid MuSK phosphorylation withconcomitant use of the commercial antibody) For myo-tubes exposed to patient antibodies to ensure that onlyphosphorylation of the antibody-bound MuSK wasassessed MuSK was immunoprecipitated by the patientIgG To adjust for this difference in the precipitationtechnique the phosphorylation signals were always nor-malized to the MuSK protein signal An example immu-noblot is shown in figure 4 Aa and results of 3ndash6experiments in figure 4 Ab Both total MuSK-MG IgG andIgG4 fractions reduced MuSK phosphorylation andphosphorylation was enhanced by NSC-87877 (figure 4Aa and Ab)

Figure 4 Ba and Bb show the effects on AChR clustersAgain both total IgG and IgG4 preparations substantiallyreduced agrin-induced AChR clustering as previouslyreported8 Moreover in each case NSC-87877 protected themyotube clusters from the inhibitory effects


Figure 1 SHP2 inhibition by NSC-87877 induces MuSK phosphorylation and AChR clustering

(A) Dose-response curve of the effects of SHP2 inhibition on AChR phosphorylation Phosphorylation increased progressively reaching a maximal effect at100 μM and decreasing at higher concentrations (B) Clustering of AChRs (labeled with Alexa Fluor 594 α-bungarotoxin) in C2C12 myotubes after 12 hoursincubation with DMEM agrin or 100 μMNSC-87877 Both agrin and NSC-87877 increased AChR clusters in native C2C12myotubes but not inMuSK knockoutcells The optimal NSC-87877 concentration was 100 μM and cluster numbers decreased at higher concentrations (C and D) The pooled results of 3ndash6independent experiments measuring MuSK phosphorylation (C) and AChR clusters (D) are shown (E) Time course of MuSK phosphorylation in C2C12myotubes after exposure to agrin (1800) NSC-87877 (100 μM) and agrinNSC-87877 In each case a similar time course was observed withmaximum effectbetween 40 and 90 minutes Phosphorylation and MuSK blots shown for NSC-87877 alone All images are 20times magnification Scale bar represents 50 μmMean + standard error of the mean are shown Results were analyzed by 2-sided t tests AChR = acetylcholine receptor MuSK = muscle-specific kinaseSHP2 = SRC homology 2 domain-containing phosphotyrosine phosphatase 2


DiscussionMuSK-MG can be a life-threatening disease that respondspoorly to symptomatic drugs such as cholinesterase inhib-itors and often requires high doses of steroids and otherimmunosuppressive drugs for long-term treatment Patientsmay have myasthenic crises with respiratory and bulbar in-volvement that often require urgent treatment in intensivecare The development of a therapy that acts specifically onthe mechanisms of the disease could achieve quick control ofsymptoms and in the longer term might help reduce thepotential side effects from immunosuppressive therapiesThus targeting the intracellular regulation of the AChRclustering pathway by inhibition of the specific phosphataseSHP2 as we show here could represent a novel and specifictherapeutic strategy for MuSK-MG The results also drawattention to the potential relevance of targeting intracellularpathways in diseases caused by extracellular antibodies orother mechanisms

There are several proteins downstream ofMuSK that could bepotential pharmacologic targets including Abl tyrosine kina-ses and guanosine triphosphatases of the Rho family How-ever increasing the activity of these regulators to enhance

AChR clustering could lead to tumor development as many ofthem such as ErbB2 and mitogen-activated protein kinaseextracellular-signal-regulated-kinase1314 play an importantrole in cell growth By contrast Src homology 2 domain-containing phosphatase 2 (SHP2) encoded by the PTPN11gene is a potentially safer target Under physiologic con-ditions through a negative-feedback loop SHP2 reducesMuSK phosphorylation providing homeostatic control ofAChR clusters at the NMJ SHP2 also enhances cell replica-tion inducing different pathways such as RasErk PI3K ser-inethreonine kinase 1 and janus kinase 2-signal transducerand activator of transcription15ndash17 thus SHP2 inhibitionshould not increase the risk of tumorigenesis Indeed phar-macologic inhibition of SHP2 has already demonstrated ef-ficacy in the treatment of a mouse glioma xenograft model18

The selective inhibition of the SHP2 phosphatase that con-trols MuSK phosphorylation by NSC-87877 was shownpreviously to increase AChR clustering in C2C12 myotubes7here we confirm that this works through increasing phos-phorylation which was clearly seen irrespective of the pres-ence of agrin or MuSK-Abs Although NSC-87877 is knownto act on both SHP1 and SHP2 only SHP2 has been detected

