Enforced MYC expression selectively redirects transcriptional programs during human plasma cell differentiation

21 MYC 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 Prec 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 drive 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 protec 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 huma 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 find 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 MYC 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 This 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 co 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 metabo 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 (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r e solve s to r e sidue W 135 . 32 33 34 35 36 37 38 39 40 41 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 3

42 The 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 tran 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 onc 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 ly 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 the 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 co 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 spre 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 poly 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 Di 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 and 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 type 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 MYC 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 MYC 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 M 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 tran 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 MYC 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 phosp 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 in 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 impli 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 d 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 t 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 s 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 the 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 targe tabl e . [39] 66 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 4 MYC 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 dereg 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 range 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 betwe 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 co 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 tran 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 ex 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 progno 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 co 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 52] 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 co mbination d r iv ing su st a in ed pop ul atio n ex pansion . [52] 77 Dif 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 pla 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 We 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 long -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 inc 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 endog 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 diff 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 reca 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 a 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 regula 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 of 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 A 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 CD40L 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 c 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 5 wa 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 CD40L 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 B-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 wou 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 tran 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 impa 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 Our 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 co 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 phy 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 secr 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 on MB0 and ar e de pe nden t on M BI I and the sing le ami no ac id W135 . 102

103 Acu 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 We 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 ac 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 wi 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 the 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 co 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 in 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 long -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 inc 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 whi 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 antig 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 sub 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 tran 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 6 diff 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 ly 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 is 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 d 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 of 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 In 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 wi 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 CD40 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 remova 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 and 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 diff 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 (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 ce 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 ce 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 pheno 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 fea 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 h 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 and 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 diff 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 marke rs. 136 M 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 PC 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 towa 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 and 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 on 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 7 of 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 tran 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 cy 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 Con 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 BCL 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 no 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 co 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 in 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 During 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 tran 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 ce 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 in 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 T 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 siz 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 diff er entia ting B-c el l s . 