Rad51 As Functional Biomarker to Select Tumors for Parp Inhibitor Treatment

ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184

ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/

WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en RAD51 AS FUNCTIONAL BIOMARKER TO SELECT TUMORS FOR PARP INHIBITOR TREATMENT

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INDEX

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======FL

======GJ

======GN

======HH

mS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSoq ),'&&&3&888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888BD 3&&/&,*,)---,&0(.),(,8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888BD

nS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSpm )')&) )/-,)'!(.!)(,*!,88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888CA @)'!(-(!(!( *,.(,-8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888CC

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pS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSpu (!-'-)!,-!-.(888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888DA !3)(88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888DG

qS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSqu &.!(/'-(-!.!0!.388888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888DH !)',%,--)( ('/..!)((&3-!-8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888E? ()'!-,-('/..!)(&-! (./,-888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888E@ 3('!!)',%,-8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888EB . ,!)',%,-88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888EC

rS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSrr )'!(.!)(1!. !( !!.),-88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888EE

======LF

3*). -!-@8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888FB 3*). -!-A8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888FB 3*). -!-B8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888FC 3*). -!-C8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888FC

======LJ

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======MN

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-.&!- '(.)*(&,)' *.!(.-9./'),-8888888888888888888888888888888888888888888888888888888888H@ &*,!(.!./'),.!0!.3!(. *(&!-.!( /!- --/-.)!;,-!-.(../'),-HB !;,-!-.(. @')&-)(). ,),., .&)--),-)(,3@'/..!)(-8888888888HD 2*,--!)()/(.!)(& 3*)'),* !@!-)),'-!---)!.1!. !,-!-.(!( @')&-8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888HD )--)DB@(BD!(!;,-!-.(. @')&-88888888888888888888888888888888888888888888888888888HG !;,-!-.(. @-,&.)),'D@(/&,)!8888888888888888888888888888888888888888888888888888@?? D@(/&,)!),'.!)((----!(/(.,../'),-'*&-(!---)!.1!. !,-!-.(!() ),.;@888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@?@ D@)!),'.!)(!(./'),-'*&-,)'*.!(.-*,!.-,-*)(-.)!88888888888888888888@?D

G ======FEM

-.&!- '(.)*(&,)'()(; *.!(.9-./'),-8888888888888888888888888888888888888888888888888@?G &*,!(.!./'),.!0!.3!(. ()(; *(&!-.!( /!- --/-.)./'), ! &3 -(-!.!0.)!888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@?G mp

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@FFD(.!./'),.!0!.3!(. *(&!-.!( /!- --/-.)./'),- ! &3-(-!.!0.)@ !( !!.!)(8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@AG -*)(-!)',%,-)@!( !!.!)(888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@B? @!( !!.!)(!(/-&,.'!.).!(.,3(,*&!.!)(-.,--!(0!0)88888888888888888888888888888888@B? @&)%!()'!(.!)(1!. !( !!.!)(-(-!.!4-@;&.,./'),-8888888888888888888888@BC &)%!()'!(.!)(1!. !( !!.!)(-(-!.!4-). ,@;&.,./'),- 88888888888888@BG

======FIF

======FJJ

@88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@DF A88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@DF B88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888@DG

======FJN

======FMF

ABBREVIATIONS

qom 6qo )1")1'683:#)1m [ )1")1' 99#::# m [ )1")1' 99#::#0#0 #8m 1/@9)93&=8)1 # 0#8) 1:@6# ;/:;8# 3//# :)31 :?):#/1')# :9)[0;::#"683:#)1.)19# 1""o[8#/:#"683:#)1.)19# 8#;1"#8:(# ;8=# m m[993 ):#" "30)1683:#)1m 8#9: 1 #8 [n [ #///@06(30n [ [ #///@06(30[#?:8/8'# 9##? )9)318#6)8 m 8#9: 1 #8'83>:(9;668#9938683:#)1m n 8#9: 1 #8'83>:(9;668#9938683:#)1n m[:#80)1/"30)1 m m[)1:#8 :)1'683:#)1[:#80)1/(#/) 9#m 3=)1#9#8;0/ ;0)1 ;883>9[(##/#8/)'10#1: m @ /)1m m @ /)1["#6#1"#1:.)19#m mn @ /)1["#6#1"#1:.)19#mn n @ /)1["#6#1"#1:.)19#n @ /) [_ `9@1:(9# p (8303"30)1(#/) 9#p m (# .63)1: )19#m m) (# .63)1: )19#m)1() ):38 m (# .63)1: )19#m n (# .63)1: )19#n 31&)"#1 # 1:#8=/ :/3';#3&930:) 0;::)319)1 1 #8 mt

306/#:#8#96319# 9:8:)318#9)9:1:6839::# 1 #8 : [:#80)1/ )1")1'683:#)1 : :[)1:#8 :)1'683:#)1 oPoU[)0)13 #1A)")1# pdPr[")0)13[n[)/)1"3 "0'#8#96319# )&&;9#/8'#[ #///@06(30 ;/ # 303")&)#"'/#90)1)0/#99#1:)/0#");0 )0#:(@/9;/&3?)"# #3?@8) 31; /#) )" [ [ :)=:#"683:#)1.)19# 3; /#9:81" 8.# 1(1 #" (#0)/;0)1#9 #1 # :(@/#1#")0)1#:#:8 #:) )" 0 8@31) /#; )1#A)66#8.)19# ;836#1#") )1#9'#1 @ 6):(#/)/[:3[0#9#1 (@0/:819):)31 9:83'#18# #6:38 n 8@:(83 /9:) 31 3'#1#n m ?31; /#9#m n 1(1 #83&A#9:#8(303/3'n msq 0)/@>):(9#7;#1 #9)0)/8):@msq0#0 #8 oq 0)/@>):(9#7;#1 #9)0)/8):@oq0#0 #8 1 31)1#0) 306/#0#1::)31'83;6 n 1 31)1#0) 306/#0#1::)31'83;6n #:/ 3=)1#9#8;0 33"1"8;'"0)1)9:8:)31 380/)1[&)?#"1"68&&)1[#0 #""#" ) 83 /9:'83>:(& :389 /;38#9 #1 #8# 3=#8@&:#86(3:3 /# ()1'

&9 &80#9()&: /@ #8/"#(@"#o[6(396(:#"#(@"83'#19# 8##1&/;38#9 #1:683:#)1 n )9:31#n #8#"):8@ 8#9:1"3=8)1 1 #8 n ;01#6)"#80/'83>:(& :388# #6:38n )'('8"#9#83;93=8)1 1 #8 o )9:31# o 303/3'3;98# 30 )1:)31"#&) )#1 @ 389#")9(6#83?)"9# 303/3'3;98# 30 )1:)318#6)8 ul #:9(3 .683:#)1ul 00;13&/;38#9 #1 # [m 19;/)1[/).#'83>:(& :38m [m 19;/)1[/).#'83>:(& :38m8# #6:38 31)A)1'8"):)31 m )=#8.)19#m 0)1 3993&(#:#83A@'39):@ 8'#[9 /#:819):)319 m #"):383&"0'# (# .63)1:m m ;/:)"8;'8#9)9:1 #683:#)1m :#81/#0 8@31) /#; )1#A)66#8.)19# -38()9:3 306:) )/):@ 306/#? [ -38()9:3 306:) )/):@ 306/#? /99 o )?#"[/)1#'#/#;.#0)683:#)1o p )?#"[/)1#'#/#;.#0)683:#)1p ;/:)6/#?/)':)31["#6#1"#1:683 #06/)&) :)31 [ #:(@/:)31[96# )&) mm #)3:) 8# 30 )1:)31mm m @#/)1819 8)6:)31 :38m nl

1 ;0 #83&8#6/) :#9

np 3");038:(3=1":# / 3");0 (/38)"# 3");0&/;38)"# ) 8)1 m )-0#'#1 8#.'#9@1"830#683:#)1m ; /#8#?638:9)'1/ n #;83&) 830)1n #?: '#1#8:)319#7;#1 )1' 31[(303/3'3;9#1"[-3)1)1' [ 319#8=:)=# /:[ /:#81:)=# ; /#8/3 /)A:)319#7;#1 # =/#") /#9#8 ( 19:):;:# X 31[3 #9#") #:) X#=#8# 30 )1#")00;13"#&) )#1 @ :;8/.)//#8 =8)1 1 #8 ""9:)3 6#18#")1'&80# 6=/;# 83 )/):@=/;# X #1) )//)1X:8#6:30@ )1 6nm 83:#)1nm 6ns 83:#)1ns 6qo 83:#)1qo n 8:1#81"/3 /)A#83&n 3/@[8) 39# 3/@[8) 39#'/@ 3(@"83/9# 3/@[8) 39#63/@0#89# m 1:#8 :)1'83:#)1m (396(:# ;&#"9/)1# (396(:# ;&#"9/)1#[>##1

839::# 1 #8 3/@0#89# ()18# :)31 [ #/:)0# 83'8#99)=#")9#9# m 83'800#""#:([/)'1"m :)#1:"#8)=#"?#13'8&: 83'8#99)31&8##9;8=)=/ ' ['/@ 3683:#)1 ' 83'#9:#831#8# #6:38 6 3:#1:)/3&(@"83'#1 o (396(:)"@/)139):3/o[.)19# (#1@/0#:(1#9;/&31@/&/;38)"# 8:)/8#96319# 83/)1#8) (0):3:) (# .63)1: 31:83/& :38 :819 :)=:)31"30)1[)1:#8 :)1'683:#)1 3/@=)1@/)"#1#")&/;38)"# tl # #6:38993 ):#"683:#)1tl #96319#=/;:)318):#8) 13/)";0389 0 3")&)#" m 6m[)1:#8 :)1'683:#)1m #//@ 1:#8#9:)1'#>#1# ")3)00;1368# )6)::)3199@ ) 31; /#) )" 0) ) 83 0 #99#1'#8 9) 0//)1:#8)1' t &)1'#8683:#)1t # #)=#836#8:)1' (8 :#8)9:) #6/) :)31683:#)1 #=#89#6(9#683:#)188@ n ) 31; /#3:)"#8#"; :9#9; ;1):n nn

9 9# 31"9 0// #///;1' 1 #8 : /#")9#9# [ 3");0"3"# @/9;/&:#63/@ 8@/0)"#'#/#/# :836(38#9)9 :1"8"#88383&:(#0#1 m 6/):(1"X96/):&33:683:#)1m m ()#/")1 306/#?9; ;1):m n ()#/")1 306/#?9; ;1):n o ()#/")1 306/#?9; ;1):o @1:(#:) #:(/):@ mm (/0)/@#0 #8mm )1'/#9:81" 8.# #/30#8) //#/) )0 /1 # 8)9 ;&#"9/)1# >##1 [β 819&380)1''83>:(& :38 #: 8)6/#1#':)=# 8#9: 1 #8 m 363)930#89#n[ )1")1'683:#)1m qo ;038683:#)1qo 8#' 8#';/:38@ [m (830 39631")1m @8@839)1# /:8=)3/#: [ 9 ;/8#1"3:(#/)/'83>:(& :38 m 0// #//683:#)1P0):3:) )1() ):38.)19# (3/#[#?30#9#7;#1 )1' )/":@6# m [8@8#6)8 8399[ 306/#0#1:)1''83;6m #//3>&/;38#9 #1:683:#)1 # m )1 &)1'#8[ 3? )1")1'(30#3 3?m # n )1 &)1'#8[ 3? )1")1'(30#3 3?n