Figure 2 Effect of SHP2 inhibition in DOK7-overexpressing C2C12s

(A) MuSK phosphorylation was already high in DOK7-overexpressing C2C12s (data not shown) but was increased further by NSC-87877 (B) Equally DOK7-overexpressing myotubes showed AChR clusters that were further increased by NSC-87877 All images are 20times magnification Scale bar represents 50 μmResults were analyzed by 2-sided t tests DOK7 = downstream of kinase 7 MuSK = muscle-specific kinase SHP = SRC homology 2 domain-containingphosphotyrosine phosphatase 2


in C2C12 myotubes719 and reassuringly conditionalknockout of SHP2 in skeletal muscle did not compromise theformation and maintenance of the NMJ suggesting thatpharmacologic inhibition of SHP2 should be well tolerated inskeletal muscle20 although other SHP2-specific inhibitorsshould also be studied

MuSK-Abs are mainly IgG4 subclass and are known to in-hibit MuSK activation and phosphorylation by blocking theinteraction between LRP4 and MuSK58 The inhibitoryeffects of MuSK-Abs on AChR clustering were present tovarying degrees in all sera studied correlating broadly withMuSK-Ab titers NSC-87877 improved phosphorylation and

Table Clinical details of patients

Patient Age at onset sex MGFA onset sampling Radioimmunoassay titer Immunotherapies at sampling

M081 67 F 3b 1 487 Aza 100 mg Pred 125 mg

M083 19 F 2b 2b 688 Pred 250 mg

M085 30 F 3b 3b 1272 None

M087 61 M 4b 1 063 Pred 50 mg

M089 59 F 3b 3b 1008 Cycl 100 mg Pred 15 mg IVIG

M092 28 F 5 1 041 Pred 30 mg

M093 63 F 4b 3b 1272 Pred 125 mg

M097 24 F 2b 1 1273 Pred 40 mg

M098 70 F 4b 2b 025 Pred 125 mg

M103 42 M 3b 0 342 Pred 25 mg

M105 67 F - 0 073 Pred 20 mg

M110 19 F 3b 2b 925 Aza 100 mg Pred 125 mg

M116 32 F 3b 0 965 Pred 175 mg

M125 36 F 5 2b 384 Pred 35 mg


M128 50 F 4b 0 1747 Pred 125 mg

M129 37 M 2b 1 415 Pred 15 mg

M133 23 F 3b 0 654 Pred 125

M147 33 F 3b 2b 124 Pred 25 mg

M149 36 F - 1 688 Aza Pred

M154 50 F 3b 1 688 Pred 125 mg IVIG

M159 28 F 4b 4b 2496 Pred 50 mg

M165 49 F 5 2b 527 Aza 100 mg Pred 625 mg

M166 29 F 2b 0 275 Pred 125 mg

M167 46 F 2b 3b 945 None

M168 49 F 2b 0 366 Pred 10 mg

M173 42 F 3b 3b 857 Pred 50 mg

M175 44 F 3b 2b 486 Pred 175 mg

M176 18 F 3b 0 469 Pred 375 mg PLEX


M181 56 F 3b 4b 437 None

Abbreviations Aza = azathioprine b = bulbar predominant disease Cycl = cyclosporin IVIG = intravenous immunoglobulins MGFA = Myasthenia GravisFoundation of America Scale PLEX = plasma exchange Pred = prednisoloneSerum dilution giving a score of 1 (range 0ndash4 representing increasing MuSK-Abs)9 All patients were studied at the University of Pisa


Figure 3 SHP2 inhibition reverses effects of MuSK-Abs on AChR clusters in C2C12s and DOK7-overexpressing C2C12s

