{"paper_id":"e8b14aba-5f67-4e82-81d1-c75fc5308bfb","body_text":"1\nEnforced MYC expression selectively redirects transcriptional programs during 1 \nhuman plasma cell differentiation  2 \n 3 \nPanagiota Vardaka 1 , Eden  P age 1 , Matt hew A  Care 2 , Sophie Stephens on 1 , Ben Kemp 1 , 4 \nMichelle U mpier r ez 1 , Eleanor O’Calla ghan 1 , Adam M abbutt 1 , Roger Owen 3 , D aniel  J  5 \nHo ds on 4 , G ina M D oody 1  and Reub en M Tooze 1,3*  6 \n 7 \n 8 \n1 Divis ion of  Haema to l o g y and Imm unology, L eed s In s t itute of  Me dical R es earch, 9 \nUniv er s it y  of  L eeds, Lee d s, LS9 7 TF, U K  10 \n2 Epidemio logy and Can cer Stat is tic s G r oup, Depar tment  of He alt h Sc iences, U niver sity of  11 \nYork, Yor k , Y O10 5DD , UK .  National Institute of H ealth R esea r ch Leeds B iomedica l 12 \nResearch Centr e.  13 \n3 Haemat ological Malignanc y  D iagn ostic Servic e, Leed s Tea ching  Hospi tals NHS Tru st, 14 \nLeeds , UK  15 \n4 W elcome MR C Cambridge Stem  C el l  Instit ute, Cambr idge, CB2 0AW, U K  16 \n 17 \n 18 \n *Cor respondence s hou l d  b e add ress e d to R .M.T. ( r.tooze@leeds.ac.uk )  19 \n  20 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 2\nAbstract 21 \nMYC pro vide s a  rh eo sta t link ing cell  gr owth and  divi sion d uring pl a sma c ell  ( PC ) di f fer enti atio n .  22 \nPrec i s e  co nt r ol o f M Y C i s c entr al t o th e netwo rk co nt r oll ing dif fe ren tia tion. De r e gula tion o f M Y C  23 \ndrive s tr an s f ormati on i n  aggre s s i ve B- ce ll neo pla s ms  and  i s o fte n acc omp a nie d by a popt otic  24 \nprotec tion c on fer r e d by BCL2 . W e  a sse s s  how  M Y C and BCL 2 der egula t io n impac t s on th e abil it y  o f  25 \nhuma n B -cell s t o com ple te PC  dif fe ren ti ation .  U nd er p e r mi ssive  c onditi on s for  P C di ff eren tia tion we  26 \nfind such de re gulati on do e s  no t tr an sfor m c ells. W hile drivi ng lo s s  of n ormal  P C s ur fac e p heno type ,  27 \nMYC dereg ula tion ha s lit tle impa ct on comp onent s  o f regul a tor y  circui t ry c ontro lling B-c ell ide n t i t y .  28 \nThis c ontra st s  w ith pro found impac t on i nitiati on  o f sec r e t o r y  ou tput  a nd s e cr eto ry reprogr amming ,  29 \nco upled t o damp eni ng o f XBP1  and i m munoglobul in gene  e nh ance men t and  a  shi ft  tow a rd di s tinc t  30 \nmetabo lic progr ams.  The  e stabli shm ent  o f  thi s abe rrant s t ate d epend s o n MY C homology  box es  31 \n(MB0 and  M BII ). Dep endenc e on M BI I  is  pr ofo u nd and r e solve s  to r e sidue W 135 .  32 \n 33 \n 34 \n 35 \n 36 \n 37 \n 38 \n 39 \n 40 \n  41 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 3\nIntroduction 42 \nThe  tran scrip tion fac tor c- MYC (MY C)  was the  fir s t  onc ogene  de r eg ula ted  by ch r omosoma l  43 \ntran s l oca tion  to  be i de n t i fi ed in  B -cel l ly mphom a.[1 -3] It i s one  o f t he mo s t  fre q uen t ly  d eregula t e d  44 \nonc ogene s i n aggr es s i ve  ly mphoid c a n c er bu t al s o ac ts a s  a c ent ral reg ula t or o f p hysiol og ical  45 \nly mphocy t e  g r ow th a n d pr o li fe rati on. [4 - 9]  MY C  a ct s  as  a s e qu ence  sp ec ific tra nscrip tion fact or o f  46 \nthe ba sic-h e lix-loo p - h e lix (bHLH ) dom ai n fa mily, oc cupy ing E -box  DNA  s e q uence  e lemen t s i n  47 \nco mplex w ith its oblig at or y  par t n e r  M A X.[10 -13] With  high MY C ex pre s si on i ts g enomic  oc cup ancy  48 \nspre ad s to sit e s  w ith non-con s en su s  E-bo x motif s ,  a nd MY C occ upanc y correla te s with RN A  49 \npoly mera s e  l o ading at  primed promo t e r s and with r e le as e o f pa u sed poly mer as es. [11 , 12 , 1 4 -17 ]   50 \nDi st in ct  model s  o f MY C functio n  s uppo rt  ac t i on  a s a globa l  enh anc er  of  pr evail in g ac tive p romot er s,  51 \nand as a more sele c tive regula t or o f sp ecific  g ene pr og ra ms  th at ove r l a p be tw een multiple cell  52 \ntype s. [11, 15-1 8 ]  Ex it fr om c ell c ycl e an d cel lular te rminal dif f eren tia tion i s link ed to r ep re s sion o f  53 \nMYC and nuc l ear exc lusion .[19 ] I n B-c el l  d iffe re nt i a tion t o  th e pla s ma  c e ll ( PC ) stag e rep r e s sio n of  54 \nMYC  ha s  b e en at tribu ted to th e tra nsc rip tional  facto r BLI MP 1/PRD M1. [20 -23] 55 \nM Y C - dr i v en  ce l l u l ar  t r ans f or m a t i on  d e p e n ds  o n D N A  b i n di n g a n d o n  t he M Y C  N - te r m i n a l 56 \ntran s a c tiva t i on  do main  (TA D ) . [13, 24 ,  2 5 ]  Thi s  TA D c o ntain s  e volu tiona r i l y con se rved r egion s, t he  57 \nMYC box e s  (MB ), t hat  are  re s p on sible  f or di stinc t co -f a ctor  i nte r a cti on s. [18, 26,  27] M BI c on t a in s a  58 \nphosp hodeg ron s e que nce c on trolling MY C degrada t i on via  t h e prote a some . [28-30]  A c r u cial  r e s idu e  59 \nin M BI  is T58 whic h is frequ ently  mut ated in  agg r e s sive lymphoma .[30 -32 ] MB0 and MBII are  60 \nimpli cated in  tr an s a ctiva t i on a nd tran s f ormation  activ ity wit h MB II id enti fi ed a s e ss enti al for MY C  61 \nd r i v en  tr a nsf or m a t io n .[ 33 - 35 ] M BI I med ia t es  r e c r u it m e nt  of  TR RA P a n d as s oc ia t e d h is t o ne  ace t y l 62 \nt r a n s f e r a s e  c o m p l e x e s . [ 2 6 ,  33 - 35 ]  A t  t h e  c o r e  o f  M B I I  i s  a  h i g h l y  c o n s e r v e d  f o u r  a m i n o  a c i d  63 \ns e qu e n ce (D CM W ) .  W 1 35 is  hi g h l y c o ns er v e d  in  M Y C f a m i ly  p r ot e i ns , [ 36,  37 ]  a n d s its  at  t h e h ea r t of 64 \nthe p redic t ed M BII  int er fac e wi th T RRA P , [38 ] an  int erf ac e tha t may  be th erap eutic a lly  65 \ntarge tabl e . [39]  66 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 4\nMYC tra ns fo r mi ng ac t i vity is hel d i n chec k by  induction  of apop to s i s . [25,  4 0-43] H ence MY C  67 \ndereg ula tion  in canc er s  i s  of ten  ac com pa nied by TP53  i na ct i v a t io n or  de r e g u la t e d  BCL 2. [44-46 ] A  68 \nrange  o f ag gre ssive  B - c ell n e opla sm s  c a rr y  o ncoge nic ev ent s  tha t a rr e st cel l s  d uring di ffe ren tia t i on  69 \nbetwe e n B -cell  a c tiva t io n a nd PC di f f e re nt i a tion. [47 ] MY C de regula t i on i s  a rec urren t even t in thi s  70 \nco nt e xt a s  a r e sult o f t ran s l oca tion or stabi liz i ng muta tion s.[1 ,  3,  31 , 3 2, 48, 49] Lymph omas w it h  71 \ntran s l oca tion o f bo th MYC  an d BCL2,  “do ubl e hit ly mphoma” , a s wel l a s ca ses wi th M Y C and B CL 2 c o-72 \nex pr e s si on, w ith out und erlyi ng t r a n s l o cation, “doubl e  expre s sing ly mphoma” , hav e an a dve r s e  73 \nprogno si s. [48, 5 0] Re ce ntly ef fic ien t tran sduc tion of prima r y  hum an B-c el ls w ith onc ogen e  74 \nco mbination s h as  pr ov ided  a ba s i s fo r  in v i t r o  mod elli ng o f human ag gre ssive  B- c ell ly mphoma. [51 ,  75 \n52] In thi s c on text MYC an d BCL2  c o-de regul a tion pr ov id ed an ex ample  of a tr an s f orming  76 \nco mbination d r iv ing su st a in ed pop ul atio n ex pansion . [52]  77 \nDif fe ren tiati on can opp ose  c ellul ar tra ns forma tion by  drivi ng c ell cyc le exit and li miting cell ula r  78 \npla stici t y . Concom ita nt w ith thi s  MYC e xpre ssion g en erally  decli n es  w i th dif fe r e n t i atio n. [15, 53, 54 ]  79 \nWe have  de velop e d mo del s of human  B-c ell ac t i va tion th at ar e p er m i ssive fo r  diffe ren tiat ion to a  80 \nlong -lived PC  s ta te .[55 -57 ] Driv en by  si gnal s  mimic king an tigen  r ece p t or liga ti on and T -cel l h el p  81 \ninc luding  tran sien t C D40L ex pos u re,  B -c ells u nd ergo a pr oce s s  of ce ll growt h a nd div is io n i n w hic h 82 \nendog enou s MY C expre s s i o n is  fir st indu ced foll ow ing a ctivatio n a nd th en  r e pr e ss ed a s t h e  83 \ndiff er entia ting  c ell s co mpl ete  c ell di vision and  t r an sitio n t o a spec iali ze d sec re tor y  sta t e ,  84 \nreca pit ulating phy siologic al P C di f fer enti ation .[55 ] A t th e hea r t o f th e p r oc e ss  of PC di f f e re nt i a tion i s  85 \na coordina t e d re organ iz a tio n o f t r an scr iption f actor s .[58 ] Ove rall  th e sequ enc e of tr an scripti onal  86 \nregula ti on coordin a te s  a MYC -a ssocia t e d  burst o f cel l growth and di vi sion, wi t h e v entual r epre s s i o n  87 \nof el ement s  o f  t h e  B-c el l sta te and a s wi tc h to s ecre tory g ene ex pr e ssion. [7, 17, 5 8]  88 \nA trigg er fo r the t r a n s iti on fr om grow t h  program to PC di ff er entia tion i s  rele as e fr om su s tain e d  89 \nCD40L  signa l s , [5 9, 60 ] whic h prov ide s p o t e n t  NF κB pathwa y activ atio n, [61, 62 ] and a  signa l  whic h  90 \nc an  de l a y a nd/ or  p r ev e n t d i ff er ent i a t ion  o f  a ct i v a te d  B- c el ls .[ 6 3- 6 5]  Sus ta in e d p r o vi s i o n of  CD 4 0 L 91 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 5\nwa s  int egral to prev iou s in  v i t ro  model lin g of human  B -ce ll l ymphoma gen e si s. [52]  By s u st aining  92 \nCD40L  s ig na ling , the app roa ch did not ad dre ss wh ethe r oncog ene d ereg ula tion su ffic ed t o t r an sf orm  93 \nB-ce ll s  und er cond it i on s  permi s s iv e fo r  PC di ff eren tia tion. Exa mining thi s i s  o f inter e st be cau s e it  94 \nwou ld te st th e i mpact of der eg ulati o n of MYC i n the co ntex t  of  an in trin sical ly r eorgani z i ng  95 \ntran s c rip tional  pr og ra m o f di ff ere ntia ti on. To addr es s t h e s e qu e st io n s, w e h a ve ev alua ted  t h e  96 \nimpa ct o f MY C d ere gulati on in  the c on t ext of  BCL2 c o -expr es s i on  a cr oss human  PC dif fe ren tiati on .  