ABSTRACT

3/@_[8) 39#`63/@0#89#_`)1() ):389_)`8##&&# :)=#1:) 1 #8"8;'9)1 1 #89 >):( "#&# :)=# (303/3'3;9 8# 30 )1:)31 8#6)8 _ `P )1 /;")1' 1 #89 >):(0;::)319)1@1"A=@:A>6>() (/93")96/@#1(1 #"9#19):)=):@:3 "0')1' (#03:(#86@ 9; ( 9 6/:)1;0 9/:9S #=#8/ 0# (1)909 3& ) 8#9)9:1 #(=# ##1"#9 8) #")1:;0389>):('#80/)1#0;::)319)1@:A_'`1" :(#8#8#/933:(#8:;0389>):(>)/":@6#@:A_131[`:(: #1#&):&830) :8#:0#1:S(#8#&38#P:(#8#)91##":3"#=#/3683 ;9: )308.#89:3 #::#89#/# : [ "#&) )#1: :;0389 1" #?:#1" :(# ;9# 3& )1() ):)31 )1 1#> )1") :)319P 9 >#// 9 )"#1:)&@ )[8#9)9:1::;03891" 9:;"@ 30 )1:)31:8#:0#1:36:)319:(:#1(1 # /)1) /#&&) @1";:)/):@3&)S ##=/;:#":(# :)=):@3&:(#)3/68) )16:)#1:["#8)=#":;038?#13'8&:9_9` &830 6:)#1:9 >):( 8#9: 38 3=8)1 1 #8P 3:( >):( 1" >):(3;: ' 0;::)31P #?() ):)1' ")&#1:)/ 8#96319# :3 )S # 9:;")#" :(# !( 0!0) 0# (1)909 3& ) 8#9)9:1 #1"9#19):)=):@)1 :(#9#03"#/91":#9:#":(#&380:)313&qm1; /#8&3 ) @)00;13&/;38#9 #1 #9 )308.#83& &;1 :)31/):@1")8#96319#)191" 83;:)1# /)1) /906/#9S#/93:#9:#":(#1:):;038 :)=):@3&:(#m)mssq1" :(# ) lmqr 9 9)1'/# '#1: 1" )1 30 )1:)31 >):( ) )1 9S (# 0#9;8#0#1:3&8#6/) :)319:8#99 )308.#89>999#99#":39:;"@:(#0# (1)9093& :)313&:(#9#:8#:0#1:9:8:#')#9S ):()1:(#'961#/P13@:A9# 31"8@0;::)319>#8#&3;1")1:(#) 8#9)9:1:03"#/9Sm1; /#8&3 )>#8#"#:# :#")19)?3;:3&:#1)[8#9)9:1:9P )1.##6)1'>):(#?68#99)313&(@630386() m)93&3809S 3993&qom1"oq >#8# )"#1:)&)#" )1 :(8## 9P 31# 3& >() ( 31 30):1:/@ #?68#99#" 1 (@630386() m683:#)1S(# 30031&#:;8#)1//9>):(68)08@38 7;)8#")8#9)9:1 # >9 :(# &380:)31 3& qm 1; /#8 &3 )S 319)9:#1:/@P / . 3& qm &3 ) >9 />@9 993 ):#">):( /)1) /8#96319#:3))16:)#1:9:8#:#" >):(:(#9#'#1:9S(#1 9:;"@)1' :(# 0# (1)909 3& ) 9#19):)=):@ )1 :(# 131[' 3(38:P @ 68303:#8(@6#80#:(@/:)311"/:#8:)319)1 [8#/:#"'#1#9>#8#&3;1")1)[ 9#19):)=# 03"#/9S ')1P :(# ;1)7;# 30031 &#:;8# )1 // 9 :(: #?() ):#" :;038

8#'8#99)31;631):8#:0#1:)9:(# 9#1 #3&qm1; /#8&3 )S(#qm99@ 3;/" #6#8&380#")1;1:8#:#"906/#91">9()'(/@")9 8)0)1:)=#3&)9#19):)=):@ =#89;9 ) 8#9)9:1 # )1 ")&#1: 3(38:9 1" 3;:6#8&380#" :(# @8)"U9 0@(3) #f '#130) :#9:S 183;:)1# /)1) /906/#9&8306:)#1:9>):((#8#"):8@ 8#9:1"3=8)1 1 #8_ `9@1"830#P// n[8#/:#":;0389>#8# /99)&)#"9 ["#&) )#1: @:(#qm9 38#S 19P)8#9)9:1 #)1m[/:#8#":;0389 3;/" #8#=#8:#";631 30 )1:)313&)>):(m38)1() ):3891" 3:( 30 )1:)31 9:8:#')#9 8#9;/:#" )1 #? #8 :#" )1"; :)31 3& 8#6/) :)31 9:8#99 _` )1 30 )1:)31[ 9#19):)=#9S ):(:(#8#9;/:93 :)1#")1:()9:(#9)9P): 1 # 31 /;"#":(:':;0389 ()#=# )8#9)9:1 # @9#=#8/0# (1)909 :(:8#9:38# &;1 :)31P//"#:# :#" @:(# 68#9#1 #3&qm1; /#8&3 )S()9&;1 :)31/99@/93#1 /#9:(#)"#1:)&) :)313& )[9#19):)=#131[':;0389)1"#6#1"#1:/@3&:(#0# (1)9093& ["#&) )#1 @P :(#8# @ #)1' 6830)9)1' )308.#8 :3 #::#8 9#/# : 6:)#1:9 &38 )1() ):)31 1" 83"#1:(#636;/:)31>(30@ #1#&):&830:()9:(#86@S;89:;"@/939;6638:9:(# /)1) /"#=#/360#1:3&) 30 )1:)3199; (9:(39#>):(m1")1() ):389 1"()'(/)'(:#":(#)1"; :)313&9:(#0-380# (1)9093& :)313&:(#9#"8;'9S

RESUMEN

39 )1() )"38#9 "# / #1A)0 3/@ _[8) 39` 63/)0#89 _)` 931 #&# :)=39 #1 #/ :8:0)#1:3 "# 1 #8#9 7;# 68#9#1:1 "#&# :39 #1 / 8#68 )41 "#/ 638 8# 30 )1 )41(304/3'_ `P)1 /;@#1"37;#//39 310;: )31#9#1/39'#1#9@ @A_@:A`P/39 ;/#968#9#1:1:0 )%1;10@389#19) )/)""7;)0)3:#86)9 7;# 683"; #1 "23 #1 #/ P 303 /9 9/#9 "# 6/:)13S # (1 "#9 8):3 ")9:)1:39 0# 1)9039"#8#9)9:#1 )/39)#1:;038#9 310;: )31#9'#80)1/#9#1@:A _'`P0)#1:897;#P6383:83/"3P#?)9:#13:839:;038#99)10;: )31#9#1@:A _13[` 7;# 9# #1#&) )1 "#/ :8:0)#1:3 31 )S 9*P #?)9:# / 1# #9)"" "# "#9883//8 ;1 )308 "38 83 ;9:3 7;# 6#80): ;1 0#-38 9#/# )41 "# :;038#9 "#&) )#1:#9 #1 68 #?:#1"#8 #/ ;93 "# /39 ) 1;#=9 )1") )31#9P / 0)903 :)#0637;#9#)"#1:)&) 1:;038#98#9)9:#1:#9/)1() ) )41"#68#9:;")8:#86)9 #1 30 )1 )417;#0#-38#1/#&) )@;93"#/39)#1/ /*1) S

1#9:#:8 -39##=/;4/ :)=)"""#/)3/68) #1?#13)06/1:#9"#:;038#9 "#8)="39"#6 )#1:#9_P*.!(.,!0./'),2() ,.-` 31 1 #8"#00; 3=8)3P:1:3' 30313['P039:81"3;18#96;#9:")#1 )//)1() ) )41 "#S1/3903"#/3999##9:;")831/390# 1)9039"#8#9)9:#1 )@9#19) )/)"" )!(0!0)69* 303/;:)/)"""#;1:% 1) "#)10;13&/;38#9 #1 )68"#:# :8 &3 391; /#8#9"#qm 303 )308 "38"#/&;1 )31/)"""# @8#96;#9: )P:1:3#19 303#10;#9:89 /*1) 9"#8;:)1S"#09P9#)1=#9:)'4/ :)=)"" 1:):;038/ "#/ )1() )"38 "# m mssq @ "#/ )1() )"38 "# lmqr #1 0313:#86)@#1 30 )1 )41 31)S#)1=#9:)'831/390# 1)9039"# )41"# #9:9 #9:8:#')9 :#86%;:) 9 ;:)/)A1"3 ")9:)1:39 08 "38#9 "# #9:8%9 8#6/) :)=3 _P ,*&!.!)(-.,--`S

1:8# /39 03"#/39 ' 8#9)9:#1:#9 ) 13 9# #1 31:8831 0;: )31#9 9# ;1"8)9 #1 @:A 303 0# 1)903 "# 8#9)9:#1 ) / :#86)S )1 #0 8'3P 9# 8#638:4/&380 )41"#&3 391; /#8#9"#m#19#)9"#/39")#A03"#/398#9)9:#1:#9 )P#1 31 38"1 ) 31/#?68#9)41"#683:#*19m()63048&) 9S1:8#9 8#9)9:#1:#9 )P 9# )"#1:)&) 4 / 6%8")" "# qom @ oq 303 0# 1)903 "# 8#9)9:#1 )P#9:1"368#9#1:#"#&380 31-;1: 31/#?68#9)41"#;1683:#*1m ()63048&) #1 ;13 "# /39 03"#/39S <1) 8 :#8*9:) 30<1 :3"39 /39 9