(A) The treatment protocols are shown above the results MuSK-Ab positive (n = 21) or healthy control sera (n = 2) were incubated with myotubes for 30minutes followed by agrin (1800) with or without NSC-87877 (100 μM) After 12 hours the AChR clusters were analyzed and expressed as a percentage ofcontrol serum results The number (Aa) of the AChR clusterswithout NSC-87877was clearly reduced by theMuSK-Abs (to 14 35 and 60at dilutions 110130 and 190 red columns) the size of the clusters was similarly reduced (Ab) In each case the effects were partially or completely reversed by NSC-87877(green columns) (B) Inverse correlation between the number of AChR clusters at 130 serum dilution andMuSK-Ab titers (nM) with or without NSC-87877 (C)Using a different protocol AChR clusters were similarly reduced and there was partial or complete protection by NSC-87877 (D) The effects of the differentserum dilutions on AChR clusters and the protection by NSC-87877 were also seen in the DOK7-overexpressing C2C12s although to a lesser extent In A Cand D comparisons at each different serum concentration were analyzed by 2-sided t tests For B linear regression was computed by GraphPad Prism Thescatter plots show results of individual sera withmean plusmn SDs AChR = acetylcholine receptor MuSK =muscle-specific kinase SHP2 = SRC homology 2 domain-containing phosphotyrosine phosphatase 2


AChR clustering at each concentration of the sera testedMoreover when the phosphorylation and AChR clusteringexperiments were conducted comparing total IgG and the IgG4subfraction from 2 patients both reduced MuSK phosphory-lation and AChR clustering and SHP2 inhibition by NSC-87877 was able to reverse their pathologic effects Of interestthe inhibitory effect of IgG4 on AChR clustering does notdepend on its monovalency and divalent monoclonal anti-bodies to MuSK also inhibit clustering2122 These recentfindings suggest that MuSK antibodies could have additionalpathogenic mechanisms other than the direct inhibition ofagrinMuSK interaction which contribute to the disruption ofthe AChR clustering pathway Nevertheless the inhibition of

MuSK phosphorylation still remains the main effect of MuSKantibodies as confirmed by our results with purified total IgGand by the effectiveness of the inhibition of SHP2

There were some limitations to the experimental procedureFor the phosphorylation experiments the patient antibody-precipitated MuSK was less than the MuSK precipitated bycommercial antibody from the control cultures There were2 reasons for this First we chose a concentration of patientMuSK antibody (05 nM) that was submaximal for in-hibition of clustering making sure that any effects on clus-tering were clearly evident but we needed to ensure that weonly measured the phosphorylation in those MuSK

Figure 4 IgG and IgG4 subclass MuSK-Abs reduce AChR clusters which are restored by NSC-87877

Agrin-stimulatedmyotubes were exposed for 40minutes to MuSK IgG or IgG4 subfraction (adjusted to 05 nMMuSK-Ab) purified from 2 patients with MuSK-MG (the total number of experimentswas IgGn =5 IgG4 n = 6) (Aa) Example of phosphorylation blotsMuSKwas immunoprecipitatedby anti-MuSKAF562 orMuSK-MG IgG fractions Pooled results (Ab) show that total IgG and IgG4 inhibitedMuSK phosphorylation and this was increased substantially by NSC-87877(100μM) (Ba) Examples of AChR clusters Pooled results (Bb) show that total IgG and Ig4MuSK-Ab fractions inhibited the formation of AChR clusters which ineach case were increased by NSC-87877 Two-sided t tests were used for comparisons at each IgG concentration AChR = acetylcholine receptor MG =Myasthenia gravis MuSK = muscle-specific kinase


molecules that were bound by patient IgG Second thecommercial antibody was applied to the cell lysate from themyotubes rather than to the live cells and thus could alsoprecipitate MuSK that was not on the surface To adjust forthese differences in MuSK immunoprecipitation the phos-phorylation was normalized to the MuSK protein signal foreach lane Further in vitro studies examining the intracellularmolecular mechanisms need to be performed and the pro-tective effects of SHP2 inhibition against MuSK antibodiesdemonstrated in vivo

Most novel therapies in MG depend on monoclonal anti-bodies targeting the immune system but given the un-derstanding of the mechanisms involved in the MuSKpathway faster pharmacologic treatments should be ex-plored Recently a monoclonal MuSK antibody was shownto stimulate MuSK phosphorylation and to preserve NMJsin a motor neuron disease model23 Using new or repur-posed drugs to enhance MuSK phosphorylation or otherdownstream components of the signaling pathway could bebeneficial in MuSK-Ab myasthenia and perhaps in otherdisorders of neuromuscular transmission where loss ofAChRs or disruption of the NMJ structure underlies themuscle weakness

Conflict of interestThe University of Oxford hold a patent for use of MuSK forantibody assays licensed to Athena Diagnostics AV receivesa proportion of royalties No other reported conflicts of interest