156 Enforc 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 ex pressio n. 158 To 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 time 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 wa 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 6 - 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 and 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 i 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 T 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 (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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 8 T 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 sub 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 progre 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 co 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 phy 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 of 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 (Fig ure 2b an d Sup . F igure 2 a ). 173 We 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 data . 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 ex 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 ex 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 T 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 TNFRS 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 Thus, 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 at tr an script l ev el. 181 P 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 ther 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 (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 we 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 diff 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 IRF4 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 obs 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 t 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 76] 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 point 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 9 also 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 ex 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 pathw ays ,[81 ] in MY C T58I - t2A -BCL2 condi t i on s (S up . Fig ure 2c ). 194 Con 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 ca 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 TRAP1 , 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 t 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 targe 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 in MY C T58I- t2A -BCL2 condi t i on s. 200 Si 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 stre 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 as 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 the 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 output 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 PC 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 the 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 damp 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 co 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 co 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 during 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 tran 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 MYC 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 To 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 and 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 co 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 10 betwe 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 module 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 2) 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 t2A- 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 that 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 diff 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 metabo 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 sta 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 repr 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 Module 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 C 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 ce 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 remain 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 co 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 we 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 regula 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 ex 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 XBP1 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 diff 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 inc 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 we 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 M15), i ncludi ng gen e s l inked t o au topha gy and c ellul ar quie sce nce . 238 Module 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 Module 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 ribo 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 diff 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 11 13 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 func 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 t2A- 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 gen 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 all 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 wa 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 This 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 diff 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 relat 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 supp 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 c 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 the 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 Ins 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 abe r r ant ex pr e s s ion sta te . 