8#9)9:#1:#9)P@9#8#9)9:#1 )68)08)3"7;)8)"P&;#/ 6 )"""#&380 )41 "#&3 391; /#8#9"#/683:#*1qmS# ;#8"3 31#9:398#9;/:"39P/;9#1 )"# &3 39"#qm9#93 )4 318#96;#9: /*1) )#10;#9:89"#6 )#1:#9:8:"9 31#9:39'#1:#9S;1"39##9:;")831/390# 1)9039"#9#19) )/)"")#1/ 3/# )41"#913['P9#3 9#8=4/68#9#1 )"#()6#80#:)/ )41"#/68303:38"# m@/:#8 )31#9#13:839'#1#98#/ )31"39 31 #103"#/399#19) /#9)S )1#0 8'3P"#1;#=3/<1) 8 :#8*9:) 30<1:3"39/3998#9631"#"38#9/ :8:0)#1:3&;#/;9#1 )"#&3 391; /#8#9"#qmS/#19@3"#qm9#6;"3 8#/)A8#10;#9:8913:8:"9@039:849#8/:0#1:#")9 8)0)1:)=3#1:8#9#19) )/)"" @8#9)9:#1 ))P9;6#81"3/ 6 )""68#") :)="#/:#9:'#1%:) 30@(3) #f "# @8)"S 1 0;#9:89 /*1) 9 "# 8;:)1 683 #"#1:#9 "# 6 )#1:#9 31 9*1"830# "# 1 #8"#00@3=8)3(#8#"):8)3_ P ,!.,3,-.()0,!((,`P:3"39 /39:;038#98#/ )31"39 310;: )31#9#1 A9# /9)&) 831 303"#&) )#1:#9#1 638#/#19@3"#qmS)1/0#1:#P9#"#039:847;#/8#9)9:#1 )/:#86) 31) #1:;038#9 31/:#8 )31#9#1@ 6;#"# 8#=#8:)89# 30 )11"3#9:39'#1:#9 31;1 )1() )"38"#m3"#P@#10 9#9:8:#')9:#86%;:) 99#8#638:4;10@38 )1"; )41"##9:8%98#6/) :)=3#1/399#19) /#9/ 30 )1 )41S

398#9;/:"393 :#1)"39#1#9::#9)9"3 :38/6#80):#1 31 /;)87;#/39:;038#9' /3'81/8#9)9:#1 ))0#")1:#")#1:#90# 1)90397;#8#9:;81/&;1 )41"# 8#68 )41"#/638 P/3 ;/6;#"#"#:# :89#638/ 6 )"""#&380 )41"# &3 391; /#8#9"#/683:#*1qmS9:##19@3&;1 )31/:0 )%16#80):#)"#1:)&) 8 :;038#913[9#19) /#9)P"#&380)1"#6#1")#1:#"#/0# 1)9037;#683"; # / "#&) )#1 ) #1 S 38 :1:3P #/ #19@3 "# qm 8#68#9#1: ;1 )308 "38 6830#:#"38 68 0#-388 / 9#/# )41 "# 6 )#1:#9 7;# 6;#"1 #1#&) )89# "#/ :8:0)#1:3 31)P9* 30306/)8/63 / )41 1")":8# ) )8#9::#86)09 //"#/39 1 #8#98#/ )31"39 310;: )31#9#1@:AS 398#9;/:"398#638:"39 #1 #9:# :8 -3 :0 )%1 )06;/91 #/ "#9883//3 /*1) 3 "# #9:8:#')9 :#86%;:) 9 7;# 30 )1#1/39) 31)1() )"38#9"#m@P"#9: 1"3/)1"; )41"##9:8%9 8#6/) :)=3 30368)1 )6/0# 1)903"# )41"##9:39&80 39S

INTRODUCTION

1.CARCINOGENESIS

;0138'1)9098# 30639#"3&0;/:)6/# #//9:(: 31:)1;3;9/@)1:#8 ::3#19;8# 1380/ &;1 :)31 1" "#=#/360#1:S 1 0:;8#P (#/:(@ (;01 #//9P ")&#1:):)31 )9 )88#=#89) /#1" #//98#6/) :#1"")#:30)1:)1:(#)1:#'8):@3&:(#")&#1::)99;#9P >() ( 8# 319:1:/@ 8#1#>#"S (#1 1 '#" #// 1 13 /31'#8 &;1 :)31 1380//@P ): :)=:#91#&&# :)=#/@9;) )"# //#"636:39)9:3")#1".##6:)99;#9(#/:(@S1:(# 3:(#8(1"P:)99;#9:#0 #//98#:)1:(# )/):@:3")&#1:):#)1:3")&#1::@6#93&0:;8# #//91"8#:(#8#96319) /#:3#19;8#:(# #//;/8:;813=#8mS

Normal cell cycle

(# #// @ /#)9()'(/@8#';/:#"683 #99 @>() ( #//")=)"#9S 1# ( @ /#3& #// ")=)9)31P :(# )9 8#6/) :#" @ 36@)1' ):9 1; /#3:)"# 9#7;#1 # )1 683 #99 :(: )9 =;/1#8 /#:3)1:83"; ##88389S;8:(#8038#P #//98# 319:1:/@#?639#":3"0')1' & :389P 3:(#?3'#13;9_8"):)31P (#0) /9`1"#1"3'#13;9_&8##8") /9`P>() (0@ ;9#"0'#:(:1##"9:3 #8#6)8#":368#=#1::(#:8190)99)311"=3)":(# ;0;/:)31 3& #//9 >):( :(#9# /#9)319S 3 '#: :()9 3 -# :)=#P 1380/ #// @ /# )9 031):38#": (# .63)1:9:(:9#19##88389)1:(#9#7;#1 #1"9:36:(#683'8#99)31 :(83;'(:(# #// @ /#;1:)///"0'#98#8#6)8#"S 3>#=#8P:(#683 #99#9)1=3/=#")1 8#6)8 8# 13: />@9 6#8&# : 1" #=#1:;//@P #//9 >):( #88389 )1 :(# P //#" 0;::)319P0@66#8S

Key cellular processes relevant for cancer

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2.DNA DAMAGE RESPONSE

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Repair NHEJ HR alt-NHEJ/ SSA ICL SSB BER TLS NER MMR pathway MMEJ repair repair IR, ROS, X-linking radiomimetics Topo I Source IR, agents, Alkylating DNA Pol X-linking inhibitors Alkylating UV, of DNA radiomimetics, replication agents, proofreading agents H O , agents alkylating Topo II inhibitors, 2 2 X-linkers errors damage alkylating agents inhibitors antimetabolites, Topo I inhibitors agents FA core Damage complex DNA XPC MSH2, MSH3, Ku70/Ku80 MRNPARP%'% MRN PARP glycosylases, PCNA DDB2 MSH6, MLH1, sensors (FANCA, B, %'% C, E, F, G, APE1 CSA PMS2 % L and M) FANCD1 )")')")'ATM, ATR, [BRCA2] Signaling/ "MK2, ?% CtIP, D2, I RAD6 XPA, XPF mediator #%DNAPK ' ' BRCA1/BARD1, CtIP J [BRIP1] ' RAD18 RPA proteins BRCA2,%! PALB2, N [PALB2] '%RPA O [RAD51C] P [SLX4] ' RAD51 "*( " MUS81/EME1 XRCC4 (!, (!,SLX1/SLX4 XRCC1 XPG XLF ')! XRCC1 RTEL1 ,' PNKP ERCC1 LIG4 !" LIG3, LIG1 Shared with EXO1 Effector BLM ! ! RAD52, POLβ As for SSB REV1, POLH POLE APLF )$%$ CtIP HR, TLS, POLD proteins TOPOIII ?% others? FEN1, TDP1 repair POLI, POLK POLD1 Artemis %$!& POLQ and NER LIG1 POLQ %$!& Aprataxin, LIG1, LIG3 PAXX %' '&!PARI LIG1, LIG3A WRN # !"RECQL5 FANCJ, BLM

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Homologous recombination repair

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ATM/ATR CHK2

exon 11 S1423 NES NLS NLS S1387 S1524 P P P P S998 RING Coiled coil

BARD1 RAD50 RAD51 PALB2 Abraxas

BRCA2 CtIP BRIP1

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3.BREAST CANCER

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Germline BRCA

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4.PARP INHIBITION

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Mechanisms of PARPi resistance

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Mechanisms of PARPi resistance that restore HRR functionality

RESISTANCE THROUGH SECONDARY MUTATIONS

# 31"8@'#1#:) 8#=#89)31#=#1:9:(:8#9:38#:(#0;::#"'#1#&;1 :)313&@38 A 0@ "#8)=# 7;)8#" 8#9)9:1 # :3 )mopPmouPmplS (#9# 9# 31"8@ )1:8'#1) 0;::)3193 ;8#):(#8 @'#1#:) #=#1:9:(: 1 #/:(#&80#9()&: ;9#" @:(#38)')1/ 0;::)311"8#9:38#"#36#1[8#")1'&80#_`38 @'#1#:) 8#=#89)313&:(#)1(#8):#" 0;::)31P8#9:38)1':/#9:1#8/@&;//[/#1':(683:#)1S(#9#9# 31"8@0;::)319(=# ##1 )"#1:)&)#" )1 6/:)1;0[ 1"X38 )[8#9)9:1: #// /)1#9 1" 6:)#1:["#8)=#" #//9mluPmmoPmouS 38#3=#8P :()9 0# (1)90 3& 8#9)9:1 # (9 ##1 )"#1:)&)#" )1 @:A[ 0;::#" :;0389 &830 6:)#1:9 >(3 (=# "#=#/36#" /)1) / 8#9)9:1 # :3 ) 8399 0;/:)6/# 1 #8:@6#9opPmntS# 31"8@0;::)319(=# ##1/93")9 3=#8#")13:(#8 '#1#9P)1 /;")1'D@PD@1" AP8#9:38)1':(#>)/":@6#9#7;#1 #1"X38 &;1 :)31 3& :(# 683:#)1 1" 6 ):@P :(#8#&38# )1"; )1' /)1) / 8#9)9:1 # :3

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RESISTANCE THROUGH HYPOMORPHIC BRCA1 PROTEINS

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RESISTANCE THROUGH REVERSION OF EPIGENETIC SILENCING

6 ):@8#9:38:)311")8#9)9:1 # 1/93 # ;9#" @#6)'#1#:) (1'#9)1 [8#/:#" '#1#9P 9; ( 9 8#=#89/ 3& @ 68303:#8 (@6#80#:(@/:)31mouS ()9 0# (1)90 13 ;8)1:(39#:;0389:(:8#68)08@9#19):)=#:3)1() ):)31:(83;'( /3>@#?68#99)31 ;9#" @#?:#19)=#68303:#80#:(@/:)31S(#/3993&68303:#8

0#:(@/:)31/#":38#[#?68#99)313&@'#1#: 3068 /#/#=#/9:3 683&) )#1: :;0389 1"P :(#8#&38#P :(#9# :;0389 # 30# )[8#9)9:1:S #:(@/:)31 0@ # "# 8#9#"98#9;/:3&1 :)=#"#0#:(@/:)31#=#1:P ;:/93&830:;038(#:#83'#1#):@ )1>() (:(#:;038 #//9>):(/#9968303:#80#:(@/:)31;1"#8'3639):)=#9#/# :)31;631 #?639;8# :3 )mouS #=#89/ 3& @ (@6#80#:(@/:)31 >9 3 9#8=#" )1 7;)8#" 6/:)1;08#9)9:1:3=8)1 1 #89P&;8:(#8"#0319:8:)1':(: 1 #8 #//90@8#[#?68#99 9)/#1 #"@98#9)9:1 #0# (1)90mpsS

RESISTANCE THROUGH REMOVAL OF A BARRIER TO DNA END RESECTION

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Mechanisms of PARPi resistance independent from HRR functionality

RESISTANCE THROUGH MITIGATION OF REPLICATION STRESS AND SLOWED CELL CYCLE PROGRESSION

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OTHER MECHANISMS OF RESISTANCE