97 \nOur  dat a argue t h a t  M Y C and BCL 2 deregul a tion do e s not suf f i ce t o tra ns for m B -c ell s whe n  98 \nco ndition s for d if f e r e n t i ati on ar e p e r mi ss ive, but t h a t MYC  div er t s  ex pr e s sion  tow a r d s a d i stinc t  n on-99 \nphy s iol ogica l pa tte r n th at al ter s  me t abo lic and grow t h -rel a te d g ene expr es sio n a nd impair s  100 \nsecr et ory output . The se tra n s c ripti onal i mpa cts o f  MYC  a r e  ind epend e nt  o f MBI but depe nd in par t 101 \non MB0 and ar e de pe nden t on M BI I and the sing le ami no ac id W135 .  102 \nResults 103 \nAcu t e  MYC an d BCL2 o verex pr e ssion d r iv es a n abe r r an t B-c ell d i ffe r e n tia tion phe n otype.  104 \nWe ai med t o te st to w ha t exten t de reg ul ated e xpr e ssion o f MY C in c ombina tion w ith BCL2 impac t e d  105 \nac utely on hu man B-ce ll di ff eren tia tion. We ini t i ally e valua t e d a T58I  variant of MYC in c ombina t io n  106 \nwi t h BCL 2, a s thi s combi nati on ha s b e e n previou sly us ed in lym phoma modell i ng.[52 ] By inc luding  107 \nthe T58 I ly mphoma -a s socia ted  MYC  sta bilizing muta tion  in the  MB I domai n, [31 , 32]  thi s  app roac h  108 \nco mbined ov er expre s s ion  an d s t a bili zati on to enha nce the pot entia l fo r M Y C im pac t .  W e  te st ed thi s  109 \nin con t e xt of our di f fer enti a t i on sy st em  w hich is p er m i s s ive f or hu man B - c ell diff er entia tion to a  110 \nlong -lived PC  sta te  ( F igur e 1a ).  Bri e f l y i n thi s mod e l sy st e m  B-c e ll s a re activ a ted by  signa l s tha t  111 \ninc lude an tige n r ec ept or  lig a tion, CD 4 0  stimul ation  and c ytokin e s IL2 and IL2 1 for 3 da ys , durin g  112 \nwhi ch B-cell s g ro w a nd b eg in to  divide  and endog en ou s  MYC  i s expre s sed . At day  3 C D40 a n d  113 \nantig en r e ce pto r ligati on a re remove d a nd N F κB signa lling i s r a pidly l os t . Activa ted  B -c ell s  114 \nsub s e quen tly divid e rapi dly while  tran si t i oning to a  pla s ma bl a st  s t ate . At d a y 6  pla s ma bl as ts are  115 \ntran sfe rred  to  IL 6 a nd A PR IL or  oth er cytok in e con tain ing co nditio n s th a t suppo rt fu rth er  116 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 6\ndiff er entia tion to the  P C s t a te . [55-57 ] A signific a nt d if f e r e nce from prev iou s  in  v i t r o  modell ing of  117 \nly mphomage ne s i s  in  whic h B -cel l s  w ere con t i nuou s l y main t a i ned in C D 40  stimula t i ng c ondition s, [52 ]   118 \nis the remov al o f C D40 stimul ation  an d NF κB a c tiva t i on  at  da y 3 in  our mo del suppo rting P C  119 \nd i f fe re n t i at i o n.[ 59]  A  d is t i n ct  pa tt e r n of  N F κB  activ a tion i s s u b seque nt l y r eint rod uced  upon  addi t io n  120 \nof A PRIL  at d ay  6 w hich suppor ts  di f fere n tiatio n to th e P C  s tag e . [57]  121 \nIn the c on text o f th is mod el  pe ripher al b l ood memory B cell s w ere  tran sduc ed on  da y 2 of a ctiva t i on  122 \nwi t h MYC  T58I -t2A -BCL2  r et r o viral vec tor (h ence forth T58I -t2 A- BCL2)  an d wer e th en r etu rned to  123 \nCD40 s timulati ng  conditi on s for 24h  befo re p rog r e s sing i nto  the  di f f e r en ti a t i on p roto col w ith  124 \nremova l of CD40  stimul ation at day  3 (F igure 1a, S up. Figur e 1a) . Tran sduc t i on e f f i cienc y wa s  hig h  125 \nand su s t a ine d exp r e s sion o f t h e  re trovir al  CD2 r epor te r was ob serv ed to d a y 20, by  w hich time  P C  126 \ndiff er entia tion ha s  be en e sta bli shed f or  t e n d ays in dif fe r e n t i at ion s i n the a b s e nc e of t r an sduc t i o n  127 \n(Fig ure 1b ). [56 ] Ac ros s  mul tiple time s  po i nt s  of  di ffe ren tia t i on, T58I -t2 A- BCL 2  c e lls  s h o we d  i n cr eas ed 128 \nce ll siz e  r e la tive to MS C V o r  u n t ran sduc e d con t r ol s (S up. F igure 1c  and d ) a cco mpanied by inc r e a se d  129 \nce ll number s at d ay 13 a nd day 20  (S up.  Fig ur e  1 b). T58I -t2 A -BCL2  w as a ssoci a te d with a c hange  i n  130 \npheno t y p e (Fi gur e 1c -f ). Thi s inc lud ed  lo ss o f CD27 a nd C D138 expr e ss i on  whic h are hallma r k  131 \nfea tur es  l inked to P C di ff ere ntia tion,  whi le al so  show ing a d ec r e a se  i n C D19 e x pression  whi ch i s a  132 \nh a l l m a r k  B - l i n e a g e  a n t i g e n  w h o s e  l o s s  o f  e x p r e s s i o n  i s  a  f e a t u r e  o f  m a l i g n a n t  P C s .  T h u s ,  M Y C  T 5 8 I  133 \nand BCL 2 ove rex pr e ssion fr om the  a ctiva t e d B -c ell  s t a ge onwa r d  r e sult ed  upon sub s e qu ent  134 \ndiff er entia tion i n i ncrea s ed c ell size a nd  number and an a b erran t  ph e no t y pe with lo s s o f ty pical  P C  135 \nmarke rs.  136 \nM Y C  a nd  B C L 2 o v er ex p r ess i on d e la ys  ce l l  c y cl e  ex i t  a nd  s ec r e to r y  o ut put . 137 \nPC d if fe rent iatio n i s  cha rac te r i z ed  by c ell  cy cle  ex it and r eprogr amming of  th e expre s s i on  st a t e  138 \ntowa r d s ecre tory ac tivity a nd  awa y from  cell  growt h and  proli fe ra tion.  MY C  dr iv es  both  c ell  g r ow th  139 \nand proli fe r a t io n in  lymphoc yte s. [5,  7, 1 7] W e ther ef or e  a sse s s e d ho w MY C d er egula t i on  impac te d  140 \non the  proli fe rativ e s t a t u s u sing a combi na t i on of EdU  label li ng an d Ki67 de tec ti on at d ay  21 and 31  141 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 7\nof di ffe r en tia t i on (19 and 29  day s , r e s pec tively , af te r tran sd uction ) in T58 I- t2A-BCL 2  or c ont r o l  142 \ntran s d uce d cel ls. During dif fe r e n t i a tion  of normal B-cell s  in the ex pe riment al culture s y stem cel l  143 \ncy cle  exit oc curs  b e t w e en da y 6 and da y  10 a s  t he c ell s tran si tion f rom pla smabl a st to PC s t a te . [56 ]  144 \nCon si sten t  w ith the ov era ll inc rea se  in c ell numbe r  EdU + Ki 67 + c ell s were r ea dily de t e ct ed in T 58I-t2A -145 \nBCL 2 b u t  n o t  i n  c o n t r o l  c o n d i t i o n s  a t  d a y  2 1 .  H o w e v e r ,  b y  d a y  31  T 5 8I -t 2A - B C L2  cond it i on s showe d  146 \nno s i gnific a nt inc re as e in  EdU + Ki67 + ex pre s s ing ce lls (Fig ur e 1g , S up. F igure 1e) . There for e, unde r  147 \nco ndition s p e r mi ssive f or B -cel l dif fe ren tiatio n to  th e P C s t at e MYC an d B CL2 d ereg ula tion re s ul te d  148 \nin a n ex t e nde d but ultim a t e ly c urta iled p rolif e r a tiv e s t a t e  during sub se quen t dif fe renti atio n .  149 \nDuring P C dif fer enti atio n , pr ol i fera tio n and secr eto ry reprogr amming  are coupl e d.[66 -68 ] Howev er ,  150 \ntran s i t i on to a s ecre tory sta te i s no t stric tly  linke d to cel l cy cle e xit. B oth sec reti n g  and non- sec reti ng  151 \nce lls have b e en repo rte d to di vide a t si mila r  ra te s. [69 ] We there for e  te sted  sec reto ry out put of  t h e  152 \nin v it r o  di ff er entia ted  c ell s.  Secr ete d Ig G  and  Ig M  n ormal ized pe r ce ll , wa s s i g nific antly reduc e d i n  153 \nT 5 8I - t 2A -B C L2  condition s  at bot h  da y 6  a nd day 13 (Fig ur e  1i ) . T hu s , ac compa nying  in crea sed c el l  154 \nsiz e  a nd  d elaye d cell  c ycl e exit,  MYC  an d BCL 2 d e r e gul ation  le d  to  re duc ed sec r e t o ry  ou tput  fr om  155 \ndiff er entia ting  B-c el l s .  156 \nEnforc ing  MYC ov erex pr e ssio n dri v es clas s i cal t arge t ge ne s a nd alte rs p at tern s  o f tran scripti on f a cto r  157 \nex pressio n.  158 \nTo furth er und er s t an d t h e  impac t  o f MYC a nd B CL2 d er e gula tion o n di f fere ntia ti on we p er f ormed  a  159 \ntime cou rse gen e ex pr e ssio n study  in c ontr ol  o r MYC T58I -t2 A- BCL2 c o nd i t i o ns .  G e ne  e x p r ess ion 160 \nwa s  a sse s sed at : day 0 - prio r to activ atio n; da y 3 –  ac tiv ated B-c e ll st a g e, 24 h a ft e r tr a n s d uc tion ; day  161 \n6  -  p l a s m a b l a s t  s t a g e ,  4  d a y s  a f t e r  t r a n s d u c t i o n ;  d a y  1 3 -  e a r l y  P C  s t a g e  1 1  d a y s  a f t e r  t r a n s d u c t i o n ;  162 \nand da y 20 -  e s tabli sh ed PC stag e 18  d ays a fte r  tra nsdu ction  (MS C V-con trol  co ndi t i on s  gen e r a t e d  163 \ni n s u f f i c i e n t  c e l l s  f o r  a n a l y s i s  a t  d a y  2 0 )  ( S u p .  T a b l e  1 ) .  C o n s i s t e n t  w i t h  a  p r o g r e s s i v e  i m p a c t  o f  M Y C  164 \nT 5 8I - t 2A -B C L2  cond it i on s  on  gene e xpre ssion, uni form manif old a pproximati on  and projec t i o n  165 \n(UM A P)  showe d  s i milar cl u ster ing o f  sa mpl es a t day 3 w ith sub sequ e nt i ncrea s e d s epa ratio n o f  M Y C  166 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 8\nT 5 8I - t 2A -B C L2  c o n d i t i o n s  f r o m  c o n t r o l s  a t  d a y  6 ,  d a y  1 3 a n d  d a y  2 0  ( F i g u r e  2 a ) .  M Y C  w a s  167 \nsub s t anti a lly exp r e s s ed i n  phy s io logic al  dif fe r e n tiati on a t d a y 3 in  ac t i vated  B - cell s and  was  the n  168 \nprogre s s iv ely r epre ss ed in co ntr ol di f fer enti ation  condi ti on s . In con tra s t  MYC T58I -t2 A-BCL 2  169 \nco ndition s s h owed a mode st in c rea se  in MYC  ex pr essi on a t d ay  3 a nd then  maintain e d su pra -170 \nphy s iol ogica l  lev el s of  MYC  ex pr e ssion th roughout  sub sequ ent  di ff eren tia tion ( F ig ure 2b ) .  