256 MB 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 The 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 ther 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 to 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 t2A- 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 co 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 F 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 BCL 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 pheno 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 t2A- 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 pheno 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 12 BCL 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 dele 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 wi 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 follow 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 To 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 day 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 BCL 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 co 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 t 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 co 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 d 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 day 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 diff 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 co 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 as 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 biol 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 M9) 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 Δ 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 wi 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 T58 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 inten 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 marke 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 PC -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 dime 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 mutant 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 ove r e xpre ssion : Δ MB I I > Δ MB0 > Δ MB I. 293 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 13 T 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 diff 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 ex 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 MYC 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 fac 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 The 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 secr 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 IgM 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 gen 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 co 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 repr 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 co 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 hig 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 diff er enc e f rom con t r ol c onditio ns . 307 The 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 Given 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 t 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 hig 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 Sub 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 co 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 mutant 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 5c , 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 ove 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 Δ 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 ( 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 14 pheno 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 cl 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 Our 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 the 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 gen 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 co 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 diff 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 Δ 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 MYCw 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 diff 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 ex 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 re 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 patt 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 s 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 betwe 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 two 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 remain 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 but 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 inc 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 and 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 the 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 the 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 ex 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 and 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 secr 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 es 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 15 co 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 su 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 in h uman B-c ell di f fer enti atio n . 347 348 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 16

349 Der 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 time 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 diff 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 addre 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 sta 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 diff 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 co 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 T58 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 lac 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 previ 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 ch 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 Di 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 ac 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 pathw 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 ce 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 co 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 drive 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 ex 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 ch 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 We 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 Gene 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 signific 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 cl 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 co 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 17 preva 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 es 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 P 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 growth 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 hea 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 ev 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 seq 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 signal 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 PRD 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 MYC ,[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 t 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 relea 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 ordinat 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 secr 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 74-76, 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 co 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 such 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 switchi 