AcknowledgmentThe authors are grateful to Dr Jonathan Cheung and DrDominic Waithe (University of Oxford) for developingImageJ macro used for automated counting of AChR clustersThey are very grateful to the Watney TrustMyawareNational Institutes of Health Research Oxford BiomedicalResearch Centre (AV SH) the European Academy ofNeurology (MC) and the Medical Research Council (MRM006824 JC DB) for their support

Study fundingThe authors are very grateful to the Watney TrustMyawareNational Institute of Health Research Oxford BiomedicalResearch Centre (AV SH) the European Academy ofNeurology (MC) and the Medical Research Council (MRM006824 JC DB) for their support

DisclosureThe University of Oxford holds a patent for use of MuSK forantibody assays licensed to Athena Diagnostics AV receivesa proportion of royalties MC AR MW PRC JC MMRR AE and DB report no disclosures Go to NeurologyorgNN for full disclosures

Publication historyReceived by Neurology Neuroimmunology amp Neuroinflammation August15 2019 Accepted in final form October 8 2019

References1 Hoch W McConville J Helms S Newsom-Davis J Melms A Vincent A Auto-

antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia graviswithout acetylcholine receptor antibodies Nat Med 20017365ndash368

2 Evoli A Tonali PA Padua L et al Clinical correlates with anti-MuSK-Abs in gen-eralized seronegative myasthenia gravis Brain 20031262304ndash2311

3 Hopf C Hoch W Dimerization of the muscle-specific kinase induces tyrosinephosphorylation of acetylcholine receptors and their aggregation on the surface ofmyotubes J Biol Chem 19982736467ndash6473

4 Wu H Xiong WC Mei L To build a synapse signaling pathways in neuromuscularjunction assembly Development 20101371017ndash1033

5 Huijbers MG Zhang W Klooster R et al MuSK IgG4 autoantibodies cause myas-thenia gravis by inhibiting binding betweenMuSK and Lrp4 Proc Natl Acad Sci USA201311020783ndash20788

6 Koneczny I Stevens JA De Rosa A et al IgG4 autoantibodies against muscle-specifickinase undergo Fab-arm exchange in myasthenia gravis patients J Autoimmun 201677104ndash115

7 Zhao XT Qian YK Chan AWS Madhavan R Peng HB Regulation of ACh receptorclustering by the tyrosine phosphatase Shp2 Dev Neurobiol 2007671789ndash1801

8 Koneczny I Cossins J Waters P Beeson D Vincent A MuSKmyasthenia gravis IgG4disrupts the interaction of LRP4 with MuSK but both IgG4 and IgG1-3 can dispersepreformed agrin-independent AChR clusters PLoS One 20138e80695

9 Huda S Waters P Woodhall M et al IgG-specific cell-based assay detects potentiallypathogenic MuSK-Abs in seronegative MG Neurol Neuroimmunol Neuroinflamm20174e357 doi101212NXI0000000000000357

Appendix Authors

Name Location Role Contribution

Saif HudaMD DPhil

Universityof OxfordUK

Author Designed and conceptualizedthe study analyzed the dataand drafted the manuscriptfor intellectual content

MichalengeloCao MD PhD



Anna De RosaMD

Universityof PisaPisa

Author Major role in acquisition ofdata and revised themanuscript

MarkWoodhallPhD


Author Helped perform CBAs forMuSK antibody subclasses

Pedro MRodriguezCruz MDDPhil


Author Provided MuSK knockout cells

JudithCossins DPhil


Author Supervision and laboratorysupport

MichelangeloMaestri MD



RobertaRicciardi MD



Amelia EvoliMD

CatholicUniversityRome


David BeesonPhD



AngelaVincent MDFRCPath FRS


Author Designed and conceptualizedthe study analyzed the datadrafted the manuscript forintellectual contentsupervision and laboratorysupport


10 Beeson D Higuchi O Palace J et al Dok-7 mutations underlie a neuromuscularjunction synaptopathy Science 20063131975ndash1978

11 Cossins J Liu WW Belaya K et al The spectrum of mutations that underlie theneuromuscular junction synaptopathy in DOK7 congenital myasthenic syndromeHum Mol Genet 2012213765ndash3775

12 Ran FA Hsu PD Wright J et al Genome engineering using the CRISPR-Cas9system Nat Protoc 201382281ndash2308

13 Leah LN Glathe S Vaisman N et al The ErbB-2HER2 oncoprotein of humancarcinomas may function solely as a shared coreceptor for multiple stroma-derivedgrowth factors Proc Natl Acad Sci 1999964995ndash5000