Expre s s i on  171 \nof CD2 and  enh anc ed a nd s u stain ed e xpr es s io n o f  BCL2 w as al so  confi rmed in tr a ns duc e d c onditi on s  172 \n(Fig ure 2b an d Sup . F igure 2 a ).  173 \nWe n ext a s s e s se d the  ex ten t t o whi ch phenotypi c ch a nge s w er e rec api tula te d  in  g en e expr es s i on  174 \ndata . We ob served  signi fic an t par alle ls with sup pre ssion of SD C 1  (C D 138),  CD 2 7 a nd CD 19  175 \nex pr e s si on a t lat er time  p oin t s  in M YC T58I- t2A -BCL 2  c onditi on s r e l ative to cont rol s. CD 38  176 \nex pr e s si on wa s n ot su bstan tia lly impa c t ed while  MS4 A1  (CD 2 0 )  e x p r ess io n  was i nc r e ase d  i n  M YC 177 \nT 5 8I - t 2A -B C L2  condi t i on s  (Fi gur e 2c). The se c ondition s wer e als o as soc iat e d w it h  suppr es se d 178 \nTNFRS F17  (B CMA ) bu t no t TNF RSF1 3B  (T ACI ) a nd CD79 A  b u t  n o t  C D79B  ex pre ssi on (S up . Figu re 2 b ).  179 \nThus, th e per turb ed phen o t y pe ob se rve d by  flow c ytometry wa s  re flec ted in c orre spondin g cha nge s  180 \nat tr an script l ev el.  181 \nP C  d i f f er e nt iat ion  is  d r i v e n b y c o or d ina t e d c h an g es  i n t ra ns c r i p t io n  fa ct or  e x p r es s io n.[ 5 8]  W e  182 \nther ef ore exa mined how  know n regula t ors of t he  B -cell  stat e and  PC di f fer enti at ion w ere expr e sse d  183 \n(Fig ure 2 d ). PA X 5  a nd EB F 1 , tra n s c ript io nal regula t or s involv ed in maint aini ng B -cell sta te s , [70 - 7 3] 184 \nwe r e  expre s s e d  in re sting  a nd  ac t i v ated B-c el ls  and  t h e n equiv alen tly  rep r e ssed  during  185 \ndiff er entia tion in con trol a n d MYC T58 I -t2A-BCL 2  c o nd i t io ns . P os it i ve  re g u l at or s  of  PC  d if f er e nt i at i o n 186 \nIRF4  and PRD M1  (BL I M P 1 ) were in d uced  up on ac t iv ati on an d di f fer entia tion w it h  modes t  redu ction s  187 \nobs erved i n m axi mal ex pre ssion for  both  fa ct or s  in  MY C T58I -t2 A- BCL 2  con dition s. XBP1 , the  prima ry  188 \nt r a ns cr i pt i o n a l  dr i ve r  of  s e cr et or y  r e p rog r a m m in g  a n d  t h e  unf ol de d  p r ot e i n  r es p on s e  of  t h e  ER, [ 7 4-189 \n76] c on t ra s ted i n showing  more  p r of ou ndl y supp r e ssed  induc ti on a t d ay 6 an d  a ll s u b s equ ent  time  190 \npoint s i n MY C T58I -t2A -BCL 2  c ondit ion s . D i ff ere ntia l regu la tion o f o ther t ra nsc ription  fac to rs w a s  191 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 9\nalso ob s erved in cludin g re pr e ssion o f RU N X1 , whic h has a rol e in c ell c ycle  entr y , [77] a nd en hanc e d  192 \nex pr e s si on o f the PC fa t e  an tag oni s t  B ACH2,[78 -80 ]  and SREBF1 , a co n t roll e r  of st erol m e t a bolic  193 \npathw ays ,[81 ] in MY C T58I - t2A -BCL2  condi t i on s  (S up . Fig ure 2c ).  194 \nCon si sten t w it h  c an onic al MYC -drive n  gene ex pr e ssion  wel l-d ef ined  MYC  ta rget s suc h  a s  TE RT , t h e  195 \nca talytic  co mpone nt o f te lomer a se, [82 ]  JAG2 , a rec epto r on  th e N OTCH s i gn alli ng pa t h way , [ 8 3]  196 \nTRAP1 ,  a  k ey mitoc ho ndrial c h ape rone, [ 84] FA BP5 ,  l inked to fat ty ac id me taboli sm and  a po ten tia l  197 \nt h e r a p e u t i c  v u l n e r a b i l i t y  i n  m y e l o m a , [ 8 5 ]  w e r e  i n c r e a s e d  ( F i g u r e  2 e ) .  A  w i d e  r a n g e  o f  o t h e r  M Y C  198 \ntarge t gene s  de fin ed in ce llula r model s i n both mous e and hum an [7] wer e al so profo undly  i nduce d 199 \nin MY C T58I- t2A -BCL2  condi t i on s.  200 \nSi nce XBP1 i s a princ ipal r egula tor o f s ecret ory rep rogramming  w e a s s e ssed k now n XBP 1 a nd E R  201 \nstre s s  re sp on se ta r g e t gene s. We found consi ste nt p a ttern s o f rep r e s s ed expre s si on for  g e ne s suc h  202 \nas HE R P UD 1 , ERL EC1,  DERL3  a nd TX N D C 5 , [ 8 6 -88] whic h sha re direc t  XBP1 pro moter occ upanc y at  203 \nthe pla smabl a s t s t age  i n ou r model sy stem (Fi gur e 2 f). [59 ]  Immun oglobu li n i s t h e ma in s ec reto r y  204 \noutput  o f P C s a nd ex pre ssion  i s  p rof o undl y de cre a sed  on cond itio n al XB P1  deleti on  in  mu r ine  205 \nPC s. [89, 90 ] W e  t her ef ore als o e xamin e d the  ex pre ssion lev el s  of immunog lo b ulin gene s . Inde e d,  206 \nthe se ge ne s compri se d some o f th e  mos t d if fe rent ially  expre s s ed  and  s howed  signi f i can tly  207 \ndamp ened ex pr e ssion p artic ul arly fo r IGH G 1 , I G HG2 , I GHG 3  a nd IG H M  i n M Y C T58I- t2A -BCL 2  208 \nco ndition s a t  la te r  time poin t s ( S up . Fig ure 2d) . Thu s , an a lysi s  a t indiv idual  g en e leve l s ug ge st e d a  209 \nco or di na ted impac t o f  MYC ov e rexpre s sion  on  the  r eg ulatio n of me taboli c and secr et ory pathw ay s  210 \nduring P C dif f eren tia tion an d a s epar atio n of thi s pert ur be d s e c ret or y  r e prog ram ming from retain e d  211 \ntran s c rip tional con trol ove r  f e atu r e s o f t he B- c e ll stat e.  212 \nMYC an d BCL2  ove r e xpre s s io n  drive s c oo rdina ted m o d ular p at tern s o f ge ne  expre s sion c ha nge .  213 \nTo te s t gen e reg ulatio n a t  a  globa l l ev el we analy s ed g ene e xpre ssion  c hang e s  i n  M Y C T58I -t2 A-BCL 2  214 \nand con trol co ndi t i o ns u s in g Pa rsimoni o us Gen e Co rrel ation  N etwork Ana ly si s (P G CN A ). [91 ] Thi s  215 \nco r rela t i on -ba se d meth od allo w s  t he shi fting pa t t ern s o f gen e e xpre ssio n a cro ss diff er entia tion an d  216 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 10\nbetwe e n condi t i on s  to  be a s se s s ed in te rms  o f modu l e s  o f c or e gul ate d g ene s.  P G C NA id enti fied  1 6  217 \nmodule s  of c oreg ulat ed ge ne s  (l a belle d  M1-M16  a cc or d ing t o number o f modul e ge ne s (S up . Table  218 \n2) w ith di stinc t pa tt ern s  o f ex pre ssion a c ross the  dif fe ren tia t i o n and betw een c on t rol a nd MYC T 58I -219 \nt2A- BCL2 con ditio ns (Fig ure  3a ) .  Gen e ontology  and  signa tur e en richme nt  ana l ysis demon s tr a t e d  220 \nthat t he  c ore gulat ed gen e mo dule s id e nt i f i ed b y PGCN A were highly  signi f i can tly  as socia t e d w ith  221 \ndiff er ent  f eatu re s rel a t e d  t o B -ce ll  di ff ere ntia tion,  ce ll  cyc le, MY C func t i o n , tran sla tion,  a nd  222 \nmetabo li s m ( F igure 3b a nd S up. T abl e 3) . Mod ule M1 wa s  enric hed for va riou s f e atur es of the B -cel l  223 \nsta te  inc luding  t arge ts  rep re s s ed  by BLI M P  and  wa s  expr e sse d a t  day  0 and 3 a nd progr e s s i vely  224 \nrepr ess ed up on di ff ere ntia tion i n a s i mi lar fa s hio n i n c ont r ol s and T58I -t2 A- BCL2  ex pre ssing c ell s .  225 \nModule  M2 w a s  e nric hed for g ene s  r eg ulat ed by NF κB re fl ec t i ng th e i mpac t  of the ini tia l  226 \nC D 4 0 : C D 4 0 L  a c t i v a t i o n  c o n d i t i o n s ,  e x p r e s s i o n  w a s  l o w  i n  d a y  0  B - c e l ls ,  i n d u c e d  i n  d a y  3 a c t i v a t e d  B -227 \nce lls and r e pre s sed on fur t her d i ffe re n t ia ti on in all conditio n s. I n con tra s t t o M1 a nd M2, t he  228 \nremain ing 14 modu le s  s howed dif fe r ential expr e ssi on be tw een con trol s  and T 5 8 I - t 2A- B C L 2 229 \nco ndition s. The s e sepa ra t e d betwe en mo dule s t ha t :  i )  w er e rep r e ssed in normal  di ff e r e n t i a tion bu t  230 \nwe r e  enhan c ed in T 5 8I - t 2A - B C L2  c ondi tion s (M4, M6 and M 1 3) , whi ch w ere en r ic hed f o r M Y C  231 \nregula te d gene signa ture s ; ii) module s tha t  w ere induc e d up on di ffe r e nt i a tion but sh owe d grea te r  232 \nex pr e s si on in con t rol th an T58 I-t2A -BCL 2 conditio n s  (M3, M5, M7, M16) , inclu ding gene s  link ed t o  233 \nXBP1 t arge t s , U PR and ER  s ec r e tory pa thway s; iii )  modul e s th at  were  ind uc ed upon  no r ma l  234 \ndiff er entia tion  but  s how ed en hanc e d expre ssi on in  T 58I -t2A - BC L2 c ondi tion s  (M8,  M10,  M 1 4) ,  235 \ninc luding  ge nes  link ed t o mit och ondr i al  metaboli sm a nd Ox P ho s  pat hwa ys; a n d iv) module s  tha t 236 \nwe r e  induc ed in cont rol d if f e r e n t i ati on  bu t  repr ess ed in T58I- t2A -BCL2 c o nd i t io n s  (M 9 ,  M 12  a nd 237 \nM15), i ncludi ng gen e s  l inked t o au topha gy  and c ellul ar quie sce nce .  238 \nModule s lin ked to MYC r eg ulat ed f ea tur es (M4, M6 an d  M13) exhi bi ted di sti nct ex pr e s si on pa tte rn s.  239 \nModule  M4 e nr i ched  f or p revi ou s ly  d efin ed signa t u r e s  o f MYC  ove re xpre ss ion, MTO RC1  an d  240 \nribo some bi ogene si s signa t ur es  w ere  s trongly induc e d by t h e  ac tiva tion  cond ition s  d riving  241 \ndiff er entia tion and we re s u st ained i n T58I- t2A -BCL2  conditi on s at  da y 6 a nd  to a les se r  e xt ent  at day  242 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 11\n13 bu t  w er e la rgel y rep re ssed  at  da y 20.  