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 The 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 burst 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 reorgani 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 ex 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 ex 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 B-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 relat 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 18 MYC 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 c 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 enha 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 posi 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 ag 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 ef 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 diff 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 Suc 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 obs 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 secr 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 gen 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 role 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 fav 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 immuno 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 arrange 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 growth 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 ex 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 materi 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 vi 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 prolif 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 ove 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 interco n nect ed f e ed for w a rd loop s (Su pp l emental Figu re 7 b) . 420 The 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 tran 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 tran 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 enha 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 19 seq 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 reduc 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 or 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 but 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 hist 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 predic 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 DCM 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 tran 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 sub 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 M 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 dro 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 the 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 dereg 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 W 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 inc 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 are 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 impa 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 suppo 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 st 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 inter 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 g 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 ef fec ts . 446 In 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 co 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 demon 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 diff 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 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 20 co 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 the 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 critic al de p endenc e on MBI I could be r e sol ved to th e DCMW mo tif a nd W135. 453 454 Data Availability Statement 455 The 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 456 457

458 We 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 suppo rt and c r i tic al r ev iew of t h i s w ork. 460 461 Funding Acknowledgements: 462 This 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 (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 1022 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 Univer 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 program 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 under 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 Natio 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 (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 Res 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 f 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 Wellc 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 Camb 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 Res 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 tho 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 Car e. F or th e purpo se o f O p en Acc e ss, t he author s ha ve app li ed a CC BY pu blic c opyright li cenc e to 477 any Author Acce pte d Manu scrip t ve rsion a r ising fr om t h i s submi s s i on . 478 479 480 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint 21

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G e nom e Re s, 202 2. 32 (4): p. 629-724 642. 725 726 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Day 6 Day 13 Day 20 0.00 0.01 0.02 0.03 0.04 10 20 30 IgM ng/ml/cell Figure 1 a b c d e f g i Day 3 Day 6 Day 13 Day 20 Untransduced MSCV-empty MYC-t2A-BCL2 Untransduced MSCV-backbone T58I-t2A-BCL2 Day 13 MSCV-backbone T58I-t2A-BCL2 CD20 CD19 Day 3 Day 6 Day 13 MSCV-backbone T58I-t2A-BCL2 CD38 CD27 Day 3 Day 6 Day 13 MSCV-backbone T58I-t2A-BCL2 CD138 CD38 Day 3 Day 6 Day 3 Day 6 Day 13 Day 20 0 50 100 150 CD2 positive cells (%) MSCV-backbone T58I-t2A-BCL2 ✱✱ ✱✱✱ ✱ ✱✱ Day 3 Day 6 Day 13 Day 20 0 50 100 150 CD19 positive cells (%) ns ns ✱✱ ✱✱✱ Day 3 Day 6 Day 13 Day 20 0 50 100 150 CD27+CD38+ cells (%) ✱ ✱✱ ✱✱✱✱ ✱✱✱ Day 3 Day 6 Day 13 Day 20 0 50 100 150 CD38+CD138+ cells (%) ns ns ✱✱✱ ✱✱✱✱ Day 21 Day 31 0 2 4 6 8 10 EdU+Ki67+ cells (%) nsp=0.09 Day 21 Day 31 0 5 10 15 EdU-Ki67+ cells (%) Untransduced T58I-t2A-BCL2 nsp=0.07 Untransduced T58I-t2A-BCL2 T58I-t2A-BCL2 MSCV-backbone Day 6 Day 13 Day 20 -1 0 1 2 3 4 IgM ng/ml/cell Untransduced MSCV-backbone MYC-t2A-BCL2 ns ns ns Day 6 Day 13 Day 20 0.000 0.002 0.004 0.006 1 2 3 4 5 IgG ng/ml/cell Figure 1. Acute MYC and BCL2 overexpression drives aberrant B cell differentiation. a, Graphical representation of the in vitro differentiation and transductions model system. This shows general phases by day of culture at the top, followed by summary culture conditions and biological processes below. Associated transcription factors (TF) are depicted on the left and phenotypic markers on the right. b, Flow cytometric quantitation of percentage CD2 positive cells for MSCV-backbone and T58I-t2A-BCL2 conditions at indicated time points. c, d, e, Representative flow cytometry plots of CD19 vs CD20, CD27 vs CD38 and CD38 vs CD138, respectively, for MSCV-backbone and T58I-t2A-BCL2 conditions at the indicated time points. f, Percentages of CD19 positive cells (left), CD27+CD38+ cells (middle), and CD38+CD138+ cells (right) for the indicated conditions at the indicated time points. g, Percentages of EdU+Ki67+ cells at the indicated time points for untransduced and T58I-t2A-BCL2 samples after 1 hour of pulse EdU incorporation. Data shown for the MSCV-backbone and T58I-t2A-BCL2 conditions are pre-gated to CD2+ populations (c, d, e, f, g) i, Quantification of total human IgG antibody secretion (left) and IgM antibody secretion (right) on day 6, day 13 and day 20 for the indicated conditions. Data are representative of at least two independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed Student’s t-test, (b, g). One-way ANOVA (f): ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. CD40L Fab2 anti-Ig IL2/IL21 B-cell state DivideGrow & PC state IL2/IL21 IL6/IL21 APRIL IL6 APRIL PAX5 MYC IRF4/BLIMP1/XBP1 CD19hi/CD20hi CD19lo/CD20lo CD27hi/CD38hi/CD138hi PhenotypeTF .