14 Steelman L Chappell WH Abrams SL et al Roles of the RafMEKERK and PI3KPTENAktmTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging Aging 20113192ndash222

15 Chan RJ Feng GS PTPN11 is the first identified proto-oncogene that encodesa tyrosine phosphatase Blood 2007109862ndash867

16 Matozaki T Murata Y Saito Y Okazawa H Ohnishi H Protein tyrosine phosphatase SHP-2 a proto-oncogene product that promotesRas activationCancer Sci 20091001786ndash1793

17 Frankson R Yu ZH Bai Y et al Therapeutic targeting of oncogenic tyrosine phos-phatases Cancer Res 2017775701ndash5705

18 Bunda S Burrell K Heir P et al Inhibition of SHP2-mediated dephosphorylation ofRas suppresses oncogenesis Nat Commun 201561ndash12

19 Chen L Sunn SS Yip ML et al Discovery of a novel Shp2 protein tyrosine phos-phatase inhibitor Mol Pharmacol 200670562ndash570

20 Dong XP Li XM Gao TM et al Shp2 is dispensable in the formation and mainte-nance of the neuromuscular junction Neurosignals 20061553ndash63

21 Huijbers MG Vergoossen DL Fillie-Grijpma YE et al MuSK myasthenia gravismonoclonal antibodies valency dictates pathogenicity Neurol NeuroimmunolNeuroinflamm 201921e547 doi101212NXI0000000000000547

22 Takata K Stathopoulos P Cao M et al Characterization of pathogenic monoclonalautoantibodies derived from muscle-specific kinase myasthenia gravis patients JCIInsight 20194e127167

23 Cantor S Zhang W Delestree N Remedio L Mentis GZ Burden SJ Preservingneuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonistantibody Elife 20187e34375


DOI 101212NXI000000000000064520207 Neurol Neuroimmunol Neuroinflamm

Saif Huda Michelangelo Cao Anna De Rosa et al SHP2 inhibitor protects AChRs from effects of myasthenia gravis MuSK antibody

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Myasthenia gravis (MG) is an autoimmune disease of theneuromuscular junction (NMJ) In a variable percentage ofpatients muscle-specific kinase antibodies (MuSK-Abs) arepresent1 In humans symptoms occur particularly in cranialbulbar and respiratory muscles with frequent respiratorycrises2 Although immunomodulatory treatments can bebeneficial in the longer term there is an unmet need for cheapfast and effective treatments

MuSK is normally activated following binding of agrin secretedfrom the motor nerve terminal to low-density lipoproteinreceptor-related protein 4 (LRP4) LRP4 then binds to MuSKleading to its dimerization and to auto- and trans-phosphorylation in the MuSK juxtamembrane region3 Thesubsequent recruitment of intracellular downstream of kinase 7(DOK7) further stimulates MuSK phosphorylation causingactivation of a phosphorylation cascade that ultimately leads toclusters of acetylcholine receptor (AChR) anchored by in-tracellular rapsyn on the postsynaptic membrane of the NMJ4

MuSK-Abs are predominantly of the IgG4 subclass and in-hibit the interaction between LRP4 and MuSK preventingMuSK phosphorylation and AChR clustering56 Considering

this mechanism increasing MuSK phosphorylation couldrepresent a potential therapeutic strategy for the developmentof specific treatments

Among several regulators of the AChR clustering pathway theSRC (Rous sarcoma gene) homology 2 domain-containingphosphotyrosine phosphatase 2 (SHP2) is a phosphatase thatreduces MuSK phosphorylation Importantly for the purposesof this study NSC-87877 an SHP2 inhibitor has been shownto enhance agrin-induced and agrin-independent AChR clus-tering in vitro7 We tested NSC-87877 for its ability to increaseMuSK phosphorylation and to reverse or prevent the effects ofMuSK-Abs in 2 in vitro models

MethodsMaterialsSerum samples were collected with informed consent from31 patients with typical MuSK-MG symptoms and immu-notherapy responses MuSK-Ab titers of patientsrsquo sera andpurified immunoglobulin G (IgG) fractions were determinedby radioimmunoassay and cell-based assay (CBA)89 Sera