Modul e  M6 link ed  to MYC targe t s  r ela te d to  mitochond ria l  243 \nfunc t i o n w a s mode stly  induce d by activ ation con d it i on s  at day  3 but w as stron g ly induc ed in T58 I -244 \nt2A- BCL2 co ndition s a t day 6 and su s t a ine d to day 20. Modul e M13 link ed to a furthe r  grou p of  245 \ngen e s  rela t e d to ribo s ome s and t r a n s l a ti on, was mod e stly ex pre sse d in re st i ng B-cel l s , r epre s sed i n  246 \nall  conditi on s upon  a ctiva ti on an d w hile fur ther  r e p r e s sed  upon d if fe renti a t io n  in  control  c onditi on s  247 \nwa s  super -induc ed bey ond ini t ia l ex pr e ss ion le vel s upon d if f e r e n t i a tion in T 58I- t2 A-BCL2 c onditi on s.   248 \nThis t im e  c ourse analy s i s i ndica t e d tha t MYC T58I ac compan ied by  BCL 2 c o -e xpres s i on div erte d  249 \ndiff er entia tion t oward a  di stinc t ex pre ssion s ta te , dr i ving modules o f met ab olic  and tr a n s la t io n  250 \nrelat ed g ene s that we re no t phy s i ol ogic a lly e xpres s ed i n PC di f fer enti ation .  At th e sam e time , while  251 \nsupp re ssing f ea t ure s  of  t h e  P C sec re tory  s t ate , MYC  T58 I did  not  int erfe re w ith  r e pre s sion o f the B  252 \nc e l l - s t a t e  d u r i n g  p l a s m a  c e l l  d i f f e r e n t i a t i o n .  T h e  i m p a c t  o f  o v e r e x p r e s s e d  M Y C  w a s  n o t  f i x e d  a c r o s s  253 \nthe t i me c ou r s e,  nor  di d i t re fl ect an a mplific atio n o f  the p hysiol ogi cal di ff ere ntiati on pr ogram s .  254 \nIns te ad, th e im pac t of  MYC d eve lop ed  acro s s  th e c our s e  of the  dif fe ren tiati o n toward a di s tinc t 255 \nabe r r ant ex pr e s s ion sta te .  256 \nMB dom a in s  o f MYC s how d i ffere nti al c ontr i b uti on s to gene  re gul atio n duri ng PC d if f e r e n tia tio n.  257 \nThe  MYC  T A D ha s be en imp lica te d a s  c ritic al in tran s f o r mi ng act iv ity in variou s  ce llular model s , we  258 \nther ef ore n ex t  a ime d to te s t  t h e  cont rib ution t hat MB0, M BI o r  MB II d omain s o f the MY C TA D made  259 \nto the d iv ergen t pr ogramming  o f pe rtu rbe d PC dif fe ren tiati on. To  thi s e nd  we ge nera ted a  MY C WT -260 \nt2A- BCL2 v ector  (i .e.  w ith T58 no t T 58I ) and v ecto rs in  whic h MB0 , MB I or  MBI I were de l et ed in thi s  261 \nco nt e xt (S up. Fi gur e 3a) . We o btain ed si mila r  tran sd uction e ffic ienc ie s t o ou r  p r evi ous  r e sult s  (Sup .  262 \nF ig u r e  3b) . M Y C WT- t2A -BCL 2 recap i t ul a ted the  phe not y pic ef fec t ob s erved  fo r MYC T 58I . ΔMBI - t2A -263 \nBCL 2 w hich remove s  t he ph o s phod eg ro n s eque nc e enc omp a ss i ng  T5 8 s howe d  li t tl e di ff ere nc e i n  264 \npheno t y p e f rom MYC WT- t 2 A-BCL 2 . By  c ontra st Δ MB0- t2A -BCL2  show ed p arti al r eve rsal a nd ΔMB II -265 \nt2A- BCL2 sh owe d s i gnific an t re ver s a l of th e MYC -a s s oc i ate d phen o t y pe an d rev ersion tow ar d  266 \npheno t y pic pa t te rn s of con trol co ndit ion s  (F igur e 4 a-c , Sup. F igur e 3e) . Over ex pre ssio n of MYC a nd  267 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 12\nBCL 2 pr otein s  in c omp ari son  to  th e M S CV -back bon e con trol  wa s v alida ted  f or a ll t h ree  MYC  M B  268 \ndele t i on muta nt s  te st ed, i ncludi ng the e nha ncem en t o f MYC ex pr e ssion ex pec t e d  for  Δ MB I -t2A -BCL 2  269 \nwi t h  pho spho deg r o n  sequ enc e remov al . S imila r  lev e ls  o f BL I M P 1  e xpre s s i on w ere  o b serve d  270 \nfollow ing d if f e r e n t i ati on in a ll c onditi on s (Sup. F igur e 3c , d).  271 \nTo furth er a s s e ss the impa ct o f MB dele tion s , w e st u di ed gen e exp r e s sio n at day  6 (pla s ma b la st) a nd  272 \nday  13 (P C stage ) w hen div e r g ent pat t e r ns of  expre s s io n  c hang e s  w ere  o b serve d i n M Y C T58I - t2A -273 \nBCL 2 exp r e s sing c ell s  (Su p. Ta ble 4 ).  Multidimen sio na l scali ng  (M DS ) s h owe d tha t the  di s tin c t  274 \nco ndition s s epar ate d in  te rms  o f di ff ere ntia t i on –  sepa r a t in g cont rol di f fer enti a tion s a t d a y 6 f rom  275 \nt h o s e  a t  d a y  1 3 –  a n d  a c c o r d i n g  t o  M Y C  t r a n s d u c t i o n  a t  e a c h  t i m e  p o i n t  ( F i g u r e  4 d ) .   T h i s  w a s  276 \nco ns i st ent  w ith th e  hi e r a r c h al ord er o f phenoty pic impac t such th at MY C WT -t 2A-BCL 2  w a s  m o s t  277 \nd i s t i n c t  f r o m  c o n t r o l s  a t  b o t h  d a y  6  a n d  d a y  1 3.  Δ MBI shi f ted fr om b e ing mo st  simila r to Δ MB0 a t  278 \nday  6 to b ec ome mo st  s i mil ar t o MY Cw t  a t  day 1 3. Δ MB0 fe ll b e t w een MYC wt and c ontro l  279 \ndiff er entia tion  c onditi on s a t bo th day 6  a nd day 1 3,  and Δ M B I I  c l u s t e r e d  i n  c l o s e s t  p r o x i m i t y  t o  t h e  280 \nco nt rol s a t bo th tim e poin ts  but wa s m ore di verg ent a t day 13 than day 6 (Fig ure 4d) .  We  ne xt  281 \nas s e ss ed c hange s a t mo dular lev el . P GC N A agai n succe ss fully r e solv e d module s  o f g ene s enric h ed fo r  282 \nbiol ogica l proce s se s  r ela te d to MY C func tion (M1 , M4,  M 8  and M10) an d PC di ff e renti ation (M7 a nd  283 \nM9) th at w e r e  di f fer enti ally  exp r e s se d b etwe en  time poi nts (Fig ur e 4e a nd Sup . T able 5) . MY Cwt a nd  284 \nΔ MBI s h o wed simila r  pa tt ern s o f  mod ul e regula t io n tha t  di ve rged from tho s e  o b s e rve d in con trol s ,  285 \nwi t h e nr i ch ed bio logic al  proce s se s map p ing onto tho s e  ob s erve d fo r  di f fer entia l r e gula tion by  M Y C  286 \nT58 I c ondition s ( S up . Table  6).   Δ M B0, while  simila r  t o MYCwt  and Δ MB I, dive rged  in t he l ev el o f  287 \ninten sity of expre s s io n of b oth MYC up - a nd  d own- regula t e d mo d ule s. In c on t r a st, Δ M B I I show e d  288 \nmarke dly reduc ed ev idenc e of MYC -a s s o c iated modul e r egulatio n  and showed  gre at er expre s s i on o f  289 \nPC -a s s o cia ted modul e s  than o t he r  MYC  condition s (Figur e 4e ). Ther ef ore , b oth in the c ont ext o f  290 \ndime ns i ona lity r educ tio n usi ng MDS and  mo dular ex pr e s sion a na lysi s  u sing  PGCN A, the MB del e t i o n  291 \nmutant s s h owed c on si s t ent f ea ture s wi t h  a hie r a r c hy of impac t  on th e c ons e quence s  o f M Y C  292 \nove r e xpre ssion : Δ MB I I  >  Δ MB0 > Δ MB I.  293 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 13\nT o  c o n s i d e r  t h i s  a t  a  g e n e  l e v e l  w e  r e v i s i t e d  t h e  e x p r e s s i o n  p a t t e r n s  o f  i n d e x  g e n e s  l i n k e d  t o  B - c e l l  294 \ndiff er entia tion,  M Y C  impac t  a nd XB P1  func t i on. Here  we o bs erved  tha t w hile MYC  a nd C D2 295 \nex pr e s si on lev el s w ere s im ilar in all c on dition s (Sup . Fig ur e  4), a hi erarchy  w a s eviden t  ac ro ss bo th  296 \nMYC r espo n s iv e and  r e pr e sse d f ea ture s at indiv idu al gen e  l evel fo r sur face  pro tein s, tr a ns c r i pt i o n  297 \nfac t o r s, MYC targe t s, UP R/ E R gen e s a nd i mmunog lobulin gene s (Fig ure  5a -d a nd S up. Figure 4 ).   298 \nThe  ob serve d c h ang e s  in secr eto ry and im munoglobul in gene ex pre ssion sugge s ted t hat funct ional  299 \nsecr et ory ac t i vity w ould be di f fer enti all y impac ted. S upe r n a t a nts we re t es ted by  EL ISA for sec re t e d  300 \nIgM a nd IgG at da y 6 (Fi gure 5e ) a nd day  13 (Fig ur e  5f) fr om  t h e  dis t in ct cond i t i o ns . Concord ant w ith  301 \ngen e ex pre ssion , se cre tory ou t p ut wa s  de t e ct ed at d ay  6 and ro s e aro und 1 0-f old b y da y 13 in 302 \nco nt rol co ndi tion s .  S e cre tory r a te s wer e s i mila rly repr e s s ed in MYCw t and Δ M BI  c ondit ion s, w it h  303 \nrepr essi on mo s t evide nt  a t t he la ter ti me point wh en hig h outp ut i s e stabli shed unde r c ont r ol  304 \nco ndition s.  S ecr et ory ou t p ut remai ne d sub sta nt i ally red uc ed i n  Δ MB0 condi t i o ns  b ut signi fican tly  305 \nhig her t ha n MY Cwt, w hile for Δ MBI I con d ition s  th e M Y C e ff ect wa s  l o s t and ther e w as no s i gnific a nt  306 \ndiff er enc e f rom con t r ol c onditio ns .  307 \nThe  DCMW  m ot i f  a nd W135 a r e  critic al f or the e ff e ct of MYC MB II o n hu ma n  PC d iffer en tia t i o n .  308 \nGiven  the  appa rent  dep en d ence o n MBI I  we ne xt  add re s s ed  to  w hat  exten t thi s could  be  at tr i bu t e d  309 \nt o  t h e cor e co ns e r ve d  s e q uen c e  of  M BI I:  th e  1 32 - 1 35 aa DC M W  m ot i f  or  W 135  a l on e ,  t he m os t 310 \nhig hly conserve d re s i du e i n M BII whic h  sit s at t h e h e art o f th e pr edic t e d  TRR AP in ter actio n .[38 ]  311 \nSub stitu tion s DC MW / AA AA or  W135A  were  ge ne rat ed in  th e  c on text of  the MYC-t2 A- BCL2  312 \nco nfigura tion (Sup .  Figure  5a )  and c omp arably  high tran sduc tion ef fic ienc y wa s  v e r i f i ed for  t he  M B I I  313 \nmutant s ( Sup. Fi gur e 5b) . MYC, B CL 2 an d BL IMP1 pr ot e i n overex pre ssi on wa s  v a lidated  (S up. Fig u r e  314 \n5c , d). BCL2 and BL IMP1 expr e ss i on  wa s  equiva l ent be tw een c ondi tion s. MY C was s ub stan tially  315 \nove r e xpre ssed  rel a t i ve to con trol s in all c ondition s bu t wa s s i gnific a ntly hig her  i n MYCwt r ela tiv e to  316 \nΔ MBI I o r MB II  point  muta nt s  sugg e sting  t ha t  MB II  ma y in pa rt  con t r ibut e t o sta bility in thi s co n t e xt  317 \n( S u p . F i g u r e 5 d ).   Th e M Y C MB II -4a a mu tan t or  th e MBI I- W135A ag ain signi fic an tly i mpact ed on  the  318 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 14\npheno t y pic c hang e s  in duced b y MYC, su ch tha t  MYC  M BI I-4 aa or M BI I- W 13 5A  expres sing c ell s more  319 \ncl os e ly re s e mbl ed con trol  di ff eren tia tio ns than  MYCwt  con diti on s  (Fi gure  6a -c and  S up . Fig ure  5 e ).  