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Figure 2 UMAP1 UMAP2 Controls MYC Day 3 Day 6 Day 13 Day 20 Day 0 MSCV-backbone T58I-t2A-BCL2 Untransduced MSCV-backbone T58I-t2A-BCL2 Untransduced MSCV-backbone T58I-t2A-BCL2 Untransduced T58I-t2A-BCL2 Untransduced 0 3 6 9 12 15 18 21 0 5 10 15 Days CD19 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days MS4A1 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days CD27 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days CD38 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days SDC1 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days CD2 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days MYC expression 0 3 6 9 12 15 18 21 0 5 10 15 Days PAX5 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days EBF1 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days PRDM1 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days IRF4 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days XBP1 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days DERL3 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days ERLECC1 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days HERPUD1 expression 0 3 6 9 12 15 18 21 0 5 10 15 20 Days TXNDC5 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days FICD expression b c d e f 0 3 6 9 12 15 18 21 0 5 10 15 Days IGLC7 expression Untransduced MSCV-backbone T58I-t2A-BCL2 Untransduced MSCV-backbone T58I-t2A-BCL2 0 3 6 9 12 15 18 21 0 5 10 15 Days IGLC7 expression Untransduced MSCV-backbone T58I-t2A-BCL2 Untransduced MSCV-backbone T58I-t2A-BCL2 0 3 6 9 12 15 18 21 0 2 4 6 8 10 Days TERT expression 0 3 6 9 12 15 18 21 0 5 10 15 Days JAG2 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days FABP5 expression 0 3 6 9 12 15 18 21 0 5 10 15 Days KISS1R expression 0 3 6 9 12 15 18 21 0 5 10 15 Days TRAP1 expression Figure 2. MYC T58I selectively perturbs gene expression in B-cell differentiation. a, Uniform Manifold Approximation Projection (UMAP) of differentially expressed genes for the indicated conditions and time points. b, c, d, e, f, Normalised RNAseq expression values (y-axis) of selected genes across the differentiation time course (x-axis days) as indicated for untransduced, MSCV-backbone and T58I-t2A-BCL2 conditions. Gene expression is shown as indicated in the figure for b, MYC and CD2; c, surface proteins linked to immunophenotyping; d, transcription factors; e, known MYC targets; and f, XBP1 targets. Data are representative of two independent experiments with a total of n=1-4 samples per time point and condition. a .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Figure 3 PGCNA: MSCV-backbone T58I-t2A-BCL2 Untransduced D0 D3 D6 D13 D20 b Figure 3. Expression network level analysis of MYC T58I on B-cell differentiation. Parsimonious Gene Correlation Analysis (PGCNA) was used to define modules of coregulated genes. a, Heat map of module level gene expression with expression patterns averaged across all genes per module on a z-score scale (-2.14 blue to +2.09 red). Modules are hierarchically clustered on the left and module number and indicative summary terms of associated ontologies are shown on the right. Time 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). Individual conditions are identified using the code as indicated at the top of the figure: yellow (MSCV-backbone), purple (T58I-t2A-BCL2), green (untransduced). b, Correlation plot of gene signature enrichment analysis for the 16 PGCNA-derived modules showing analysis for MsigDB Hallmark signatures. Enrichment is illustrated on a z-score scale from blue to red. With hierarchical clustering for enriched signatures and module enrichment patterns above and to the right. Module numbers and summary designations are identified to the right. a .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Figure 4 Donor 1 Donor 2 Donor 3 Donor 4 Day 6 Day 13 Untransduced MSCV-backbone WT ΔMB0 ΔMBI ΔMBII MYC- t2A-BCL2 3.01.50.0-1.5-3.0 Untransduced MSCV-backbone MYC WT-BCL2 MYC ΔMB0-BCL2 MYC ΔMBI-BCL2 MYC ΔMBII-BCL2 Untransduced MSCV-backbone MYC WT-BCL2 MYC ΔMB0-BCL2 MYC ΔMBI-BCL2 Day 6 Day 13 MYC ΔMBII-BCL2 ΔMB0 ΔMBII ΔMBI MDS1 MDS2 MSCV-backbone WT ΔΜΒ0 ΔΜΒΙ ΔΜΒΙΙ MYC- t2A-BCL2 Untransduced Day 13 0 50 100 150 CD27+CD38+ cells (%) Untransduced MSCV-empty MYCisoform-t2A-BCL2 delMB0 delMBI delMBII b c d e Day 13 0 50 100 150 CD27+CD38+ cells (%) ✱ ✱✱✱✱ ✱ ✱✱✱✱ ✱✱✱✱ Day 13 0 20 40 60 80 100 CD38+CD138+ cells (%) ✱✱ ns ns ✱✱ ✱✱✱✱ Day 13 MYC-t2A-BCL2 WT ΔMB0 CD38 CD27 ΔMBI ΔMBIIMSCV-backbone Day 13 MYC-t2A-BCL2 WT ΔMB0 CD138 CD38 ΔMBI ΔMBIIMSCV-backbone Figure 4. Deletion of MYC TAD MB0, MBI and MBII have differential effects on MYC driven phenotypic and expression features. a, b, Representative flow cytometry plots at day 13 for control MSCV-backbone, MYCwt, MB0, MBI and MBII conditions as shown for a, CD27 vs CD38, and b, CD38 vs CD138. c, Summary of flow cytometrically defined percentages of CD27+CD38+ cells (left) and CD38+CD138+ cells (right), at day 13 for the indicated conditions. Data are representative of three independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed Student’s t-test: ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001. Data shown for the transduced conditions are pre-gated to CD2+ populations (a, b, c,). d, Multidimensional Scaling (MDS) of differentially expressed genes at the day 6 (green) and day 13 (purple) time points, and controls, for the indicated samples as illustrated in the figure. e, PGCNA defined modules of coregulated genes shown as a heat map of module level gene expression with expression patterns averaged across all genes per module on a z-score scale (-3 blue to +3 red). Modules are hierarchically clustered on the left and module number and indicative summary terms of associated ontologies are shown on the right. Time points are indicated as grey day 6 and black day 13. Samples from different donors are illustrated in the blue to orange color code, and for individual conditions with color code identified in the figure. a .