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9)1() ):)31 /3 .9:(##1A@0:) :)=):@3&#1A@0#9P"# 8#9#"#?68#99)313& :(#9#683:#)19P:3'#:(#8>):(0;::)319)1:(# :/@:) P[38"8;'[ )1")1'"30)19P 0@ #13:(#80# (1)903&8#9)9:1 #:3)mntPmppS3>18#';/:)31380;::)319)1 m8#03=#:(# )/):@:3:86m31:3P:(#8#&38# 31X)1'8#9)9:1 #)1 :;0389mssPmstS)0)/8/@P0# (1)909:(:)1 8#9#@/:)313&#1A@0#9P9; (9/399 3&P0@/#":3)8#9)9:1 # @"# 8#9#":866)1':(83;'(8#/#9#3& m&830mntS

(# (/&0)/@0#0 #8mm_ mm`(9 ##1683639#"968#") :)=# )308.#83& 9#19):)=):@:3)0313:(#86@)190// #///;1' 1 #8_ `msuS 1& :P()'(683:#)1 /#=#/93& mm68#") :#"9#19):)=):@:36/:)1;0 9#"'#1:91"))168# /)1) / 03"#/9 1" /)1) / :8)/9mtlPmtm 1"P :(#8#&38#P mm )1 :)=:)31 1 31 ) 8#9)9:1 #P683 /@:(83;'()1 8#9#""#6#1"#1 #31:(#[ m (# .63)1:msuPmtnPmtoS

)1//@P#?:#19)=#:;038"#9036/9)(9/93 ##19;''#9:#"90# (1)903&) 8#9)9:1 #)1:;038 #//9>):(3;:8#=#89)313& 6 ):@P1"(9 ##1993 ):#" >):( (#038#9)9:1 #1"9; 36:)0/"8;';6:.#)16839::# 1 #8_`mpsS

#!( &*&'$"#& &'(#&(#" # -%#!#&%819 #+"'(&! (&"' (#" "((#" 38=84

)("( %&#(" ,#" '% " )("( " )"(#" " 38884

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*&,%&''#" # #&! '(&#! '!#% '( '(&#!

%#30 LY $,%1+1 -" 0 1%12, 2- %,$%%2-01= #9)9:1 # :3 ) 1 # )1(#8):#" 38 7;)8#"1":(#63:#1:)/0# (1)9093&)8#9)9:1 # 1 # /99)&)#")1:(8##0)1 :#'38)#9R _)`8#9:38:)313&(303/3'3;98# 30 )1:)318#6)8_ `:(83;'( 3:(")8# :1")1")8# :0# (1)909P _))`0):)':)313&8#6/) :)319:8#99_` 30031/@:3'#:(#8>):(9/3>#8 #// @ /#683'8#99)31P1"_)))` 0# (1)90913: ;88#1:/@8#/:#">):(6:(>@ ;:9:)///:#8:(#8#96319#:3)S9#"31mntS

PARPi beyond BRCA

9)(=#"#0319:8:#" /)1) /#&&) @)1':;0389P8#9#8 ((9&3 ;9#"31 )"#1:)&@)1'""):)31/6:)#1:636;/:)319>(30@ #1#&):&830:(#9#"8;'9mtpPmtqS9/[ /).#1"@[0;::#":;0389(=# /)1) 36:(3/3') /1"03/# ;/89)0)/8):)#9P 9; (99#19):)=):@:36/:)1;0[ 9#" (#03:(#86@P0;::)319)1DBP")0)1)9(#"m 683:#)1 #?68#99)31tnPmtr 1" 6::#81 3& '#130) )19: )/):@ (8 :#8)A#" @ //#/) /399ssPmtsP939)0)/8#:)3/3'@)9683639#"&38:(#9#'83;693&tmPmtt\mulS30#3&:(#9# 9)0)/8):)#98#8#/:#">):(:(#68#9#1 #3&(303/3'3;98# 30 )1:)31"#&) )#1 @_ ` /93)1131[&0)/)/ 1 #89S()96(#13:@6#)9 //#"1#99P>() (8#Y:39638") _'#80/)1#@:A>)/":@6#` 1 #899(8)1':8):9>):(&0)/)/:;0389P)168:) ;/8 :(# "#&# :tnPmtqP >() ( 1 8)9# :(83;'( 81'# 3& '#130) P #6)'#1#:) 38 639:[ :819/:)31//:#8:)319tnS

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5.BIOMARKERS OF RESPONSE TO PARPi

8# )9)31 0#") )1# 6683 (#9 (=# :(# 63:#1:)/ :3 )0683=# 1 #8 :8#:0#1:9 @ )"#1:)&@)1' 68)38) :(# 9; 9#: 3& 6:)#1:9 039: /).#/@ :3 #1#&): &830 96# )&) :8'#:#" :(#86)#9P)1 /;")1')nltS 1:()99#19#P:(#"#=#/360#1:3& )308.#89:(:)1:#883':# &38 :8#:0#1: 8#96319# 683=)"# 83 ;9: :33/ &38 /)1) / "# )9)31S 1 :(# &)#/" 3& )1() ):389P/:(3;'('#80/)1#38930:) @:A0;::)319(=# ##1;9#":3#18) (&38 8#9631"#89P :(#8# 8# 6:)#1:9 >):( @:A[0;::#" 1 #89 :(: "3 13: 8#9631" :3 )S38#3=#8P9#?6/)1#"P:(#8#)9/939; 9:1:)/1;0 #83&@:A>)/":@6# 6:)#1:9>(3/ .:(#9#0;::)3191"0@ #1#&):&830)0313:(#86@mntS(#8#&38#P =/)":#":#9:9:(://3>68#") :)1:):;038 :)=):@8#1)0638:1: /)1) /1##"P ;::(#8#)9 ;88#1:/@13 ;8:# )308.#893&8#96319#:3)mppS#=#8/6683 (#9 :3 68#") : (9 ##1 683639#"P >):( ")&#1 #9 )1 :(#)8 6 )/):@ )1 68#") :)1'

8#96319#:3)1"63:#1:)/ /)1) /;:)/):@_* G`mppPnllPnluS

Platinum sensitivity

# ;9#9#19):)=):@:36/:)1;01")8# 3:(993 ):#">):( "#&# :9P6/:)1;0 9#19):)=):@ (9 ##1 ;9#" 9 9;883':# 3& ) 8#96319#nlu 1" 930# :8)/9 (=# "#0319:8:#" #1#&): :3 ) :8#:0#1: )1 // 6/:)1;0[9#19):)=# 3=8)1 1 #89nmlS 3>#=#8P ): )9 .13>1 :(: 6/:)1;0 1" ) 8#96319)=#1#99 8# 13: />@9 3=#8/66)1'nmmS 38#?06/#P:;0389>):("#&# :9)1:(#1; /#3:)"##? )9)318#6)8_` 6:(>@8#9#19):)=#:36/:)1;0:(#86@ ;:8#0)18#9)9:1::3)1() ):)31nmnP1" :(#8#8#/936/:)1;0[8#9)9:1: 1 #89:(:8#9631":3)mmoS

Biomarkers based on gene mutation analysis

Germline BRCA1/2 mutations

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Targeted sequencing of HRR-related genes

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Genomic scars and mutational signatures

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Limitations of genomic assays

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Dynamic biomarkers

Transcriptional profiles and protein expression

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Functional assays

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Other biomarkers

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6.TARGETING DDR IN COMBINATION WITH PARPi

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Combination with DDR inhibitors

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Effect is manifest M G1

G2 S Maximize DNA damage Prevent repair

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WEE1 inhibition

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HYPOTHESIS AND OBJECTIVES

Hypothesis 1

$ 0 12-02%-,-" .%27%1"0 /3 ,2+ $,%1+-"0 1%12, 2-%,$%%2-01 %,# 0+*%, @+322 23+-01=

3:#9::()9(@63:(#9)9P:(#&3//3>)1'3 -# :)=#9>#8##9: /)9(#"R

• #=#/3661#/3&6:)#1:"#8)=#"?#13'8&:9_`&830 8#9:1"3=8)1 1 #8 6:)#1:9>):('#80/)1#0;::)319)1@:A'#1#9_'` • 1=#9:)':# :(# 1:):;038 :)=):@ 3& :(# ) 3/68) )1 :(# 61#/ 3& ' 03"#/9 • (8 :#8)A#:(#0# (1)9093&8#9)9:1 #:3))1:(#61#/3&'03"#/9

Hypothesis 2

$ 0 12-02%-, -" .%27 %, # 23+-01 , + 130 7 2$ %++3,- 2 2%-, -" JF ,3* 0 "-% , 2$%1 117 , 31 1 "3,2%-,* %-+0) 0-"%0 1.-,1 =

3:#9::()9(@63:(#9)9P:(#&3//3>)1'3 -# :)=#9>#8##9: /)9(#"R

• #=#/36 1 )00;13&/;38#9 #1 # 99@ :3 "#:# : qm 1; /#8 &3 ) &380:)31 )1 &380/)1&)?#"68&&)1#0 #""#"_`:;038906/#9 • /)":#:(# 6 ):@3&:(#qm)00;1399@:368#") :)8#96319#)1:;038 906/#9&83091"6:)#1:9

Hypothesis 3

$ JF 117 **-51 2$ % ,2%"%2%-, -" @ "%% ,2 23+-01 5%2$ ,- +322%-,12$2+7 , "%2"0-+%,$%%2%-,;**-5%,#2- 62 ,2$ 31 -"% 20 2+ ,2 7-,2$ #-,%2%-,=

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• #=#/3661#/3&9&8306:)#1:9>):(>)/":@6#@:A_131['` • 1=#9:)':#:(#1:):;038 :)=):@3&:(#)3/68) )1:(#61#/3&131[' 03"#/9 • (8 :#8)A# :(# 0# (1)909 3& 9#19):)=):@ :3 ) )1 :(# 61#/ 3& 131[' 03"#/9 • /)":#:(#qm99@9 )308.#8:368#") :1:):;0388#96319#:3) • 3068# :(# 68#") :)31 6 ):@ 3& :(# qm 99@ >):( 13:(#8 ;88#1:/@ ;9#" '#130) )308.#83&)8#96319# • :;"@ :(# &#9) )/):@ 3& :(# qm 99@ )1 83;:)1# :;038 906/#9 &830 6:)#1:9P )1 /;")1' n[8#/:#" 8#9::;0389

