Report  format  1.   Title  of  the  research  project  funded  by  the  Mizutani  Foundation  Regulation  of  Natural  Killer  cell  function  by  O-­GlcNAc  modification.    
2.   Name  of  the  principal  investigator  and  collaborators.  
PI-­Reshmi  Parameswaran  Collaborator-­  Parameswaran  Ramakrishnan    3.   Institution.  
Case  Western  Reserve  University,  Cleveland,  USA.    
4.   Abstract  of  objectives,  methods,  and  results.    
O-­GlcNAc   modification   (O-­GlcNAcylation)   is   a   reversible   post-­translational  modification   which   involves   the   addition   of   N-­acetylglucosamine   to  serine/threonine  residues  of  cytosolic  and  nuclear  proteins.1,  2  An  elevated  cellular  level   of   O-­GlcNAc   is   best   described   under   chronic   hyperglycemic   conditions.  
Although  the  number  of  proteins  found  to  be  O-­GlcNAcylated  in  immune  cells  is  growing,   knowledge   on   the   functional   significance   of   O-­GlcNAcylation   in  controlling   immune   cell   function   remains   rudimentary.   We   are   interested   in  studying  the  role  of  O-­GlcNAcylation  in  innate  immune  system,  specifically  its  role  in   Natural   Killer   (NK)   cells.   We   found   that   global   inhibition   of   O-­GlcNAcylation  increases   NK   cell   cytotoxicity   against   cancer   cells.   We   identified   the   pool   of   O-­GlcNAcylated  proteins  in  NK  cells  by  mass  spectrometry  and  found  that  perforin  and   granzyme-­B   are   O-­GlcNAcylated   in   NK   cells,   which   are   the   key   effector  molecules   for   NK   cell   mediated   cytolysis.   We   also   observed   elevated  perforin/granzyme  release  after  O-­GlcNAc  inhibition,  which  might  account  partially  for   the   observed   increase   in   cytotoxicity.   This   study   reveals   a   novel   role   of   O-­GlcNAcylation  in  NK  cells  and  contributes  to  improve  the  NK  cell  mediated  killing  of  cancer  cells  and  to  develop  a  better  therapeutic  approach  for  NK  cell  adoptive  therapy.  
We  chose  Acute  myeloid  leukemia  (AML)  as  the  cancer  model.  AML  is  the  most  common   acute   leukemia   in   adults.   Unfortunately,   the   current  AML   therapeutics  have  poor  efficacy  and  high  toxicities.  Only  20%  of  patients  over  age  56  survive  two   years.3   New   therapeutic   approaches   are   urgently   needed.   One   potential  strategy   is   immunotherapy   with   Natural   Killer   (NK)   cells.   NK   cells   are   cytotoxic  lymphocytes   of   the   innate   immune   system.4   The   advance   of   NK   cell  immunotherapy  has  been  hampered  in  large  part  by  a  lack  of  suitable  expansion  systems  that  generate  large  amounts  of  functional,  highly  active  cytotoxic  NK  cells  in  doses  suitable  for  administration  in  humans.  We  used  a  novel  NK  cell  expansion  system   using   artificial   antigen   presenting   cells   (aAPCs)   transfected   with  membrane-­bound  IL-­21  and  other  NK  cell  stimulating  molecules.5  Peripheral  blood  NK  cells  grown  in  this  system  can  rapidly  expand  in  3  weeks  up  to  47,000-­fold  and  exhibit   higher   cytotoxic   activity.5   These   expanded   NK   cells   are   capable   of  decreasing  tumor  burden  in  xenogeneic  models  of  neuroblastoma  and  myeloma  and  are  now  being  used  in  phase  1  clinical  trials  for  multiple  malignancies.  Though  this  NK  cell  expansion  system  has  promise,  these  cells  still  exhibit  suboptimal  NK  cell   killing   of   target   cancer   cells.   We   have   found   that   targeting   O-­GlcNAc  modification  dramatically  increases  the  ability  of  NK  cells  to  kill  cancer  cells.  Thus  the  combination  of  aAPC  expansion  and  stimulation  by  O-­GlcNAc  inhibitors  might  result  in  clinically  relevant  doses  of  highly  cytotoxic  NK  cells  with  better  therapeutic  potential.   Based   on   this,   we   propose   to   further   characterize   these   O-­GlcNAc  inhibited  NK  cells  as  well  as  develop  a  platform  to  manufacture  these  NK  cells  for  clinical  use.      5.   Progress  after  the  granted  period  To   confirm   the   O-­GlcNAcylation   in   perforin   and   granzyme   proteins,   we  overexpressed   perforin   and   granzyme   (V5   Tagged)   in   NK   cells   by   viral  transductions  and  immunoprecipitated  perforin  and  granzyme  from  NK  cells.  We  performed  western  blotting  using  anti  O-­GlcNAc  antibody  to  confirm  perforins  and  Granzymes  are  indeed  O-­GlcNAcylated  in  NK  cells.  
Next,  we  investigated  a  pathologically  relevant  condition  that  elevates  cellular  O-­GlcNAcylation  with  potential  to  alter  NK  cell  function.  Hyperglycemic  conditions  are  known  to  elevate  OGlcNAcylation.  Hence,  we  isolated  NK  cells  from  blood  of  diabetes   patients   as   a   relevant   model   to   study   the   pathological   role   of   O-­GlcNAcylation   in   NK   cell   function.   NK   cells   from   diabetic   patients   are   known   to  exhibit  significant  functional  defects.6  However,  specific  causes  for  this  dysfunction  are   less   clear.   Type   1   diabetic   NK   cells   reported   to   exhibit   decreased  NKG2Ddependent   cytotoxicity   and   interferon-­γ   secretion,7   while   type   2   diabetic  patients   are   highly   susceptible   to   infection   and   have   an   increased   incidence   of  some  tumors,  possibly  due  to  NK  cell  dysfunction.  