320 \nOur  pr ev iou s  an aly se s i ndica t e d tha t ph eno t y pic e ff ec ts o f M Y C c onditi on s wer e cl osel y r e la ted to  321 \nthe ex t e n t  o f  ge ne ex pre ssion c h ange  in  t h e mod el.  I nd eed,  in m ul tidimen sion ali ty  s c aling  of  day 1 3 322 \ngen e ex pr e ssion dat a Δ MBI I a nd th e t w o MBI I mutan ts c lu s te red tog eth er with untra ns duce d  323 \nco nt rol s and sepa r a te fr om MYCw t c ondition s (Fi g. 6d ). Ab solu te numbe rs  o f signi ficantl y  324 \ndiff er ential ly exp r e s s ed gene s even a t  l en ient f o ld -chan ge t hr e shol ds wer e pro foundly  redu c ed fo r  325 \nΔ MBI I and the  two M BII mu tan t condi t i o ns  (Sup .  T able 7 ). P GC NA  c on firmed  th at a t mo dula r leve l  326 \nMYCw t  c ondi tion s aga in  dif fe red p ro f oundly  from  contr ol s  (Fig . 6e ) with c ons i s ten t biolog ica l  327 \ndiff er enc e s  to pr ev iou s  re sul t s  (S up . Tab le  8 and 9). Whil e  in Δ M BII a nd the M BII  mutant c ondi tion s  328 \nex pr e s si on of modul e s rel ate d to P C d iffe re nt i a tion an d immunog lobu lin ge ne expre s sion w ere  329 \nre st o red (Fi g . 6e) , module s  link ed with MYC- as so ciat ed g ene s w er e le s s  induc e d (Fig . 6e) . A s imi lar  330 \npatt ern w a s ob s e rve d for  gene s  i ndic a t iv e o f MY C impac t (Supple m enta l F i gure 6) . Inde ed no  331 \ns i g n i f i c a n t l y  di ff er ent ia l l y  e x p r ess e d g e ne s  w er e o bs er ve d  b e t w een  Δ M BII a nd e i ther MB II mut ant o r  332 \nbetwe e n the MB II mut ant condi tio n s  in direc t compa r i s on. Wh en c omparing Δ M BII or ei the r of th e  333 \ntwo MBI I poin t mutan t c ondi tion s to un tran sduce d cont rol condi t i on s  only  s ma l l number s o f  g ene s  334 \nremain ed di f fere nt i ally ex pre ssed. XB P1  remain ed mod estly  red uce d rela tiv e t o  c ont rol c ondi tion s  335 \nbut w as  al so s i gnific an tly re sto r e d rel ativ e to MYCw t  (F igure 7a ) . Po ten tial XBP1 targe t g ene s  336 \ninc luding  imm unoglobul in s  ge n e s  we re  not signi fic antly di f fer ent  be tween Δ MB II o r MB II mu tan t s  337 \nand control con dition s i ndic a t i ng th at fun ctio n of s e cr et or y  repr ogrammin g w as  re t a in ed unde r  338 \nthe se con d it i on s  (F igure 7 a) . S imila r ly , al most all MYC  in duce d gen e e xpre s s ion c hang e s  w ere l os t  i n  339 \nthe con tex t of Δ MB II or M BII  sub sti tut i o ns  wi th only  v ery f e w ge ne s s how ing retai ned s i gni fic an t  340 \nex pr e s si on di ff ere nc e s  (F igu re 7b and  S u p. Fig ure 6 a) . C on si s ten t  w ith t he  simi lar ity betwe e n Δ M B II 341 \nand MBII mu ta nt  c ondi tion s at gene e xpre s s ion le vel, EL ISA dem on stra ted comp arable Ig M  a nd IgG  342 \nsecr eti on in the se c ondi tion s rel a tive t o control a t day  6 and day  13 whil e in MY Cwt c ondition s t he  343 \nes timat ed p er c ell secr eto ry out put w a s a gain pr o foun d ly rep re s s ed  a t day 13 (Fi gure 7 c and  d).   W e  344 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 15\nco nclude  tha t eith er a  fou r amino ac id D CMW /AAAA s ub sti tu tion o r  a s i ngl e W135A s ub s titu t i o n  345 \nsu ffic ed to ph enoc opy d e le tion of  MBI I and ab roga te fea t u re s a s s oc ia ted  wi t h MYC  ov er expre s s i o n  346 \nin h uman B-c ell di f fer enti atio n .  347 \n  348 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 16\nDiscussion 349 \nDer egula t i on of MYC i s one o f th e  key dr i vers  o f  aggre s s i ve B-c e ll mal ignanc ie s. [3, 4, 9 2] At  th e s am e  350 \ntime pr ec i s e  con trol  o f MY C ex pre ssi on i s e ssenti a l to  co -ordi nat e c ell  grow t h  an d proli fer atio n w ith  351 \ndiff er entia tion in fun c tional lymph ocyt e expa ns io n of th e immune re s pon se .[ 5-7] H e r e  we have  352 \naddre s sed the ac ute impac t o f M Y C o v erexpre s s i o n du r in g h uman B -cell  di f fer entia t io n t o th e P C  353 \nsta ge. W e hav e ass e s s e d t h e  e xten t to whic h M Y C overex pr e ssion pr even ts o r  pe r t urb s P C  354 \ndiff er entia tion an d the ex t e n t  to whic h t h e i mpact of MY C overexp re s sion chan g es  a s the cellu l ar  355 \nco nt e xt prog res s e s  al on g t he dif fe ren ti ation  tr ajec t ory .  W e te s t ed thi s both  f o r MYC  ca rryi ng t h e  356 \nT58 I m ut a t i on found in aggre s sive ly mphoma, w hich sta bilize s MY C e xpre s s ion ,  a nd wild type  MY C  357 \nlac king  t h is  mut ation . [2 9, 32, 93] MY C d ereg ula tion dr ove g ene ex pres si on cha n ges con si sten t  w ith  358 \nprevi ou s  mo d el s  o f lympho ma  and  o the r c anc er  t y pe s .[7 ]  H o weve r th e e f fect  o f MYC ov e rexpre s s i o n  359 \nch anged a s  t he  und erlyi ng c ellula r di ffe ren t i a tion sta t e  s hi ft ed t ow a rd s  t he  PC s ta t e .  360 \nDi st in ct m o del s o f gen e  regu l ation  by o verex pr e ssed M Y C h ave  bee n prop os ed , s ug ge s t i ng eit her  361 \nac tion as  a  glob al tran sc ription al  ac t i vator  or  a s a  mor e spec i fic re gulat or o f d i stinc t bi olog ica l  362 \npathw ays . [11, 15-17 ] In ou r  mode l MY C expre ssi on is  su sta ined a t high  l evels fro m t he  activ ate d B-363 \nce ll sta ge onw ard and c or r ela t e s  w it h pr o gre ssive ly low er endogen o us  MY C l evel s in c ontr o l  364 \nco ndition s. Our  exp er i m ent s  incl uded BCL 2 co-ex pre ssi on to provid e r e s c ue f rom poten tial M Y C  365 \ndrive n apop to si s , [45,  46] and did  no t d ir e ctly di s tingu i s h t he po ten t ia l e ff ect s of B CL2 over -366 \nex pr e s si on al one . H owe ver,  th e v ario u s  M Y C mu tan t s  d emon st r ate d t ha t t h e  gen e expr e ssi on  367 \nch anges d epend e d on eleme n ts of MYC and the impa c t o f BCL2 on g en e exp r e s s ion was negl igibl e .  368 \nWe ca n the r e for e a ss ign the di st i nc t expre s s i on s ta te s to MYC a ctivity  w ith r e a s on a ble co n f i dence .  369 \nGene s that we re enh a nced  or  suppr e s s ed b y ove r ex pr e ssed MYC du ring  diff ere ntia tion w ere  370 \nsignific antly li nked  to known  b i ology an d  e nr i che d f or hallma rk  MYC  targ et s ge nes  and tho s e  w i th  371 \ncl assic al E -box  moti f s . How eve r, t he ex p re s s ion pa t t ern s linke d t o  M Y C over ex pre ssio n we r e n eit her  372 \nco nstant over t he cou rs e o f di ff eren tia tion nor  re adi ly expla ined as enha n ced  expre s s i on o f t h e  373 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 17\npreva iling pat te rn s  of ge ne expr es s i on in the con trol di ff ere nt i a tion s.  Rat her overex pr e ssed M Y C  374 \nes tabli sh ed a  di s tin ct exp r e s sion sta t e  du ring PC  di ff ere ntia tion .  375 \nP h y s i o l o g i c a l l y  M Y C  h a s  b e e n  i d e n t i f i e d  a s  a  r h e o s t a t  l i n k i n g  t h e  e x t e n t  o f  B - c e l l  a c t i v a t i o n  t o  c e l l  376 \ngrowth an d the  sub s equen t ca paci t y  for  sequ enti al  c ell div ision .[6 -8 ] In t h i s  se tti ng MYC s i ts a t t h e  377 \nhea rt of a  tra n s c r i pt i onal c ircui try w hich coordina te s a  bu rst o f c ell g r o w th  and division w i th  378 \nev entual  d i ffe r e n t i a tion an d se cre tory reprogr ammi ng (S up plemen tal  Fig ur e  7 a). [58 ] D u r ing  thi s  379 \nseq uenc e of  e vent s ex te rnal s i gnal s d r i ve B -cel l ac tiva tion and expre s s i on o f M YC. [7 , 9 4 -96] Th e se  380 \nsignal s  al so induc e e xpre ssion o f IRF4 wh ich in turn c ooper ate s with STAT3 t o  dr i ve exp r e ssi on of  381 \nPRD M1/BLI MP1 .[97 ,  98 ] Accu mulating  e xpre s s ion  o f  BLI MP1  ultima t e ly  le a ds  to  rep re s s i on  o f  382 \nMYC ,[20 ] c ur tai ling th e prolif era tive  bu rst . A t th e s a m e time BLIMP1 suppr e sse s t he  B -c ell ide n t i t y  383 \nt r a ns cr i pt i o n  f a c t or  PAX5 and  o the r fe at ure s linke d t o the ma ture B -c ell sta t e . [7 0, 71] Los s o f  P A X 5  384 \nrelea se s XB P 1  f rom P AX5 mediat ed rep re ssion. [23, 71 , 99 ]  Acting  toge t h e r  BLI M P 1 a nd XBP 1 c o -385 \nordinat e t h e high -l evel expre s s i o n of i mmu noglobuli n gen e s , t h e t r a n s i t i o n f r o m membrane  to  386 \nsecr et ed immunog lobulin enc oding tr an scrip t s , and the expa ns i o n o f the sec re tor y  a ppa ratu s. [23,  387 \n74-76,  89, 90,  100] The  s uppr e s s i on of MYC  a n d  e x p r e s s i o n  o f  n e g a t i v e  c e l l  c y c l e  r e g u l a t o r s  388 \nco or di na te s  the  sec reto ry tr an sitio n t o c ell  cyc le ex it. [21, 101,  1 02 ]  A d di t i onal  t ran scripti on fac tor s  389 \nsuch a s BA CH2 and BCL 6 c an add fur ther  leve ls  o f  c ont rol t o delay  di ff eren tia tion  and  facil i t a te  cla s s  390 \nswitchi ng or in terp o se th e c omplex  biolo g y of the g erminal ce ntr e re spo nse. [ 7 8 -8 0, 103-1 06]  391 \nThe  enfo rc ed e xpre s s io n of MYC that we hav e modelled  oc cur s at t he c ritical  junc ture w hen t h e  392 \nburst of p hy siologic al MYC  expre s s i on i s at it s  pe ak a nd then beg in s  t o be cur tail e d by the underlying  393 \nreorgani zing  t ran sc r ip tion  fa ctor n etw ork.[5 9 ] H e re d ere gulat ed MY C expre s s i on s u sta in s gene  394 \nex pr e s si on link ed  to  t h e  c ell g row t h  a nd me tabolic  p rogram s.  