Hypothesis 4

$ %,$%%2%-,-"F,-2$ 0.0-2 %,1%,0 1 12$ * 4 *1-"0 .*%2%-,120 11 ,%+.0-4 1,2%23+-00 1.-,1 2-%5$ ,31 1-+%,2%-,1202 #7=

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METHODS

Generation of patient-derived xenograft (PDX) models

8#9(:;038906/#9&830 8#9:383=8)1 1 #86:)#1:9>):(_1zmm`38>):(3;:_1zmp` ' 0;::)31 >#8# 3//# :#" 68396# :)=#/@ &38 )06/1::)31 )1:3 1;"# 0) # : // "U # 831 19:):;:#3&1 3/3'@_ P6)1`;1"#81)19:):;:)31/8#=)#> 38"_ `[ 6683=#"683:3 3/1":(#993 ):#")1&380#" 319#1:P38 @:(#:)31/#9#8 (:() 9 #8=) #P0 8)"'#9()8#n_8##1 #1;0 #8RltX loltXmst`S(##?6#8)0#1:9 >#8# 31"; :#"&3//3>)1':(#;836#11)31U91)0/ 8#")8# :)=#_nlmlXroX`1" >#8#6683=#" @:(#:() /300)::##3&1)0/?6#8)0#1::)313&:(#//"U # 831 #9#8 ( 19:):;:#S8#9(68)08@380#:9::) (;01 8#9::;0389>#8#3 :)1#"&830 6:)#1:9::)0#3&9;8'#8@38 )369@1")00#"):#/@)06/1:#")1:3:(#0008@&:6" _9;8'#8@906/#9`38:(#/3>#8&/1._0#:9::) 906/#9`3&r[>##.[3/"�/#:(@0) 9"6 R [3?1m1; _ 8/1 38:38)#9` 38 S'[8." 9 )" /n8':0m-/XA _(8/#9 )=#8`0) #S1)0/9>#8# 31:)1;3;9/@9;66/#0#1:#">):(mmsβ[#9:8")3/_)'0[ /"8) (` )1 :(#)8 "8)1.)1' >:#8S 631 '83>:( 3& :(# #1'8&:#" :;0389P :(# 03"#/ >9 6#86#:;:#" @9#8)/:8196/1::)3131:3:(#/3>#8&/1.S 1# (699'#P&/9([&83A#1 1" &380/)1[&)?#" 68&&)1 #0 #""#" _` 906/#9 >#8# :.#1 &38 '#13:@6)1' 1" ()9:3/3') / 9:;")#9S (8## 03"#/9 _n P mou 1" omr 1" m [639):)=# P nlm` >#8# '#1#8:#" )1 XP 0#0 #8 3& :(# ;8 31938:);0 _(::6RXX>>>S#;836"?S#;`P 9 68#=)3;9/@ 8#638:#"nruS 3;8 03"#/9 >):( 7;)8#" )[ 8#9)9:1 #>#8#'#1#8:#")1:(#/ 38:38@_9## #/3>`S

In vivo experiments of PDX sensitivity to PARP, WEE1 and ATM inhibitors

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Analysis of the BRCA1/2 genes and loss of heterozygosity in PDX tumors

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06/)&) :)31 3& >):( #?31 m 3/)'31; /#3:)"# qU[[oU 1" #?31 ml 8#=#89# 3/)'31; /#3:)"# qU[ [oUS 30#1 /:;8# 3& =8)1:9 >9 38")1' :3 :(# ;01 #130# 8):)31 3 )#:@S ()9 1/@9)9 >9 6#8&380#")1 3// 38:)31>):(:(#1 3'#1#:) 983;6: S

Analysis of BRCA1 mRNA expression

>9#?:8 :#"&830906/#9_mq[ol0'` @;9)1':(##8&# :;8#)99;#.): _q8)0#`S(#6;8):@1")1:#'8):@>#8#99#99#" @:(#')/#1:nmll)31/@A#89@9:#0 1" >93 :)1#";9)1':(#8)0# 8)6:#'#1:.):_.8`S;1:)::)=#[ >96#8&380#")1sull 9:#/[)0#@9:#0_66/)#")39@9:#09`;9)1'71 1)=#89/9:#8)? _66/)#")39@9:#09`1"68#"#9)'1#"(;0196# )&) 68)0#891" 71 683 #9 _ 9uuuuuult^0m &38 P 9uuuuuulo^0m &38 1" 9lmqqrmuo^0m&38m`S(# 3068:)=#0#:(3">9;9#"&38":1/@9)9P)1 >() ('#380/'38):(09>#8#66/)#":39#/# ::(#039:9: /@#?68#99#"(3;9#.##6)1' '#1#9_1"`1"'#30#:8) 0#19>#8# / ;/:#":33 :)11380/)A#" =/;#9nsnS

Exome sequencing (PDXs cohorts 1 and 2)

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38/=)//#P P`S)8#"#1"mql 69#7;#1 )1'>96#8&380#"311 //;0)1 )#7 plll;9)1'8;#78#'#1:9_ //;0)1`>):(6683?)0:#/@ml 66#8906/#&38cnll[ &3/" =#8'# 9#7;#1 # "#6:(S : 1/@9)9 &3//3>#" 9:1"8" 0#:(3"3/3')#9S 8)#&/@P 9#7;#1 )1' 8#"9 >#8# /)'1#" :3 3:( (;01 ('mu 1" 03;9# 00ml '#130#9 ;9)1' ;883>9[(##/#8/)'10#1:_`P:(#103;9#["#8)=#"9#7;#1 #9)1:(#(;01S 0&)/# >#8#8#03=#";9)1')90 )';:#nsoS8)1:9>#8# //#")1:(#(;01S 0&)/#9;9)1' 8)8# :nspS36@1;0 #81/@9)9>96#8&380#";9)1'#7nnsqS

BRCA1 promoter methylation

>9)93/:#"9"#9 8) #"1"9#1::3:(#)=)9)313&3/# ;/8:(3/3'@1"1 #8 #130) 9 : (# #:(#8/1"9 1 #8 19:):;:#S @ 68303:#8 0#:(@/:)31 >9 0#9;8#" ;9)1' #:(@/:)31[96# )&) _[ ` _ 3//1"P 09:#8"0P :(# #:(#8/1"9` 38")1':301;& :;8#8U9)19:8; :)319S(#:>3?#13'8&:9'#1#8:#")1 X _mou 1" nlm` (" ##1 68#=)3;9/@ :#9:#" ;9)1' 8#"; #"[ 8#68#9#1::)31 )9;/&):# 9#7;#1 )1' _`nru 1" &;8:(#8 =/)":#" ;9)1' [ S 39):)=# 31:83/93&@68303:#8(@6#80#:(@/:#">#8#;9#"nsr_mns1"Xmrn`S

Immunofluorescence experiments

(# &3//3>)1' 68)08@ 1:) 3")#9 >#8# ;9#" &38 )00;13&/;38#9 #1 # _ `R 8 ): 1:)[ qm_1:8;A)3:# (13/3'@9 [topumRnql`P8 ):1:)[qm_ 0 mooqopP mRmlll`P 03;9# 1:)['#0)1)1 _3=39:8 [ P mRmll )1 906/#9P mRrl )1 6:)#1: 906/#9`P8 ):1:)['#0)1)1_83:#)1# (mltln[m[PmRpll`P03;9#1:)[m_1: 8;A )3:# (13/3'@ 9 [ruqpP mRql`P 03;9# 1:)[m _ 0 mrstlP mRnll`P 03;9# 1:)[γ n _)//)638# ilq[rorP mRnll`P 8 ): 1:)[qom _#// )'1//)1' ipuosP mRmll`P 8 ): 1:)[6(396(3[on pXt _6 pXtP #:(@/P oll[npqP mRqll`P 8 ): 1:)[ 6(396(3[[ nlqr_6[ P 0 mtmunPmRnll`P8 ):1:)[6(396(3[()9:31# oml_6 oP#//)'1//)1'iuslmPmRmll`S3:1:)[8 ):/#?&/;38qrtP'3:1:)[ 03;9#/#?&/;38pttP"31.#@1:)[03;9#/#?&/;38qrt1"'3:1:)[8 ):/#?&/;38 tl

ptt_ 1=):83'#1QmRqll`>#8#;9#"99# 31"8@1:) 3")#9S38:8'#:1:)'#18#:8)#=/P 9# :)319>#8#0) 83>=#"&38p0)1;:#9:mmlE)1 1:)'#1#:8)#=/;&V uSl _6 rSl &38 qom 1" 6 o 9:)1)1'` )1 X 0;/:)&;1 :)31/ ) 83>=# )9:3683 #9938_)/#9:31#`S# :)319>#8# 33/#""3>1)1")9:)//#">:#8&38q0)1;:#9P :(#16#80# )/)A#">):( 9(;&_ 31:)19>##1nl`&38q0)1;:#9P&3//3>#" @)1 ; :)31)1 /3 .)1' ;&_ 9(;&>):(mx 3=)1#9#8;0/ ;0)1`&38q 0)1;:#9S8)08@1:) 3")#9>#8#")/;:#")1 1:) 3"@)/;#1:1")1 ; :#":8330 :#06#8:;8#&38m(3;8S# :)319>#8#>9(#"&38q0)1;:#9)1 9(;&&3//3>#" @q0)1;:#9)1 /3 .)1' ;&S# 31"8@1:) 3")#9>#8#")/;:#")1 /3 .)1' ;&1" )1 ; :#"&38ol0)1;:#9:8330:#06#8:;8#S(#n[9:#6>9()1'>98#6#:#"&3//3>#" @ q 0)1 )1 ; :)31 )1 ")9:)//#" >:#8S #(@"8:)31 >9 6#8&380#" >):( )1 8#9)1' 31 #1:8:)319 3& #:(13/S # :)319 >#8# 03;1:#" >):( 83 31' 3/" 1:)&")1' 8#'#1:1"9:38#":[nlES 00;13&/;38#9 #1 #)0'#9>#8# 7;)8#";9)1'/@06;9 sn0) 839 36#1"'#1#8:#";9)1'#//#191:8@93&:>8#S

Immunofluorescence scoring

38)1'>96#8&380#" /)1"/@31:3/)&#)0'#9;9)1'rl?[)00#89)313)//#19S:/#9:n )3/3') /8#6/) :#93&# (03"#/1":8#:0#1:>#8#1/@A#"S ; /#8 &3 )3&lSpn :3 mSmq μ0 ")0#:#8 >#8# 7;1:)&)#" 31 :;038 906/#9S 38 qm 9 38)1'P :(# 6#8 #1:'#3&'#0)1)1[639):)=# #//9>):(q38038#qm1; /#8&3 )>97;1:)&)#"S #0)1)1>9;9#"9 3;1:#89:)1)1':308.&38Xn[ #// @ /#6(9#nssS1#[(;1"8#" '#0)1)1[639):)=# #//9&830:/#9:o8#68#9#1::)=#8#93&# (906/#>#8#1/@A#"S 38:(#6(80 3"@10) 9:;")#9P:(#/#=#/93&"0'#1"8#6/) :)319:8#99>9 7;1:)&)#"31:;038906/#9 @9 38)1':(#6#8 #1:'#3&'#0)1)1[639):)=# #//9>):( γ n 38 6on pXt 1; /#8 &3 )P 8#96# :)=#/@P 9 "#9 8) #" &38 qm 9 38)1'S #6/) :)319:8#99/#=#/9>9/937;1:)&)#" @9 38)1':(#6#8 #1:'#3& #//9>):(61[ 1; /#8γ n9:)1)1'S(#6#8 #1:'#3& #//9)10):39)938Xn[6(9#3&:(# #// @ /# >9 7;1:)&)#" @ 9 38)1' :(# 6#8 #1:'# 3& #//9 >):( 6 o 38 '#0)1)1 9:)1)1'P 8#96# :)=#/@S