GlossaryAChR = acetylcholine receptor CBA = cell-based assay DOK7 = downstream of kinase 7 FACS = fluorescence-activated cellsortingKO = knockout LRP4 = low-density lipoprotein receptor-related protein 4MG =myasthenia gravisMuSK =muscle-specific kinase NMJ = neuromuscular junction SHP2 = Src homology 2 domain-containing phosphotyrosine phosphatase 2SK = specific kinase






































M081 67 F 3b 1 487 Aza 100 mg Pred 125 mg

M083 19 F 2b 2b 688 Pred 250 mg

M085 30 F 3b 3b 1272 None

M087 61 M 4b 1 063 Pred 50 mg


M092 28 F 5 1 041 Pred 30 mg

M093 63 F 4b 3b 1272 Pred 125 mg

M097 24 F 2b 1 1273 Pred 40 mg

M098 70 F 4b 2b 025 Pred 125 mg

M103 42 M 3b 0 342 Pred 25 mg

M105 67 F - 0 073 Pred 20 mg


M116 32 F 3b 0 965 Pred 175 mg

M125 36 F 5 2b 384 Pred 35 mg


M128 50 F 4b 0 1747 Pred 125 mg

M129 37 M 2b 1 415 Pred 15 mg

M133 23 F 3b 0 654 Pred 125

M147 33 F 3b 2b 124 Pred 25 mg

M149 36 F - 1 688 Aza Pred

M154 50 F 3b 1 688 Pred 125 mg IVIG

M159 28 F 4b 4b 2496 Pred 50 mg

M165 49 F 5 2b 527 Aza 100 mg Pred 625 mg

M166 29 F 2b 0 275 Pred 125 mg

M167 46 F 2b 3b 945 None

M168 49 F 2b 0 366 Pred 10 mg

M173 42 F 3b 3b 857 Pred 50 mg

M175 44 F 3b 2b 486 Pred 175 mg

M176 18 F 3b 0 469 Pred 375 mg PLEX


M181 56 F 3b 4b 437 None





























Appendix Authors


Saif HudaMD DPhil






Anna De RosaMD



MarkWoodhallPhD






JudithCossins DPhil






RobertaRicciardi MD



Amelia EvoliMD



David BeesonPhD

































Reprints








































M081 67 F 3b 1 487 Aza 100 mg Pred 125 mg

M083 19 F 2b 2b 688 Pred 250 mg

M085 30 F 3b 3b 1272 None

M087 61 M 4b 1 063 Pred 50 mg


M092 28 F 5 1 041 Pred 30 mg

M093 63 F 4b 3b 1272 Pred 125 mg

M097 24 F 2b 1 1273 Pred 40 mg

M098 70 F 4b 2b 025 Pred 125 mg

M103 42 M 3b 0 342 Pred 25 mg

M105 67 F - 0 073 Pred 20 mg


M116 32 F 3b 0 965 Pred 175 mg

M125 36 F 5 2b 384 Pred 35 mg


M128 50 F 4b 0 1747 Pred 125 mg

M129 37 M 2b 1 415 Pred 15 mg

M133 23 F 3b 0 654 Pred 125

M147 33 F 3b 2b 124 Pred 25 mg

M149 36 F - 1 688 Aza Pred

M154 50 F 3b 1 688 Pred 125 mg IVIG

M159 28 F 4b 4b 2496 Pred 50 mg

M165 49 F 5 2b 527 Aza 100 mg Pred 625 mg

M166 29 F 2b 0 275 Pred 125 mg

M167 46 F 2b 3b 945 None

M168 49 F 2b 0 366 Pred 10 mg

M173 42 F 3b 3b 857 Pred 50 mg

M175 44 F 3b 2b 486 Pred 175 mg

M176 18 F 3b 0 469 Pred 375 mg PLEX


M181 56 F 3b 4b 437 None





























Appendix Authors


Saif HudaMD DPhil






Anna De RosaMD



MarkWoodhallPhD






JudithCossins DPhil






RobertaRicciardi MD



Amelia EvoliMD



David BeesonPhD

































Reprints






























Appendix Authors


Saif HudaMD DPhil






Anna De RosaMD



MarkWoodhallPhD






JudithCossins DPhil






RobertaRicciardi MD



Amelia EvoliMD



David BeesonPhD

































Reprints






























Reprints




shp2 inhibitor protects achrs from effects of …serum dilutions with agrin but without the shp2...

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