I mpo r ta n t l y  deregula t e d M Y C  395 \nex pr e s si on ha s lit tle impa ct o n the ex pr e ssio n of IR F 4  o r  BL IM P1 or th e tran sc r i ption al r egula tor s o f  396 \nB-ce ll  i den tity PA X 5  o r  EBF1.  Hence  th e r e gul ato ry circu itr y  c on t ro ll ing chang es in B -cell  ide n t i t y  397 \nrelat ed ge n e s  i s a t  mo st ma r g inally  a ff e cted .[22 , 70-73] Thi s con tra s t s  with a pr ofo un d im pact  o f  398 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 18\nMYC dere gulati on on th e initia tion o f s e cr et ory outpu t a nd secr et ory repr ogramm ing, w hich  is  399 \nc ou p l e d  wi t h da m p e n i n g of  XB P1  ex pr e s si on and  s uppre s s i on o f i mmunoglob ulin gen e  400 \nenha nceme nt .  MYC  h a s  p rev iou s l y been  id enti fied a s cap a ble o f bindi n g t o  th e XBP 1 p r om ot er  and 401 \nposi tivel y reg ulati ng exp r e s sion in c anc er  model s . [107 , 108 ] Inde e d, MYC  i s o f ten der egul a ted i n  402 \nag gressive  PC malig na nci e s  in whic h XBP 1 i s e xpre ssed . [10 9]  The con tra st with t he  supp re ssive  403 \nef fec t of  MYC ov erexpr es s i on  on  XB P1  a nd s ecre tory pr ogramming  t ha t  w e h ave  observ ed in  B -cel l  404 \ndiff er entia tion c ould b e e xplai ne d by diff ere ntial re crui tment o f c o fact or s  by MYC in t hi s  s e t ting .  405 \nSuc h diff eren tial  r e c ruitmen t would  have p arallel s in th e c omplex  web  of poten t ial MYC i nt e ra ction s  406 \nobs erved  acro s s ot h e r  c ell  s y stem s. [1 8] W e h a ve n o t  ad dre sse d to what  e x tent the  del ay i n  407 \nsecr et ory pr ogrammi ng i s d riven by  repr essio n o f XB P 1 a s  oppo se d to an impa c t on immun oglobuli n  408 \ngen e expre s sion and pro tein l o ad. Howe v er, murine c onditi onal  knock out model s argue  f o r  a  critica l  409 \nrole of XBP1  in tra n s c r i pt i onal enh anc e ment o f  immunog lobulin  ge ne ex pr e s s i on.[90 ] Henc e,  we  410 \nfav or  a mo del  in whi ch de regul a ted MYC s upp re s se s  XB P1  w hic h i n t urn  lea d s  to r educe d  411 \nimmuno globuli n gene e xpre s s ion a n d dela y in sec ret ory r eprogrammi n g. This regul a tor y  412 \narrange m ent w ould be c on s i st ent with the r ol e of MY C in the pha s e of lymph o cyte ac tivation a n d  413 \ngrowth prio r t o  dif fe ren tia t i o n, [5-8 , 1 7] with MY C de regula tion  simult ane o us l y bia sing gen e  414 \nex pr e s si on to ward an abol ic pa thwa ys a nd  aw ay from p athway s  a s s oc ia ted wit h ex por t o f cellu l ar  415 \nmateri al  a s  s ecre ted i mmunoglob ul in. I f the tran sc r ip tio nal ci r c ui tr y  cont rolling  P C dif fe ren tiati on i s  416 \nvi ewed as t w o int er c onn ec ted fe edf o r w ard loop s, o ne  r eg ulating the pul s e of g r ow t h  a nd  417 \nprolif era tion a nd the  ot her  t h e  de lay ed t r a n s iti on to a  sec re t o ry fa te , t h e n the  de regula t e d  418 \nove r e xpre ssion  of  MYC  in ac tivated  B - cells d isrupt s dif fe ren tiati on by un cou plin g both of th ese  419 \ninterco n nect ed f e ed for w a rd loop s (Su pp l emental Figu re 7 b) .  420 \nThe  MYC TAD harb or s  evo luti onarily  c o ns e r v ed amino ac id seq uenc e s e ssenti a l  for MYC -media t e d  421 \ntran sforma tion . [18,  2 6, 27 ]  O ur  d ata d emons tr a t e  a s i milar  de p endenc e o n MB domain s fo r t h e  422 \ntran s c rip tional  i mpact  o f MYC  during  B -cell di ffe r e n t i a tion.  In  thi s  se tting  Δ M B I re s u lt e d i n  r e lat i v e l y  423 \nenha nced M Y C p rote in ex pr e ssi on simila r to T 58I and  con si ste nt  wi th abl a tion of the pho s p hode gr on  424 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 19\nseq uenc e .[30 ]  Δ MB0  led  t o a  par tial  suppre s s i on  of  XBP1  a n d  s e c r e t o r y  r e p r o g r a m m i n g  a n d  a  425 \nreduc ed abili t y  t o  induce  a se lect sub s e t of g ene s po s i tiv ely regu lat ed by MYCw t . By co nt ra st Δ M B II 426 \nor se lectiv e amino a cid mut ation s , inc luding the singl e point s ub sti tuti on W135 A, rend ered MY C al l  427 \nbut non func tional .  Th e MB II doma in i s  p art i cula r ly  not able f o r r ec r ui t m e nt  of TR RAP and a ssoci a t e d 428 \nhist one ac etyl t ran s f e ra s e c omplex e s. [26 , 33, 34 ]  Th e D CMW mo t i f a nd W135 sit at t h e  he art of t h e  429 \npredic te d in terfa ce  with TRR AP . [38 ] Ne ve r thel e s s , th e d egre e t o whic h MB II  d el eti on, mu tati on o f  430 \nDCM W ,  or W 1 35 i mpact ed wa s  unex pe cted.  Whil e the MB II d omain i s  c r itic al for MYC medi a t e d  431 \ntran sforma tion  in fi brobla s t s ,  th e W135 A mutatio n had lit t l e e ffe ct in thi s con t ex t  and  an  W135E  432 \nsub s ti tu tion wa s ne eded  to ph enoc opy MBII d ele tion. [35 ] MB II mu tant s  c a n bi n d to phy siolog ica l  433 \nM Y C  t a r g e t s  i n  U 2 O S  c e l l s . [ 37 ]  F u r t h e r m o r e ,  Δ M BII MY C  c ould par tially  compens at e for MY C wt i n  434 \ndro s o phila dev el opmen t. [110 ] T ha t a ll t hr e e ver s i on s  o f MYC ta r g eting MB II in our model produc e  435 \nthe s ame e ff ec t provide s ev idenc e fo r  a critic al depe nd enc e on MBI I in the  contex t  of M Y C  436 \ndereg ula tion during PC  di ffe r en tia t i on. Othe r stu die s h a ve iden ti fied  the  int er action o f M Y C w it h  437 \nW D R 5  v i a  t h e  M B I I I b  r e g i o n  o f  t h e  M Y C  T A D  a s  c r i t i c a l  f o r  r e c r u i t m e n t  o f  M Y C  t o  c h r o m a t i n ,  438 \ninc luding  in Burki tt lymp homa . [111-11 4 ] The MB II Ib r eg ion alo n g with the MY C DNA bindi ng domai n  439 \nare i ntac t in  Δ M BII  and  MBI I  mutan t s.  Howe ver,  i t re main s to b e te s ted w h ethe r the  pr o f o un d  440 \nimpa ct on  ove r e xpre s s ed MY C in B-c ell  diffe ren tiati on is ex plain ed by  a  requir emen t  f or MBI I to  441 \nsuppo rt rec r uitmen t o f  exc e ss MYC  t o t a rget g ene s or  to d riv e sub seq uen t gen e r eg ulation . A r ec ent  442 \nst u d y ha s  s h o wn tha t in U 2OS cel l s  hi gh leve l MYC  st a bili ze s  and extend s lo ng-range  c h r oma t i n  443 \ninter ac t i on s.[11 5 ] I f MYC DNA bi nding i s retai ned  in t he MB II mut an ts, a po s sible e xplana tion fo r t h e  444 \ng l ob a l  i m p a ct  of  M BI I m u t a nts  c o u ld be  a n es s e nt i a l  r o l e f o r  MB I I i n  me d i at i n g  s u c h  c hr o m a t in 445 \nef fec ts .  446 \nIn summary we hav e te s t e d a mode l  that allow s  the acu te e f f ec t o f MYC  ove r e xpre ssion i n  447 \nco opera tion with BCL2 t o be studie d  a s  hum an B -ce ll s  di ff eren tia te to t h e P C s tat e. Thi s  448 \ndemon str ate d t ha t  MYC ove rex pre ssio n  did not t ran s for m  B -c ell s un d er  c ondi ti ons p e r mi s sive fo r 449 \ndiff er entia tion .  In st ead, MYC over ex pr e s sion s el ec tive ly impac t e d on  th e meta boli c and s ec r eto r y  450 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\n   \n \n 20\nco mponent s o f r eprogram ming,  le aving  t he  eleme n t  of  di ffe ren tia ti on ass oc iat e d with r epr e ss i on o f  451 \nthe B -ce ll s t a t e  larg ely  intact. T he TA D d omain MB0  and MBI I we r e  nec e ss ary for  MYC ef fec ts , and a  452 \ncritic al de p endenc e on MBI I could be r e sol ved to th e DCMW mo tif a nd W135.  453 \n 454 \nData Availability Statement 455 \nThe  pr i mary da t a set s a re av aila bl e at the  Gene  Expre s s i on O m nibu s GSE262809 456 \n 457 \nAcknowledgements: 458 \nWe tha nk  Erica  Wilson  fo r  t e ch nic al su pp ort and a dvic e a nd U lf K lein a n d Richa r d  Bayli ss f or advi ce, 459 \nsuppo rt  and c r i tic al r ev iew of t h i s  w ork.  460 \n 461 \nFunding Acknowledgements: 462 \nThis work wa s  s u p ported  by  the  Ell a D ic kin son Cha ritabl e  F o undati on Sc holar s h ip P r og ra m  463 \n(sch ola rship  recip i ent s: P . V.,  B.K. , A .M. ; inve s t iga t o rs : R .O., G.  D.,  R .T .)  a nd  an I ntercal a te d Deg r e e  464 \n1022  Award, P atho logi cal Soc ie ty of Great Br it ai n and N or the rn Ir ela nd and  EXSE L scho lar s hip ,  465 \nUniver sity o f L e ed s S chool  of  M e d icine  to E. P. . Thi s w ork wa s  suppor ted  by C ance r  R e sea rch UK  466 \nprogram gran t  (C78 45 / A29 212) (M. C, G. D .,  a nd R .T) and C a ncer R e sea rch  U K a nd FC AEC C an d AIR C  467 \nunder th e Acc ele rat or Award Program ( C355 / A2681 9 ). Thi s r e s earc h i s  fund e d i n part (M .C .) by  t h e  468 \nNatio nal In st i t ut e fo r  Healt h a nd Ca re  Res earch  (N I HR) Leed s Biom edic al Re sea r c h C e nt r e (BR C )  469 \n(NIHR2 03331). R . T., G. D., a re suppo rt e d in p art  by the Na tiona l In s t i t u te f o r  Hea lth and C a r e  470 \nRes earch  ( NIH R) L eed s Biome di cal Re se arc h Cen tr e  ( BRC ) (N IH R2 03331) .  D. J.H . was s u ppo rted by  a  471 \nf e l l ows hi p f ro m  Ca n ce r R ese a r c h U K  ( CR U K ) ( RC CFE L ∖ 10 0072) a nd  rece ive d c ore funding  fr om  472 \nWellc ome (2031 51 / Z / 16 / Z) to the W ell c ome-M RC Cam br id ge S tem C e ll In sti t u te  a nd from th e CR UK 473 \nCamb r idg e Cen tre (A2511 7) . D .J. H  i s  s upport ed by t he  Na t i on al In sti tu te f or H e alth and C ar e  474 \nRes earch  ( N IHR) Cambri dg e Biomedi ca l Resea r c h Cen tr e (BR C -1215 - 2 0014 ). Th e view s  expre s s ed  are  475 \ntho se o f t he  au thors an d not nec es sarily  t h ose of the N IHR o r  th e De par tment o f Heal th and S ocia l  476 \nCar e. 