ATM immunohistochemistry

389:)1)1'P:(#90#683:3 3/9&38 99@>96#8&380#")1 3// 38:)31>):(

9:8#1# S)99;#9>#8#""):)31//@ /3 .#")1#1"3'#13;96#83?)"9# /3 ._ox nn

)1" n`&38ml0)1S ):1:)[_ 0 onpnlPmSn'X0 `>9;9#"968)08@ 1:) 3"@S 1:) 3"@ "#:# :)31 >9 6#8&380#" >):( 1=)9)31 &38 ol 0)1 >):( =)9;/)A:)31 ;9)1' 93/;:)31 &38 ml 0)1 1" 3;1:#89:)1)1' )1 8AA)U9 (#0:3?@/)1S>99 38#" @)0'#1/@9)9;9)1' 1") 9 P @;:30:#" :)99;# /99)&) :)313&:;0388#')3191"1/@9)93&6#8 #1:'#639):)=#:;0381; /#)S

Reverse phase protein array (RPPA) analysis

1/@9)9>96#8&380#")1:(#1"#89311 #8#1:#89"#9 8) #"nstPnsuS :;038683:#)19>#8#"#1:;8#" @mx_>X=`)1:(#68#9#1 #3&G[0#8 6:3#:(13/ 1""-;9:#":3&)1/ 31 #1:8:)313&m0'X0/S06/#9>#8#")/;:#")1&)=#9#8)/n[&3/" ")/;:)319)1")/;:)31 ;&_/@9)9 ;& 31:)1)1'mx`1"88@#"311):83 #//;/39#[ 3:#"9/)"#9_8 #)3/ `;9)1'1;9(31npsl88@#8_;9(31)3@9:#09`S (9/)"# >9 683 #" >):( =/)":#" 68)08@ 1:) 3"@ 6/;9 )3:)1[ 31-;':#" 9# 31"8@ 1:) 3"@S(#9)'1/>906/)&)#";9)1'.3@:30:)31\ :/@A#"9@9:#0_.3`1" =)9;/)A#" @ 3/38)0#:8) 8# :)31S/)"#9>#8#9 11#"P1/@A#"1"7;1:)&)#";9)1' ;9:30)A#"[93&:>8#) 83=)'#1#_)'#1## ( 1 S`:3'#1#8:#963:)1:#19):@S

PDX for RAD51 assay validation (PDX cohort-3)

&830 3(38:[o>#8#'#1#8:#":;8)# 19:):;:#_8)9P81 #`1"3/)/0#::# 19:):;:#_89#)//#P81 #`;1"#86683=#")1&380#" 319#1:S(#0-38):@3&:(#9#9 >#8#68#=)3;9/@6; /)9(#"ntlPntmS ( 0!0) #?6#8)0#1:9 >#8#6#8&380#": #1:# (S (#1 :;0389 8# (#" 9)A# 3& sl[nql 00oP 0) # >#8# 81"30/@ 99)'1#" :3 (303'#1#3;9 '83;693&q:3ml1)0/91">#8#:8#:#"6S3S>):(1)868) _ql0'X.'38sq0'X.'`P 3/68) _ql0'X.'38mll0'X.'`38=#/)68) _mll0'X.'`")/@&38nt"@9S;038=3/;0# >9#=/;:#" @0#9;8)1' )>##./@:;038")0#:#89>):( /)6#8S tn

HRD score

@8)"d90@(3) #f #9:>96#8&380#":@8)"#1#:) 931#?:8 :#"&830 93& 3(38:[oS#?:8 :)31>96#8&380#":#1:# (;9)1':(#; /#331"mll .):_ (#8#@['#/`S

73-gene profiling of PDX cohort-3

;::)31683&)/)1'3&so'#1#9031':(#039:&8#7;#1:/@0;::#")1 1 #8 38")1':3 :(# ": 9# >9 6#8&380#" : _#)-)1'P ()1` 31 '#130) @ #?31 :866)1' >):( )0 /##1 0) 8388@ &3//3>#" @ "##6 9#7;#1 )1' @ ;9)1' //;0)1U9 )#7:# (13/3'@P>):(:/#9:ql#&&# :)=#0#1"#6:(&38# (906/#S()99:;"@>9 6#8&380#")1 3// 38:)31>):(#1:# (S

Cell lines

(#&3//3>)1'!(0!.,)9:;")#9>#8#6#8&380#"::(##130#: )/):@ 38:38@: "# ;% # #9#8 ( #1:#8 )1 3// 38:)31 >):( #1[=#9 931P 1"@ ; @ 1" 7;#9)08"Sn39:#398 30 #//9_ [ur`>#8#6;8 (9#"&8300#8) 1@6# ;/:;8#3//# :)31_`1"0)1:)1#")1 3@U9q_) 3`9;66/#0#1:#">):(mlx #:/3=)1##8;0_`1"mx6#1) )//)1X9:8#6:30@ )1_X`S # #//9>#8#0)1:)1#" )10#");0_381)1'`9;66/#0#1:#">):(mlx1"mxXS// #///)1#9>#8# 83;:)1#/@:#9:#":3 #0@ 36/90&8##S

Cas9/mClover-LMNA homologous recombination assay

(# 0/3=#8[ (303/3'3;9 8# 30 )1:)31 99@ >9 "6:#" &830ntnPntoS 1 8)#&P n #//9>#8#9##"#":msqPlll #//96#8>#//)1r[>#//6/:#9:3 #:819&# :#"r(3;89 /:#8>):( 31:83/38 A9):&)1/ 31 #1:8:)313&ql1;9)1' )63&# :0)1# )_ 1=):83'#1`S>#1:@[&3;8(3;89639:[:819&# :)31Pm?mlr #//96#8 31"):)31>#8# 6#//#:#" 1" 8#9;96#1"#" )1 mllK 306/#:# 1; /#3&# :38 93/;:)31 _ #// )1# p[ to

; /#3&# :38g ):P 31A` :3 >() ( m K' 3& 6nSm[/3=#8 "3138P m K' 6ool[ 'P m K' 3& :(# )1") :#" A 319:8; :P lSm K' 3& 6)rsl[m _;9#" 9 :819&# :)31 31:83/`P1"nllK03/3&9)>9""#"S1 #:819X#":3mllK/ 31A #8:)&)#" ;=#::#P #//9>#8#:819&# :#";9)1':(#p[; /#3&# :38[;1):P683'80[ mlpP)00#"):#/@8#9;96#1"#")1 ;/:;8#0#")1":819X#":3mll00")9(&38rp (3;89S(#1PqllPlll #//9>#8#6/:#"31:3'/99 3=#89/)69>()/#:(#8#0)1)1'>9/@9#" &38>#9:#81 /3::)1'9"#9 8) #" #/3>S3=#89/)69>#8#&)?#">):(px68&380/"#(@"# 1" 1/@A#" &38 /3=#8 #?68#99)31 @ &/;38#9 #1 # 0) 839 36@ :3:/ 3& sn ( 639: 1; /#3&# :)31S

Localization of PALB2 to laser-induced DSBs

(##?6#8)0#1:9>#8#6#8&380#"9"#9 8) #")1ntpS8)#&/@P # #//9>#8#:819&# :#" >):( [ n[ 38 [ n[6Snur&9 1" 0) 83)88"):#" /31' :8 . )1 #// 1; /#)S(#8# 8;):0#1:3&[ n:3/9#8[)1"; #""0'#9):#9>9031):38#" 3=#8:)0# @)0')1' #//9#=#8@n0)1&38m(S(#&/;38#9 #1 #)1:#19):@3&:(#)1") :#" 319:8; :9:"0'#9):#98#/:)=#:31;1)88"):#"8#>97;1:)&)#"1" 6/3::#"3=#8:)0#S

Protein extraction and western blotting

/9([&83A#16)# #93&:;038>#8#/@9#")1) #[ 3/" ;& 31:)1)1'8)9\ /6 sStnl 003/X P/mos003/X P6 tSln003/X PplmxP'/@ #83/mlxP9;66/#0#1:#"

>):( ml 003/X P #;6#6:)1 ml 0'X0 P np nll 003/X P q 003/X P 1" 683:)1)1 _)'0[/"8) (`S 303'#1)A:)31 >9 6#8&380#" 31 ) # >):( f 9@9:#0mnll_ )1#0:) `S @9:#9>#8# #1:8)&;'#":moSlll860pE";8)1'ol0)1 1":(#9;6#81:1:9>#8# 3//# :#"S83:#)1 31 #1:8:)31>9 / ;/:#";9)1' 83:#)199@_)3["`S:3:/3&ol'3&683:#)1>#8#9#68:#"31mnx[: mll1":819X#":31):83 #//;/39#0#0 81#&38mSq(:mllS#0 81#9>#8# /3 .#"&38m()1qx0)/.)18)9[ ;&#"9/)1#_`[>##11":(#1(@ 8)")A#";9)1' tp

:(#68)08@1:) 3")#9)1qx[>##1R8 ):1:)[qm_1:8;A)3:# (13/3'@ [unPmRqll`P8 ):1:)[(;01 _ 0 mntumqPmRqlll`P03;9#1:)[:; ;/)1 _)'0[/"8) ( [ulnrP mRqlll`S 3;9# 1" 8 ): (389#8")9( 6#83?)"9# _ `[ 31-;':#"9# 31"8@1:) 3")#9_ #/:( 8#PmRnlll`>#8#")/;:#")1qx0)/.)1[ >##1 1" 683:#)19 >#8# "#:# :#" >):( 003 )/31 #9:#81 (#0)/;0)1)9 #1: 9; 9:8:#_)//)638#`S 0'#9>#8# 6:;8#">):( [plll 0#89@9:#01" 7;1:)&)#">):( 0'# _(::6RXX89 S)1&3S1)(S'3=X)-X`S

38mP:;038/@9:#9>#8#0"#)1 /@9)9 ;& 306/#0#1:#">):(n306/#:# 683:#9# )1() ):38 3 .:)/ _3 (#` 1" #& /3 _3 (#Q m0'X0/`S #9:#81 /3:9 >#8# 6#8&380#"9"#9 8) #"mpn @:(#)=)9)313&3/# ;/8:(3/3'@1"1 #8#130) 9: (##:(#8/1"91 #8 19:):;:#S8)#&/@P683:#)1906/#9 31:)1)1'pl'683:#)18# (#:#"ml0)1:slEP/3"#"31:3;'#o[tx8)9 #::#'8")#1:'#/_ 1=):83'#1 losqn`1"8;1&38n[o(:mll)13=#?8)9[ #::#8;11)1' ;&_ 1=):83'#1 llpm`9;66/)#">):(;6'#1:)3?)"1:_lllq`S(#683:#)1906/#9>#8#:819X#" 310#0 81#_)//)638# lllmlolQol968#>#::#")10#:(13/`)1ql08)9P or0'/@ )1# 31:)1)1'lSlmxP";8)1'mr(:mll0S#0 81#9>#8# /3 .#"&38 m(>):(qx 131[&:"8@0)/.63>"#8_06)1`X[>##11")1 ; :#"3=#81)'(: :ph>):(68)08@1:) 3"@_1:)[mP#//)'1//)1'iulmlPmRmlll)1mx X`S 3:1:)8 ):[ [ 31-;':#"9# 31"8@1:) 3"@_ PmRnlll`>9")/;:#")1mx X 1" )1 ; :#" &38 m ( : 8330 :#06#8:;8#S (# 9)'1/ )9 "#:# :#" @ #/# :83 (#0)/;0)1#9 #1 #_ `[;9)1' /;9_0#89(0nmonX)#8 #lltlmur`S