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Figure 5 a e Day 6 Day 13 4 6 8 10 12 14 CD19 expression Day 6 Day 13 4 6 8 10 12 14 16 MS4A1 expression Day 6 Day 13 9 10 11 12 13 14 15 CD27 expression Day 6 Day 13 4 6 8 10 12 14 16 CD38 expression Day 6 Day 13 0 5 10 15 SDC1 expression Day 6 Day 13 2 4 6 8 10 12 SDC1 expression Untransduced MSCV-backbone WT-t2A-BCL2 ΔΜΒ0-t2A-BCL2 ΔΜΒΙ-t2A-BCL2 ΔΜΒΙΙ-t2A-BCL2 MSCV WT ΔΜΒ0 ΔΜΒΙ ΔΜΒΙΙ MYC- t2A-BCL2 Untransduced Day 6 Day 13 4 6 8 10 12 PAX5 expression Day 6 Day 13 5 6 7 8 9 10 EBF1 expression Day 6 Day 13 10 11 12 13 14 15 PRDM1 expression Day 6 Day 13 11 12 13 14 15 16 17 XBP1 expression Day 6 Day 13 11 12 13 14 15 16 IRF4 expression f MSCV-backbone WT ΔΜΒ0 ΔΜΒΙ ΔΜΒΙΙ MYC- t2A-BCL2 Untransduced Day 13 0 50 100 150 CD27+CD38+ cells (%) Untransduced MSCV-empty MYCisoform-t2A-BCL2 delMB0 delMBI delMBII b c d Day 6 0.000 0.002 0.004 0.006 0.008 0.010 IgG ng/ml/cell ns ✱ ns ✱✱✱ ✱✱ Day 13 0.00 0.02 0.04 0.06 0.08 0.10 IgG ng/ml/cell ns ✱✱ ns ✱ ✱✱✱✱ Day 6 0.000 0.005 0.010 0.015 0.020 0.025 IgM ng/ml/cell ns ✱ ns ✱ ns Day 13 0.00 0.05 0.10 0.15 0.20 0.25 IgM ng/ml/cell ns ✱✱ ns ✱ ✱✱ Day 6 Day 13 0 2 4 6 8 10 12 TERT expression Day 6 Day 13 2 4 6 8 10 12 JAG2 expression Day 6 Day 13 4 6 8 10 12 SORD expression Day 6 Day 13 2 4 6 8 10 KISS1R expression Day 6 Day 13 8 9 10 11 12 13 TRAP1 expression Day 6 Day 13 8 10 12 14 16 DERL3 expression Day 6 Day 13 9 10 11 12 13 ERLEC1 expression Day 6 Day 13 10 12 14 16 HERPUD1 expression Day 6 Day 13 6 7 8 9 10 FICD expression Day 6 Day 13 13 14 15 16 17 18 TXNDC5 expression Figure 5. MB0, MBI and MBII deletion impacts on indicative gene regulation and on functional secretory output. Violin plots of log2 normalised RNAseq expression values of individual genes plotted at day 6 (left side of graphs) and day 13 time points (right side of graphs) for the indicated conditions (top right of figure). Genes shown are indicated to the left of each graph for: a, surface antigens; b, transcription factors; c, MYC targets; and d, XBP1 targets . Data are representative of two independent experiments with a total of n=4 samples per time point and condition. Quantification of e, IgM and f, IgG antibody concentration normalized per cell at day 6 (left graph) and day 13 (right graph) for conditions as indicated to the lower right of the figure. Data are representative of two independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed Student’s t-test: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint Figure 6 1.50.0-1.5 Donor 1 Donor 2 Donor 3 Untransduced WT ΔMBII MYC-t2A-BCL2 MBII-4aa mut MSCV-backbone MBII-W135ADay 13 PGCNA: Untransduced MSCV-backbone WT ΔMBII MBII-4aa mut MYC- t2A-BCL2 MBII W135A Day 13 MDS1 MDS2 d WT ΔΜΒII MBIΙ-4aa mut MYC- t2A-BCL2 MSCV-Backbone Untransduced MBIΙ-W135A Day 13 0 20 40 60 80 CD38+CD138+ cells (%) Untransduced MSCV-empty MYCisoform-t2A-BCL2 delMBII Mut 31 Mut 33 ns ✱✱✱ ns ns ns ✱ ✱ ✱✱ Day 13 0 50 100 150 CD27+CD38+ cells (%) ns ✱ ✱ ✱ ✱ Day 13 0 20 40 60 80 100 CD38+CD138+ cells (%) ns ✱✱ ✱ ✱✱ ✱✱✱ Day 13 MYC-t2A-BCL2 WT ΔMBII CD138 CD38 MBII-4aa mutMSCV-backbone MBII-W135A Day 13 MYC-t2A-BCL2 WT ΔMBII CD38 CD27 MBII-4aa mutMSCV-backbone MBII-W135A b c e Figure 6. Point mutation of the DCMW motif and W135 phenocopy MBII deletion. Representative flow cytometry plots at day 13 for the indicated conditions above each dot plot for: a, CD27 vs CD38; and b, CD38 vs CD138. c, Flow cytometric quantification of percentage data CD27+CD38+ cells (left), and CD38+CD138+ cells (right) at day 13 for the conditions indicated to the right of graphs. Data are representative of at least three independent experiments. Bars and error represent mean and standard deviation (SD); Unpaired two-tailed Student’s t-test: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Data shown for the conditions tested, apart from the untransduced, are pre-gated to CD2+ populations (a, b, c). d, Multidimensional Scaling (MDS) of differentially expressed genes at day 13 for the indicated samples. e, PGCNA defined modules of differentially coregulated genes at day 13 shown as a heat map of module level gene expression with expression patterns averaged across all genes per module on a z-score scale (-1.5 blue to +1.5 red). Modules are hierarchically clustered on the left and module number and indicative summary terms of associated ontologies are shown on the right. Samples from different donors are illustrated in the blue to orange color code and for individual conditions with color code identified above the figure. a .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint WT ΔΜΒII MBIΙ-4aa mut MSCV-Backbone Untransduced MBIΙ-W135A Day 13 6 8 10 12 CD19 expression Untransduced MSCV-backbone WT-t2A-BCL2 ΔΜΒII-t2A-BCL2 MBII-4aa mut-t2A-BCL2 MBII-W135A-t2A-BCL2 b Day 13 0.00 0.02 0.04 0.06 0.08 IgG ng/ml/cell Untransduced MSCV-backbone WT-t2A-BCL2 ΔΜΒII-t2A-BCL2 MBII 4aa mut-t2A-BCL2 MBII W135A-t2A-BCL2 ✱✱ ✱✱✱ WT ΔΜΒII MBIΙ-4aa mut MSCV-Backbone Untransduced MBIΙ-W135A Day 6 0.000 0.001 0.002 0.003 0.004 IgG ng/ml/cell ✱✱ ✱✱ Day 13 0.00 0.02 0.04 0.06 0.08 IgG ng/ml/cell ✱✱ ✱✱✱ Day 6 0.00 0.01 0.02 0.03 0.04 0.05 IgM ng/ml/cell ns ns Day 13 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 IgM ng/ml/cell ns ns Day 13 3 4 5 6 7 8 9 10 DEPTOR expression Day 13 10 11 12 13 14 15 LAPTM5 expression Day 13 4 6 8 10 12 14 ASS1 expression Day 13 14 15 16 17 18 19 EEF1A1 expression Day 13 13.0 13.5 14.0 14.5 15.0 15.5 16.0 RPL3 expression Day 13 12 13 14 15 16 XBP1 expression Day 13 16 18 20 22 24 IGHM expression Day 13 14 16 18 20 22 IGHG1 expression Day 13 8 12 16 20 24 IGHG2 expression Day 13 12 15 18 21 IGHG3 expression Day 13 16 18 20 22 24 IGHA1 expression Day 13 12 14 16 18 20 22 IGHA2 expression Day 13 10 12 14 16 18 20 22 IGLC1 expression Day 13 12 14 16 18 20 22 IGLC2 expression Day 13 10 12 14 16 DERL3 expression Day 13 8 10 12 14 ERLEC1 expression Day 13 11 12 13 14 15 16 17 HERPUD1 expression Day 13 6 8 10 12 FIDC expression Day 13 13 14 15 16 17 18 TXNDC5 expression Day 13 14 16 18 20 22 IGKC expression Figure 7 Figure 7. Point mutation of the DCMW motif and W135 phenocopy MBII deletion and retain only minimal impact on secretory reprogramming and MYC target gene regulation. Violin plots of log2 normalised RNAseq expression values of genes 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, immunoglobulin genes and XBP1 targets; and b, MYC targets. Data are representative of two independent experiments with a total of n=3 samples per time point and condition. Quantification of c, IgM and d, IgG, antibody concentration normalized per cell at day 6 (left graph) and day 13 (right graph). Data are representative of two independent experiments. Bars and error represent mean and standard deviation (SD); One-way ANOVA: ns, not significant; ** P < 0.01; *** P < 0.001. a c d .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 21, 2024. ; https://doi.org/10.1101/2024.04.18.589889doi: bioRxiv preprint