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Ranun colo, S .M. , et a l ., Bcl -6 me dia t e s  th e germin al center B cell  phe no ty pe an d 704 \nly mphom age ne sis  thro ug h  tr a nsc r ip t io na l repres s i o n o f th e DN A- da mag e s e n sor A TR.  Nat 705 \nImmunol , 2007.  8 (7 ): p. 70 5 -14.  706 \n107.  Zha o, N., et al . , P harm ac ologic al t arge tin g o f MYC -r e g ula ted I RE1/XB P1 pa thw ay su ppre s s e s 707 \nMYC-driv e n bre as t canc er.  J Cli n Inve st , 2018 . 128(4): p .  1283-129 9.  708 \n108.  Xie,  H . , e t al.,  I R E 1 a l pha  RN as e- d e p e nd ent  l i p i d  ho m eo st as is  p r om ot es  su r v i v al  in  M yc -709 \ntran sforme d  canc ers.  J Cli n In ve st ,  2018 . 128 (4): p . 1300-13 16.  710 \n109.  Sha f f e r, A . L. , e t  a l ., IR F 4 ad dic tion i n mul t iple myelo ma .  Na tur e, 2008 . 454(720 1 ): p. 226-31.  711 \n110.  Sc hwink endorf , D. and P . Galla n t , The c o nser ved M y c b ox 2 a nd Myc  box  3 region s  are 712 \nimpo rtan t, b u t  n ot e ssen tia l , for Myc fun ction in vi v o.  G ene ,  2009. 436(1 -2): p . 9 0- 100. 713 \n111.  Thoma s , L.R ., e t  al. , In te r ac tio n w ith W DR5 pr o mo te s  t ar ge t  g ene rec o gni tion  and  714 \nt u m o r i g e n e si s b y  M Y C .  Mol Cell, 2 015 . 58 (3) : p . 440-52 .  715 \n112.  Thoma s , L.R ., e t  al. , Th e MYC- WDR5 N e xus a nd Ca nce r .  Ca nce r  R e s, 2 015. 75 (19) : p. 401 2-5.  716 \n113.  Sun , Y., e t  a l., W DR5 Sup por t s an N -Myc  Transcrip tion al  Com ple x Tha t  Drive s  a  717 \nProt umori gen ic Ge ne  Exp r e ssi on S ign at ur e in N eu rob la stom a.  Canc er R e s , 2015. 75 (23): p . 718 \n5143-54.  719 \n114.  Thoma s , L.R ., e t  al. , In te r ac tio n o f th e on cop r ote i n tr a ns c r i p tion fac tor MYC  with i ts 720 \nch r om ati n co fa ct or WDR5 is  e ssen tial  f or t um or  m ain ten anc e.  P r oc  N atl Aca d Sc i U  S A, 20 19. 721 \n116(5 0): p . 2526 0-25268.  722 \n115.  Se e, Y.X. ,  K. Che n,  and M .J. F ullwo od , MY C ov erexpres si on lea d s  to increa se d c hro mati n 723 \ninterac ti on s  a t  supe r-e n ha nce r s a nd  MYC  bind i ng site s .  G e nom e Re s, 202 2. 32 (4): p.  629-724 \n642.  725 \n 726 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nDay 6 Day 13 Day 20\n0.00\n0.01\n0.02\n0.03\n0.04\n10\n20\n30\nIgM ng/ml/cell\nFigure 1\na b\nc d e\nf\ng i\nDay 3 Day 6 Day 13 Day 20\nUntransduced\nMSCV-empty\nMYC-t2A-BCL2\nUntransduced\nMSCV-backbone\nT58I-t2A-BCL2\nDay 13\nMSCV-backbone T58I-t2A-BCL2\nCD20\nCD19\nDay 3\nDay  6\nDay 13\nMSCV-backbone T58I-t2A-BCL2\nCD38\nCD27\nDay 3\nDay  6\nDay 13\nMSCV-backbone T58I-t2A-BCL2\nCD138\nCD38\nDay 3\nDay  6\nDay 3 Day 6 Day 13 Day 20\n0\n50\n100\n150\nCD2 positive cells (%)\nMSCV-backbone\nT58I-t2A-BCL2\n✱✱ ✱✱✱ ✱ ✱✱\nDay 3 Day 6 Day 13 Day 20\n0\n50\n100\n150\nCD19 positive cells (%) ns ns ✱✱ ✱✱✱\nDay 3 Day 6 Day 13 Day 20\n0\n50\n100\n150\nCD27+CD38+ cells (%) ✱ ✱✱ ✱✱✱✱ ✱✱✱\nDay 3 Day 6 Day 13 Day 20\n0\n50\n100\n150\nCD38+CD138+ cells (%) ns ns ✱✱✱ ✱✱✱✱\nDay 21 Day 31\n0\n2\n4\n6\n8\n10\nEdU+Ki67+ cells (%) nsp=0.09\nDay 21 Day 31\n0\n5\n10\n15\nEdU-Ki67+ cells (%) Untransduced\nT58I-t2A-BCL2\nnsp=0.07\nUntransduced\nT58I-t2A-BCL2 T58I-t2A-BCL2\nMSCV-backbone\nDay 6 Day 13 Day 20\n-1\n0\n1\n2\n3\n4\nIgM ng/ml/cell Untransduced\nMSCV-backbone\nMYC-t2A-BCL2\nns ns ns\nDay 6 Day 13 Day 20\n0.000\n0.002\n0.004\n0.006\n1\n2\n3\n4\n5\nIgG ng/ml/cell\nFigure 1. Acute MYC and BCL2 overexpression drives aberrant B cell differentiation. a, Graphical representation of the in\nvitro differentiation and transductions model system. This shows general phases by day of culture at the top, followed by\nsummary culture conditions and biological processes below. Associated transcription factors (TF) are depicted on the left\nand phenotypic markers on the right. b, Flow cytometric quantitation of percentage CD2 positive cells for MSCV-backbone\nand T58I-t2A-BCL2 conditions at indicated time points. c, d, e, Representative flow cytometry plots of CD19 vs CD20, CD27\nvs CD38 and CD38 vs CD138, respectively, for MSCV-backbone and T58I-t2A-BCL2 conditions at the indicated time points. f,\nPercentages of CD19 positive cells (left), CD27+CD38+ cells (middle), and CD38+CD138+ cells (right) for the indicated\nconditions at the indicated time points. g, Percentages of EdU+Ki67+ cells at the indicated time points for untransduced and\nT58I-t2A-BCL2 samples after 1 hour of pulse EdU incorporation. Data shown for the MSCV-backbone and T58I-t2A-BCL2\nconditions are pre-gated to CD2+ populations (c, d, e, f, g) i, Quantification of total human IgG antibody secretion (left) and\nIgM antibody secretion (right) on day 6, day 13 and day 20 for the indicated conditions. Data are representative of at least\ntwo independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed Student’s\nt-test, (b, g). One-way ANOVA (f): ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.\nCD40L \nFab2 anti-Ig\nIL2/IL21\nB-cell state\nDivideGrow & \nPC state\nIL2/IL21 IL6/IL21\nAPRIL\nIL6\nAPRIL\nPAX5\nMYC\nIRF4/BLIMP1/XBP1\nCD19hi/CD20hi\nCD19lo/CD20lo\nCD27hi/CD38hi/CD138hi\nPhenotypeTF\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nFigure 2\nUMAP1\nUMAP2 Controls\nMYC\nDay 3\nDay 6\nDay 13\nDay 20\nDay 0\nMSCV-backbone\nT58I-t2A-BCL2\nUntransduced\nMSCV-backbone\nT58I-t2A-BCL2\nUntransduced\nMSCV-backbone\nT58I-t2A-BCL2\nUntransduced\nT58I-t2A-BCL2\nUntransduced\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nCD19 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nMS4A1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nCD27 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nCD38 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nSDC1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nCD2 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nMYC expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nPAX5 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nEBF1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nPRDM1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nIRF4 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nXBP1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nDERL3 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nERLECC1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nHERPUD1 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\n20\nDays\nTXNDC5 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nFICD expression\nb\nc d e f\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nIGLC7 expression Untransduced\nMSCV-backbone\nT58I-t2A-BCL2\nUntransduced\nMSCV-backbone\nT58I-t2A-BCL2\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nIGLC7 expression Untransduced\nMSCV-backbone\nT58I-t2A-BCL2\nUntransduced\nMSCV-backbone\nT58I-t2A-BCL2\n0 3 6 9 12 15 18 21\n0\n2\n4\n6\n8\n10\nDays\nTERT expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nJAG2 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nFABP5 expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nKISS1R expression\n0 3 6 9 12 15 18 21\n0\n5\n10\n15\nDays\nTRAP1 expression\nFigure 2. MYC T58I selectively perturbs gene expression in B-cell differentiation. a, Uniform Manifold Approximation\nProjection (UMAP) of differentially expressed genes for the indicated conditions and time points. b, c, d, e, f, Normalised\nRNAseq expression values (y-axis) of selected genes across the differentiation time course (x-axis days) as indicated for\nuntransduced, MSCV-backbone and T58I-t2A-BCL2 conditions. Gene expression is shown as indicated in the figure for b,\nMYC and CD2; c, surface proteins linked to immunophenotyping; d, transcription factors; e, known MYC targets; and f,\nXBP1 targets. Data are representative of two independent experiments with a total of n=1-4 samples per time point and\ncondition.\na \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nFigure 3\nPGCNA:\n MSCV-backbone T58I-t2A-BCL2 Untransduced\nD0 D3 D6 D13 D20\nb\nFigure 3. Expression network level analysis of MYC T58I on B-cell differentiation. Parsimonious Gene Correlation Analysis\n(PGCNA) was used to define modules of coregulated genes. a, Heat map of module level gene expression with expression\npatterns averaged across all genes per module on a z-score scale (-2.14 blue to +2.09 red). Modules are hierarchically\nclustered on the left and module number and indicative summary terms of associated ontologies are shown on the right.\nTime points are indicated in the grey to black bars at the top of the figure going from Day 0 (D0 left) to Day 20 (D20 right).\nIndividual conditions are identified using the code as indicated at the top of the figure: yellow (MSCV-backbone), purple\n(T58I-t2A-BCL2), green (untransduced). b, Correlation plot of gene signature enrichment analysis for the 16 PGCNA-derived\nmodules showing analysis for MsigDB Hallmark signatures. Enrichment is illustrated on a z-score scale from blue to red.\nWith hierarchical clustering for enriched signatures and module enrichment patterns above and to the right. Module\nnumbers and summary designations are identified to the right.