38 nP>#9:#81 /3:9>#8#6#8&380#" @:(##130#: )/):@ 38:38@: "# ;% # #9#8 (#1:#8Sn #//9>#8#8#9;96#1"#")1) #[ 3/"/@9)9 ;&_ql08)9[ /P6 sSpPqll0/PlSqx[pl` 31:)1)1'683:#9#1"6(396(:9#)1() ):389

__m0`P683:)1)1_p'X0/`P/#;6#6:)1_m'X0/`P_q0`1"op_m0``S 83A#1:;03893&# (_1zm[o8#6/) :#9`>#8#/@9#")1qll/3&) #[ 3/"/@9)9 ;& _pl08)9[ /6 sSpPmx8):31[[mllPpl0#:['/@ #836(396(:#Pqx/@ #83/Pmll 0/Pm0Pql0P1"683:#9#1"6(396(:9#)1() ):3899 3=#`6#8 mll 0' 1" :(#1 8;9(#" ;9)1' 9:#8)/# 6#9:/# _?@'#1`S n 1" /@9:#9 >#8# )1 ; :#"&38ol0)131) #1"931) :#"ol9Z&38ml @ /#9>):()38;6:38

_)'#13"#`S 193/; /# 0:#8)/ >9 8#03=#" @ ()'([96##" #1:8)&;':)31 : ph 1" 683:#)1 31 #1:8:)31>9"#:#80)1#" @:(#8"&38"99@S3:/93/; /#683:#)1#?:8 :9 >#8# 9#68:#" @ [ 1" :819X#" 31:3 1):83 #//;/39# 0#0 81#9 _ #/:( 8#`S#0 81#9>#8# /3 .#"&381(3;8:8330:#06#8:;8#>):(qx131[&: "8@0)/.)11"683 #"3=#81)'(:PpEP>):(8 ):1:)[ n1:) 3"@_#:(@/olm[ nprPmRnlll`1"03;9#1:)[ 1:) 3"@_):A'#8/"ml[mluPmRmrllll`)1:(# /3 .)1' 93/;:)31S [ 31-;':#" 9# 31"8@ 1:) 3")#9 _ .931 00;13#9#8 (P mRmllll`>#8#")/;:#")1&38m(:8330:#06#8:;8#&3//3>#" @"#:# :)31;9)1':(# #9:#81 )'(:)1'(#0)/;0)1#9 #1 ##'#1:/;9_#8.)1/0#8`S

gBRCA patient cohort

(# 3(38: 319)9:#"3&'0#:9::) 6:)#1:9:8#:#"::(#//"U # 8311)=#89):@ 396):/>):()1 /)1) /:8)/9:#9:)1')1() ):389)10313:(#86@38 30 )1:)31P>):( 0:#8)/8#68#9#1::)=#3&:(#")9#9#1"9)'1#" [6683=#")1&380#" 319#1: &380S 1/@9)91"qm7;1:)&) :)31>96#8&380#"9"#9 8) #"&38:;038 906/#9S

HBOC patient cohort

(# 3(38: 319)9:#"3& 8#9: 1 #86:)#1:9&830:(#//"U # 8311)=#89):@ 396):/P >):( 0:#8)/ 8#68#9#1::)=# 3& :(# ")9#9# 1" 9)'1#" [6683=#" )1&380#" 319#1:&380S;#:36#8931/38&0)/@()9:38@P1"&:#88;/)1'3;:@:A 0;::)319P 6:)#1:9>#8#:#9:#"&38'#80/)1#0;::)319/)1.#":3 8#9: 1 #89;9 #6:) )/):@>):()1 8#9#8 (683:3 3/S 1/@9)91"qm7;1:)&) :)31>96#8&380#"9"#9 8) #"&38 :;038906/#9S

Statistical analysis

#'8")1':(#906/#9)A# / ;/:)31P:()9#?6/38:38@9:;"@)1=3/=#"901@906/#99 6399) /#";8)1':(#9:;"@:)0#&80#S:>91/@A#">):(86("8)90=#89)31sSlS 8838 89 8#68#9#1: :(# :1"8" 8838 3& :(# #1 _` 3& : /#9: :>3 )3/3') / 8#6/) :#9P;1/#993:(#8>)9#9::#"S(6)83[)/.:#9:>9;9#":399#991380/):@3&": ")9:8) ;:)319S::)9:) /:#9:9>#8#6#8&380#";9)1'6)8#"38;16)8#":>3[:)/#".[:#9: _&38:>3'83;69 3068)9313&m1"qom683:#)1/#=#/9P)1:#19):@_x`)1:(# 99@1":(#6#8 #1:'#3&γ nX'#0)1)1[639):)=# #//9P'#0)1)1[639):)=# #//91" 6 o[639):)=# #//9`Q11[():1#@[:#9:_&38:>3'83;69 3068)9313&:(#6#8 #1:'# 3& qmX'#0)1)1[639):)=# #//9P 6on pXtX'#0)1)1[639):)=# #//9 1" 61[ γ n[ 639):)=# #//9`Q3831#[>@_&38:(8##38038#'83;69 3068)9319`S38:8#:0#1: #&&) @ 3068)93193&:(#!(0!0)#?6#8)0#1:/":P:>3[>@>9;9#"S#8931 388#/:)31>9;9#":31/@A#:(# 388#/:)31 #:>##1:(#qm9 38#1":(#:;038 =3/;0# (1'# ;631 3/68) :8#:0#1:S 38 :(# =/)":)31 3& :(# :>3 1:)[qm 1:) 3")#9P6#801 388#/:)31>9;9#"S(#>9 / ;/:#":3#9:)0:#:(# 68#") :)31 6 ):@ 3& 1" qm 9 38#9 :3 ) 8#96319#S 38 ;8=# 3068)931P:>3[9)"#" 33:9:86:#9:>9;9#" @0#193&9::)9:) /6 .'#6)1 93&:>8#=#89)31oSpSmS3 / ;/:#:(#993 ):)31 #:>##1 A0;::)311":(# qm9 38#/3')9:) 8#'8#99)3103"#/>9&)::#":3#9:)0:#:(#3""98:)3_`>):( uqxS #=#1#d9 :#9: )9 ;9#" :3 :#9: )& '83;69 3& 3068)931 (=# #7;/ =8)1 #9 _(3039 #"9:) ):@`S 3 #=)"#1 # (9 ##1 &3;1" :3 8#-# : :(# (@63:(#9)9 3& (3039 #"9:) ):@)13;8":S319#7;#1:/@P:(#9::)9:) / 3068)931(9 ##1 88)#" 3;:;1"#8:(#(@63:(#9)93&9)0)/8=8)1 # #:>##1'83;69S

RESULTS

PART 1 A RAD51 assay identifies restoration of homologous recombination repair as the major mechanism of PARP inhibitor resistance in germline BRCA1/2 mutated breast cancer.

The establishment of a PDX panel from gBRCA patients’ tumors

3)1=#9:)':#:(#0# (1)9093&)8#9)9:1 #)1(;01:;0389P61#/3&6:)#1: "#8)=#"?#13'8&:9_`&830'0;::)31 88)#89>):( 8#9:383=8)1 1 #8>9 "#=#/36#"S 38 :()9 6;8639#P &8#9( :;038 906/#9 &830 6:)#1:9 >#8# 68396# :)=#/@ 3//# :#"1")06/1:#")1:31;"#0) #S:3:/3&:>#/=#03"#/9_mm'm1"m

'n` >#8# #9: /)9(#" _* H`S 6# )&) //@P #)'(: 9 "#8)=#" &830 'm 0#:9::) :;0389 &830 ")&#1: 38'19 1" &3;8 9 >#8# #9: /)9(#" &830 68)08@ 8#9::;0389S)=##1'8&:#"03"#/9>#8#"#8)=#"&8306:)#1:9>):(0#:9::) ")9#9# 68:) )6:)1')1 /)1) /:8)/9>):()S&13:#P:(8##906/#9>#8#3 :)1#"68)38:3 3/68) :8#:0#1:&8306:)#1:9>(39(3>#"68:)/8#96319#_P1zm`38138#96319#:3 :(# :8#:0#1: _68)08@ 8#9)9:1 #P 1zn`S >3 ""):)31/ 906/#9 >#8# "#8)=#" &830 0#:9::) 'm 6:)#1:9 >(3 683'8#99#" :3 :(# ) 3/68) &:#8 9(3>)1' 9;9:)1#"68:)/8#96319#_ 7;)8#"8#9)9:1 #P1zn`S

#80/)1#0;::)319)1@:A>#8# 31&)80#")1//03"#/9 ;:nspP1":(#@>#8#

993 ):#">):(/3993&:(#>)/":@6#//#/#_.. ,%6%#=F`S6# )&) //@Pnsp/ .#":(# '#80/)1#@ Snmm|0;::)31)1#?31q";#:313;:[3&[&80#"#/#:)313&#?319o :3s3&@)1:(#6:(3'#1) //#/#P;1=#)/#" @099)=#/@68//#/9#7;#1 )1'1" S ()9"#:# :)31>9 31&)80#"::(#930:) /#=#/)1:(#38)')1/6:)#1:U9:;038P0#1)1' :(:):>913: 7;)8#")1:(#S

#<;2 #<;2 =* B;8;

#<;2 #<;2 ;

0;::)31P68)38 #<;2 #<;2 #<;2 #<;2 1292A6<; <:=92A2 @:A =# B;8;

%& "3*&.3 ! %?6:.?F %?6:.?F 0>B6?21 0>B6?21 '2;@6A6C2 );A?2.A21 :% );A?2.A21 :% );A?2.A21 :% );A?2.A21 :& !$ ?2@6@A.;02 ?2@6@A.;02 ?2@6@A.;02 ?2@6@A.;02 &20/.2&3/,"0"1*# P:8)6/#1#':)=#S#:P0#:9:9)9S6P66)//8@SP3=8 :)31)1 /;"#:)0#63)1:3&)06/1::)31P8#96319#:3)P/399 6?6;

3 3 3 3 3 3 3 3 3 3

%&%6

6:=9.;A21.A=?<4?2@@6<;A<%&%6 6:=9.;A21=?6

1&"3&% *4.3 *31&"3&% * 8# #6:38639):)=#S"-P"-;=1:Q#3"-P1#3"-;=1:SP9 * H @:A6 #&&# :)9)1") :#"S P ;01#130#8):)313 )#:@S :(#86)#91"8#96319#:3)>(#1=)/ /#S(#)1&380 31&"3-&.3 un