Interestingly,  we  found  that  NK  cells  from  diabetic  patients,  who  are  known  to  have  elevated  O-­GlcNAcylation  levels,  exhibited  a  reduced  ability  to  kill  target  AML  cells  as   compared   to   normal   donor   NK   cells.  This   supports   our   in   vitro   results   using  PUGNAc   (PUGNAc   inhibits   O-­GlcNAcase,   a   beta-­exo-­N-­acetylhexosaminidase  which  cleaves  beta-­O-­linked-­N-­acetylglucosamine  residues  from  glycoproteins)  to  elevate   O-­GlcNAcylation,   and   suggests   that   pathological   hyperglycemia   and  associated  increase  in  O-­GlcNAcylation  might  be  a  mechanism  that  compromises  NK  cell  function.  To  further  confirm  that  the  impaired  NK  cell  function  in  diabetic  patients  is  indeed  due  to  increase  in  O-­GlcNAcylation,  we  treated  the  diabetic  NK  cells  with  DON  (6-­Diazo-­5-­oxo-­L-­norleucine,  a  glutamine  analogue,  is  used  also  as  an  OGT  inhibitor)  and  studied  their  cytotoxic  function.  As  expected,  DON  treated  diabetic   NK   cells   showed   enhanced   efficacy   in   killing  AML   cells.   These   results  suggest  that  high  O-­GlcNAc  modification  of  proteins  may  directly  contribute  to  NK  cell  function.  
6.   Future  perspective.  
We  will  identify  the  O-­GlcNAcylation  sites  in  perforin  and  granzyme  and  will  mutate  these  sites  in  NK  cells  to  further  explore  biochemical  and  functional  consequence  of  those  mutations.  We  will  also  analyze  the  endogenous  O-­GlcNAc  transferase  (OGT,   the   enzyme   which   adds   O-­GlcNAc   to   proteins)   binding   to   overexpressed  perforin/granzyme.   Next,   we   will   examine   signal-­induced   binding   of   OGT   to  perforin/granzyme   and   analyze   different   deletion   mutants   of   perforin/granzyme  and  OGT  to  identify  the  binding  region.  We  will  also  study  perforin/granzyme  -­OGA  interaction  in  a  similar  manner  as  described  above.  These  studies  will  identify  the  discrete  binding  domains  in  OGT,  OGA  and  perforin/granzyme  and  we  will  employ  these  short  peptides  to  modulate  perforin/granzyme  O-­GlcNAcylation  in  vivo.  We  will   develop   a   specific   monoclonal   antibody   to   detect   mutant   O-­GlcNAcylated  perforin/granzyme.  This  could  be  a  valuable  tool  to  explore  the  biochemistry  of  O-­GlcNAcylation-­dependent   perforin/granzyme   signaling   in   NK   cells.   These  molecules  could  prove  valuable  tools  to  inhibit  perforin/granzyme  O-­GlcNAcylation  in  vivo  and  thereby  glycosylation  dependent  perforin/granzyme  functions  like  the  enhanced  secretion  and  tumor  apoptosis.      7.  Publications  (a  maximum  of  10  references)  should  be  given  using  the  following  style.      1)  Smith  AB,  Jones  CD.  Title,  J.  Biol.  Chem.  (168):  1758-­1764,  1976    References    1.   Hart  GW,  Housley  MP,  Slawson  C.  Cycling  of  O-­linked  β-­N-­acetylglucosamine  on  nucleocytoplasmic  proteins,  Nature.  (446):1017–1022,  2007  2.   Hart   GW,   Slawson   C,   Ramirez-­Correa   G,   Lagerlof   O.   Cross   talk   between   O-­GlcNAcylation  and  phosphorylation:  Roles  in  signaling,  transcription,  and  chronic  disease...  Ann.  Rev.  Biochem.  (80):825–858,  2011  3.   Estey  E,  Dohner  H.  Acute  myeloid  leukaemia.  Lancet.  (368):1894–1907,  2006.  
4.   Vivier  E,  Tomasello  E,  Baratin  M,  Walzer  T,  Ugolini  S.  Functions  of  natural  killer  cells.  Nat  Immunol.  (9):503–510,  2008  5.   Denman  CJ1,  Senyukov  VV,  Somanchi  SS,  Phatarpekar  PV,  Kopp  LM,  Johnson  JL,   Singh   H,   Hurton   L,   Maiti   SN,   Huls   MH,   Champlin   RE,   Cooper   LJ,   Lee   DA.  
Membrane-­bound  IL-­21  promotes  sustained  ex  vivo  proliferation  of  human  natural  killer  cells.  PLoS  One.  (7)  2012  6.   Qin   H,   Lee   IF,   Panagiotopoulos   C,   Wang   X,   Chu  AD,   Utz   PJ,   Priatel   JJ,  Tan   R.  
Natural   killer   cells   from   children   with   type   1   diabetes   have   defects   in   NKG2D-­
dependent  function  and  signaling.  Diabetes.  60(3):857-­66,  2011  7.   Berrou   J,   Fougeray   S,   Venot   M,   Chardiny   V,   Gautier   JF,   Dulphy   N,   Toubert   A,  Peraldi  MN.  Natural  killer  cell  function,  an  important  target  for  infection  and  tumor  protection,  is  impaired  in  type  2  diabetes.  PLoS  One.  8(4):2013