\na \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nFigure 4\nDonor 1\nDonor 2\nDonor 3\nDonor 4\nDay 6\nDay 13\nUntransduced\nMSCV-backbone\nWT\nΔMB0\nΔMBI\nΔMBII\nMYC-\nt2A-BCL2\n3.01.50.0-1.5-3.0\nUntransduced\nMSCV-backbone\nMYC WT-BCL2\nMYC ΔMB0-BCL2\nMYC ΔMBI-BCL2\nMYC ΔMBII-BCL2\nUntransduced\nMSCV-backbone\nMYC WT-BCL2\nMYC ΔMB0-BCL2\nMYC ΔMBI-BCL2\nDay 6\nDay 13\nMYC ΔMBII-BCL2\nΔMB0\nΔMBII\nΔMBI\nMDS1\nMDS2\nMSCV-backbone\nWT\nΔΜΒ0\nΔΜΒΙ\nΔΜΒΙΙ\nMYC-\nt2A-BCL2\nUntransduced\nDay 13\n0\n50\n100\n150\nCD27+CD38+ cells (%)\nUntransduced\nMSCV-empty\nMYCisoform-t2A-BCL2\ndelMB0\ndelMBI\ndelMBII\nb \nc d \ne\nDay 13\n0\n50\n100\n150\nCD27+CD38+ cells (%)\n✱\n✱✱✱✱\n✱\n✱✱✱✱\n✱✱✱✱\nDay 13\n0\n20\n40\n60\n80\n100\nCD38+CD138+ cells (%)\n✱✱\nns\nns\n✱✱\n✱✱✱✱\nDay 13 MYC-t2A-BCL2\nWT ΔMB0\nCD38\nCD27\nΔMBI ΔMBIIMSCV-backbone\nDay 13 MYC-t2A-BCL2\nWT ΔMB0\nCD138\nCD38\nΔMBI ΔMBIIMSCV-backbone\nFigure 4. Deletion of MYC TAD MB0, MBI and MBII have differential effects on MYC driven phenotypic and expression\nfeatures. a, b, Representative flow cytometry plots at day 13 for control MSCV-backbone, MYCwt, MB0, MBI and MBII\nconditions as shown for a, CD27 vs CD38, and b, CD38 vs CD138. c, Summary of flow cytometrically defined percentages of\nCD27+CD38+ cells (left) and CD38+CD138+ cells (right), at day 13 for the indicated conditions. Data are representative of\nthree independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed\nStudent’s t-test: ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001. Data shown for the transduced conditions are\npre-gated to CD2+ populations (a, b, c,). d, Multidimensional Scaling (MDS) of differentially expressed genes at the day 6\n(green) and day 13 (purple) time points, and controls, for the indicated samples as illustrated in the figure. e, PGCNA\ndefined modules of coregulated genes shown as a heat map of module level gene expression with expression patterns\naveraged across all genes per module on a z-score scale (-3 blue to +3 red). Modules are hierarchically clustered on the left\nand module number and indicative summary terms of associated ontologies are shown on the right. Time points are\nindicated as grey day 6 and black day 13. Samples from different donors are illustrated in the blue to orange color code,\nand for individual conditions with color code identified in the figure.\na \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nFigure 5\na \ne\nDay 6 Day 13\n4\n6\n8\n10\n12\n14\nCD19 expression\nDay 6 Day 13\n4\n6\n8\n10\n12\n14\n16\nMS4A1 expression\nDay 6 Day 13\n9\n10\n11\n12\n13\n14\n15\nCD27 expression\nDay 6 Day 13\n4\n6\n8\n10\n12\n14\n16\nCD38 expression\nDay 6 Day 13\n0\n5\n10\n15\nSDC1 expression\nDay 6 Day 13\n2\n4\n6\n8\n10\n12\nSDC1 expression Untransduced\nMSCV-backbone\nWT-t2A-BCL2\nΔΜΒ0-t2A-BCL2\nΔΜΒΙ-t2A-BCL2\nΔΜΒΙΙ-t2A-BCL2\nMSCV\nWT\nΔΜΒ0\nΔΜΒΙ\nΔΜΒΙΙ\nMYC-\nt2A-BCL2\nUntransduced\nDay 6 Day 13\n4\n6\n8\n10\n12\nPAX5 expression\nDay 6 Day 13\n5\n6\n7\n8\n9\n10\nEBF1 expression\nDay 6 Day 13\n10\n11\n12\n13\n14\n15\nPRDM1 expression\nDay 6 Day 13\n11\n12\n13\n14\n15\n16\n17\nXBP1 expression\nDay 6 Day 13\n11\n12\n13\n14\n15\n16\nIRF4 expression\nf\nMSCV-backbone\nWT\nΔΜΒ0\nΔΜΒΙ\nΔΜΒΙΙ\nMYC-\nt2A-BCL2\nUntransduced\nDay 13\n0\n50\n100\n150\nCD27+CD38+ cells (%)\nUntransduced\nMSCV-empty\nMYCisoform-t2A-BCL2\ndelMB0\ndelMBI\ndelMBII\nb c d \nDay 6\n0.000\n0.002\n0.004\n0.006\n0.008\n0.010\nIgG ng/ml/cell\nns\n✱\nns\n✱✱✱\n✱✱\nDay 13\n0.00\n0.02\n0.04\n0.06\n0.08\n0.10\nIgG ng/ml/cell\nns\n✱✱\nns\n✱\n✱✱✱✱\nDay 6\n0.000\n0.005\n0.010\n0.015\n0.020\n0.025\nIgM ng/ml/cell\nns\n✱\nns\n✱\nns\nDay 13\n0.00\n0.05\n0.10\n0.15\n0.20\n0.25\nIgM ng/ml/cell\nns\n✱✱\nns\n✱\n✱✱\nDay 6 Day 13\n0\n2\n4\n6\n8\n10\n12\nTERT expression\nDay 6 Day 13\n2\n4\n6\n8\n10\n12\nJAG2 expression\nDay 6 Day 13\n4\n6\n8\n10\n12\nSORD expression\nDay 6 Day 13\n2\n4\n6\n8\n10\nKISS1R expression\nDay 6 Day 13\n8\n9\n10\n11\n12\n13\nTRAP1 expression\nDay 6 Day 13\n8\n10\n12\n14\n16\nDERL3 expression\nDay 6 Day 13\n9\n10\n11\n12\n13\nERLEC1 expression\nDay 6 Day 13\n10\n12\n14\n16\nHERPUD1 expression\nDay 6 Day 13\n6\n7\n8\n9\n10\nFICD expression\nDay 6 Day 13\n13\n14\n15\n16\n17\n18\nTXNDC5 expression\nFigure 5. MB0, MBI and MBII deletion impacts on indicative gene regulation and on functional secretory output. Violin\nplots of log2 normalised RNAseq expression values of individual genes plotted at day 6 (left side of graphs) and day 13 time\npoints (right side of graphs) for the indicated conditions (top right of figure). Genes shown are indicated to the left of each\ngraph for: a, surface antigens; b, transcription factors; c, MYC targets; and d, XBP1 targets . Data are representative of two\nindependent experiments with a total of n=4 samples per time point and condition. Quantification of e, IgM and f, IgG\nantibody concentration normalized per cell at day 6 (left graph) and day 13 (right graph) for conditions as indicated to the\nlower right of the figure. Data are representative of two independent experiments. Bars and error represent mean and\nstandard deviation (SD); Unpaired two-tailed Student’s t-test: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001;\n**** P < 0.0001.\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nFigure 6\n1.50.0-1.5\nDonor 1\nDonor 2\nDonor 3\nUntransduced\nWT\nΔMBII\nMYC-t2A-BCL2\nMBII-4aa mut\nMSCV-backbone\nMBII-W135ADay 13\nPGCNA:\nUntransduced\nMSCV-backbone\nWT\nΔMBII\nMBII-4aa mut\nMYC-\nt2A-BCL2\nMBII W135A\nDay 13\nMDS1\nMDS2\nd\nWT\nΔΜΒII\nMBIΙ-4aa mut\nMYC-\nt2A-BCL2\nMSCV-Backbone\nUntransduced\nMBIΙ-W135A\nDay 13\n0\n20\n40\n60\n80\nCD38+CD138+ cells (%)\nUntransduced\nMSCV-empty\nMYCisoform-t2A-BCL2\ndelMBII\nMut 31\nMut 33\nns\n✱✱✱\nns\nns\nns\n✱\n✱\n✱✱\nDay 13\n0\n50\n100\n150\nCD27+CD38+ cells (%)\nns\n✱\n✱\n✱\n✱\nDay 13\n0\n20\n40\n60\n80\n100\nCD38+CD138+ cells (%)\nns\n✱✱\n✱\n✱✱\n✱✱✱\nDay 13 MYC-t2A-BCL2\nWT ΔMBII\nCD138\nCD38\nMBII-4aa mutMSCV-backbone MBII-W135A\nDay 13 MYC-t2A-BCL2\nWT ΔMBII\nCD38\nCD27\nMBII-4aa mutMSCV-backbone MBII-W135A\nb \nc \ne \nFigure 6. Point mutation of the DCMW motif and W135 phenocopy MBII deletion. Representative flow cytometry plots at\nday 13 for the indicated conditions above each dot plot for: a, CD27 vs CD38; and b, CD38 vs CD138. c, Flow cytometric\nquantification of percentage data CD27+CD38+ cells (left), and CD38+CD138+ cells (right) at day 13 for the conditions\nindicated to the right of graphs. Data are representative of at least three independent experiments. Bars and error\nrepresent mean and standard deviation (SD); Unpaired two-tailed Student’s t-test: ns, not significant; * P < 0.05; ** P <\n0.01; *** P < 0.001. Data shown for the conditions tested, apart from the untransduced, are pre-gated to CD2+ populations\n(a, b, c). d, Multidimensional Scaling (MDS) of differentially expressed genes at day 13 for the indicated samples. e, PGCNA\ndefined modules of differentially coregulated genes at day 13 shown as a heat map of module level gene expression with\nexpression patterns averaged across all genes per module on a z-score scale (-1.5 blue to +1.5 red). Modules are\nhierarchically clustered on the left and module number and indicative summary terms of associated ontologies are shown\non the right. Samples from different donors are illustrated in the blue to orange color code and for individual conditions\nwith color code identified above the figure.\na \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint \n\nWT\nΔΜΒII\nMBIΙ-4aa mut\nMSCV-Backbone\nUntransduced\nMBIΙ-W135A\nDay 13\n6\n8\n10\n12\nCD19 expression Untransduced\nMSCV-backbone\nWT-t2A-BCL2\nΔΜΒII-t2A-BCL2\nMBII-4aa mut-t2A-BCL2\nMBII-W135A-t2A-BCL2\nb \nDay 13\n0.00\n0.02\n0.04\n0.06\n0.08\nIgG ng/ml/cell\nUntransduced\nMSCV-backbone\nWT-t2A-BCL2\nΔΜΒII-t2A-BCL2\nMBII 4aa mut-t2A-BCL2\nMBII W135A-t2A-BCL2\n✱✱\n✱✱✱\nWT\nΔΜΒII\nMBIΙ-4aa mut\nMSCV-Backbone\nUntransduced\nMBIΙ-W135A\nDay 6\n0.000\n0.001\n0.002\n0.003\n0.004\nIgG ng/ml/cell\n✱✱\n✱✱\nDay 13\n0.00\n0.02\n0.04\n0.06\n0.08\nIgG ng/ml/cell\n✱✱\n✱✱✱\nDay 6\n0.00\n0.01\n0.02\n0.03\n0.04\n0.05\nIgM ng/ml/cell\nns\nns\nDay 13\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\n0.6\n0.7\nIgM ng/ml/cell\nns\nns\nDay 13\n3\n4\n5\n6\n7\n8\n9\n10\nDEPTOR expression\nDay 13\n10\n11\n12\n13\n14\n15\nLAPTM5 expression\nDay 13\n4\n6\n8\n10\n12\n14\nASS1 expression\nDay 13\n14\n15\n16\n17\n18\n19\nEEF1A1 expression\nDay 13\n13.0\n13.5\n14.0\n14.5\n15.0\n15.5\n16.0\nRPL3 expression\nDay 13\n12\n13\n14\n15\n16\nXBP1 expression\nDay 13\n16\n18\n20\n22\n24\nIGHM expression\nDay 13\n14\n16\n18\n20\n22\nIGHG1 expression\nDay 13\n8\n12\n16\n20\n24\nIGHG2 expression\nDay 13\n12\n15\n18\n21\nIGHG3 expression\nDay 13\n16\n18\n20\n22\n24\nIGHA1 expression\nDay 13\n12\n14\n16\n18\n20\n22\nIGHA2 expression\nDay 13\n10\n12\n14\n16\n18\n20\n22\nIGLC1 expression\nDay 13\n12\n14\n16\n18\n20\n22\nIGLC2 expression\nDay 13\n10\n12\n14\n16\nDERL3 expression\nDay 13\n8\n10\n12\n14\nERLEC1 expression\nDay 13\n11\n12\n13\n14\n15\n16\n17\nHERPUD1 expression\nDay 13\n6\n8\n10\n12\nFIDC expression\nDay 13\n13\n14\n15\n16\n17\n18\nTXNDC5 expression\nDay 13\n14\n16\n18\n20\n22\nIGKC expression\nFigure 7\nFigure 7. Point mutation of the DCMW motif and W135 phenocopy MBII deletion and retain only minimal impact on\nsecretory reprogramming and MYC target gene regulation. Violin plots of log2 normalised RNAseq expression values of\ngenes identified on the y-axis of each graph at day 13 for the conditions as indicated to the top right of the figure: a, XBP1,\nimmunoglobulin genes and XBP1 targets; and b, MYC targets. Data are representative of two independent experiments\nwith a total of n=3 samples per time point and condition. Quantification of c, IgM and d, IgG, antibody concentration\nnormalized per cell at day 6 (left graph) and day 13 (right graph). Data are representative of two independent experiments.\nBars and error represent mean and standard deviation (SD); One-way ANOVA: ns, not significant; ** P < 0.01; *** P <\n0.001.\na \nc d \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}