()961#/3&9//3>#";9:36#8&380!(0!0)#?6#8)0#1:91"(=#;1/)0):#" #99:3 :;0380:#8)/&830:(#68#[):8#:0#1:P";8)1'[:8#:0#1:1"639:[)8#9)9:1 # 7;)9):)319 #18)39S9)1':(#9#:;038906/#9>#6#8&380#"'#130) 1/@9)9:3 "#:# : 3:(9# 31"8@0;::)319)1@:A1"3:(#8'#130) /:#8:)319P&;1 :)31/ :#9:93&8#6)808.#89_)1 /;")1'qomPmPγ n1"qm&3 )`1"/93 683:#30) 9:;")#93&")&#1:683:#)19)06/) :#")1)8#96319#S

Olaparib antitumor activity in the gBRCA PDX panel distinguishes a subset of PARPi-resistant tumors

(#1:):;038 :)=):@3&:(#)3/68) >999#99#")1:(# 3//# :)31_%#=N`S (8##'03"#/9#?() ):#"1:):;038 :)=):@>):(3/68) :8#:0#1:999#99#" @ 0 _9##0:#8)/91"0#:(3"9`R 306/#:#8#96319#_P1znRlsm1"nol` 389: /#")9#9#_P1zmRmnp`S(#8#0)1)1'1)1#'03"#/9>#8#8#9)9:1: :33/68) :8#:0#1:_P683'8#99)=#")9#9#`S&13:#P//:(#)[8#9)9:1:98# @0;:1:1"9#=#13;:3&1)1# 0#&830:(#"=1 #"0#:9::) 9#::)1'S031' :(# 03"#/9 &830 ' 68)08@ :;0389P :>3 3;: 3& &3;8 _qlx` 9(3>#" S (# 8#9)9:1 #:3)[:8#:0#1:)1:(#9)06/1:#"&:#86:)#1:683'8#99)31:3)P

10#/@mur1"ntlP>9 31&)80#")1:(#9_%#=N`S38#3=#8P:(#9#19):)=):@ :3)[:8#:0#1:)1:(#9&8300#:9::) 6:)#1:968#=)3;9/@:8#:#">):()

_mnsPmsu1"mnp`0)8838#":(#6:)#1:9U /)1) /8#96319#_%#=N`S

1#""):)31/8#9)9:1:03"#/>9'#1#8:#"&:#8683/31'#"#?639;8#_|mll"@9`1" 9:##6683'8#99)31:33/68) _nol`&830):9)[9#19):)=# 3;1:#868:_nolP

%#=N`S()91#>'m 7;)8#"[8#9)9:1:>99: /)9(#" @)06/1:)1':(#:;038 3=#8'83>)1' >):( 3/68) :8#:0#1: _ / . :8)1'/#9 )1 %#=N`S (# !( 0!0) :)=):@ 3& 3/68) >999#99#")1:(#nolP683=)1':(::(#1#>03"#/)98#9)9:1::3) :8#:0#1:_%#=N`S/:3'#:(#8P 3//# :)313&mo'mXn03"#/9_ //#" 3(38:[m` >9'#1#8:#"1"P>(#1.13>1P8# 6):;/:#96:)#1:9U8#96319#:3)S(;9P:()9 61#/ 8#68#9#1:9 :33/ &38 031):38)1' :(# 1:):;038 :)=):@ 3& ) 1" 9;): /# 6/:&380:39:;"@ /)1) //@8#/#=1:0# (1)9093&)8#9)9:1 #!(0!0)S

700 200

600 PDX Patient 500 150

400

300 100

200

100 50 % Tumor volume change volume % Tumor Time to progression (days) progression Time to 0

-100 0 PDX127 PDX274 PDX221 STG316 PDX179 PDX280 PDX252 PDX196 PDX236 PDX124 PDX230 PDX071 PDX127 PDX274 PDX221 STG316 PDX179 PDX280 PDX252 PDX196 PDX236 PDX124 PDX230 PDX071 PDX230OR PDX230OR

1200

Vehicle n=5 1000 Olaparib n=4

800

600

400 Tumor Volume Change (%) 200

0 0 20 40 60 80 100 120 140 160 180 200 Day of treatment

%#30 NY$ ,2%23+-02%4%27-"-*.0%%,2$ -$-02@F% ,2%"% 1131 2-"#@+322 %@0 1%12,223+-01=B:#8&//6/3:9(3>)1':(#6#8 #1:'#3&:;038=3/;0# (1'# 3068#" :3:(#:;038=3/;0#31"@mSynlx1"[olx8#08.#" @"3:#"/)1#9:3)1") :#:(#81'#3&P 1"S(# 3?;1"#81#:(9;008)A#9")&#1: (8 :#8)9:) 93&# (03"#/1":(# /)1) / 31:#?: : :(# 030#1: 3& )06/1::)31S P 8)6/# #':)=# 8#9: 1 #8Q yP 9:83'#1 # #6:38639):)=#8#9:1 #8QP=8)11 #8QP68)08@QP0#:9:9)9S8838 89)1") :# &830)1"#6#1"#1::;0389SB86(9(3>)1':(#9#19):)=):@3&:(#=9S:(#6:)#1:U9 /)1) /8#96319# :33/68) S)0#:3)683'8#99)313&6:)#1:91"9)99(3>1S/;# 89P:)0#63)1::>() (:(# x3&:;038=3/;0# (1'#>9nlx&3//3>)1'3/68) :8#:0#1:)1:(#03"#/9S:)#1:9U:)0#31 ):(#86@;1:)/683'8#99)31)96/3::#")1/)'(:'8#@ 89_:mnsP:msu1":mnp`S/;# 899(3> 0#1yX[3&:/#9:o)1"#6#1"#1::;0389SB 7;)9):)313&)[8#9)9:1 #)1nol&:#8 683/31'#"#?639;8#:33/68) S(#6#8 #1:'#3&:;038=3/;0# (1'#";8)1'3/68) :8#:0#1:)9 6/3::#"S38=#() /#[:8#:#":;0389P:(#0#1yX[)98#68#9#1:#"_97;8#9`S(#)1")=)";/:;0389 3&:(#3/68) 808#9(3>1)1'8##1/)1#9_")&#1::8)1'/#6::#819`S

PARPi-resistant gBRCA1 PDX models do not harbor target loss or secondary BRCA1 mutations

=)1'9: /)9(#":(#61#/3&'03"#/91":(#!(0!0)1:):;038 :)=):@3& 3/68) P>#)0#":3)1=#9:)':#:(#0# (1)9093&8#9)9:1 #:3))1:(#9#9S 1 38"#8 :3 =#8)&@ :(: // :;0389 #?68#99 :(# "8;' :8'#:P m 683:#)1 /#=#/9 >#8# "#:#80)1#" @S#9;/:9 31&)80#":(://'9#?68#99#"")&#1:/#=#/93&

m9)0)/8:(13:(#8:;0389>):(13'0;::)31_%#=FE`P1"3/68) [8#9)9:1: 9")"13:9(3>#"/3>#8/#=#/93&m_%#=FE`S(#1P)138"#8:3)1=#9:)':#>(#:(#8 '#1#:) 8#=#89)31)9"30)11:0# (1)90&38 /)1) /8#9)9:1 #:3))13;861#/3& '9P099)=#/@68//#/#?30#9#7;#1 )1'>999#99#":39:;"@:(#68#9#1 #3& 9# 31"8@0;::)319)1@:A'#1#9S;868)9)1'/@P13&80#9()&:[ 388# :)31138'#1#:) 8#=#89)313&:(#)1(#8):#"0;::)313 ;88#")1:()9636;/:)313&)[8#9)9:1::;0389P 9)1 #//03"#/9 ;:nsp(8 38#":(##?6# :#"'#80/)1#0;::)31S(#3;:[3&[&80# "#/#:)313&@#?319o[s)1nsp>968#") :#":3 ;9#68#0:;8#9:36 3"31 1":8;1 :#"683:#)1_.. ,%6%#=F 1"* H`S

/:3'#:(#8P:(#9#8#9;/:9"#0319:8:#":(:1#):(#8:8'#:/3991389# 31"8@0;::)319 8#68#9#1:#"0# (1)903&)8#9)9:1 #)1 3(38:[mS

The expression of functional hypomorphic BRCA1 isoforms is associated with PARPi resistance in gBRCA1 PDX models

#1#?:1/@A#:(##?68#99)31/#=#/3&@ @0#9;8)1':(#/#=#/93&@0

031' :(# ' 61#/ _%#=FE`S 9 #?6# :#"P ntlP >() ( (8 38#" /8'# "#/#:)31#1 30699)1':(# 306/#:#@'#1#P9(3>#" 9#1:@0S//:(# 8#0)1)1''03"#/99(3>#"=8) /#@0#?68#99)31S =)1' 31&)80#" @ 0 #?68#99)31 )1 // 03"#/9 ;: ntlP ># )1=#9:)':#" :(# 63:#1:)/ #?68#99)313&(@630386() m)93&38091"):98# 8;):0#1::3"0'#9):#9S38 :()9 6;8639#P ># 31"; :#" 1 )00;13&/;38#9 #1 # _ ` 9:)1)1' 3& 683:#)19 )1 906/#9 &830 )[8#9)9:1: 0- )[9#19):)=# 9S 9 >#8# "#:# :#" 9 6(396(38@/:#" ()9:31# n _γ n` 1; /#8 &3 )ntrQ qm 1; /#8 &3 ) >9 ;9#" 9

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639):)=#P.. ,%6%#=G`"#0319:8:#":(#96# )&) ):@3&:(#99@S

#:(#16#8&380#":(# 99@:3"#:# :m1; /#8&3 ))1:(#'61#/P;9)1' :(#A0;:1:lsm9639):)=# 31:83/&38:(#99@_m1; /#8&3 )>#8# "#:# :#">):( 3:(m1:) 3")#9`1"ntl91#':)=# 31:83/";#:3 306/#:# "#/#:)311"130#?68#99)313&@S):(:()999@Pm1; /#8&3 )>#8#/93 "#:# :#")19#=#1""):)31/'m03"#/9P#):(#8 @;9)1':(#m[38:(#m[ 1:) 3")#9P)1 31 38"1 #>):(:(##?68#99)313&")&#1:(@630386() m)93&3809S mnp1"mur(8 380;::)31)1:(##?31mm3&@_ Smurm"#/`1"#?68#99 :(#m[Δmm796/) #)93&380_6S#8nrp^/@morr"#/`mpqS9#?6# :#"Pm1; /#8&3 )

>#8#"#:# :#">):( 3:(m[1"m[1:) 3")#9_%#=FF`S 1:(#[:#80)1/m 0;:1:9msuPomr1"nspPm1; /#8&3 )>#8#3 9#8=#"#? /;9)=#/@>):( :(#m[1:) 3"@P 319)9:#1:>):(m [/#99683:#)1)93&380 #)1'#?68#99#"mpo _%#=FF`S >3 3;: 3& :(# :(8## [:#80)1/ m 0;:1: 03"#/9P 10#/@ nnm 1" norP9(3>#"m1; /#8&3 )&:#89:)1)1'>):(:(#m[1:) 3"@ ;:13:>):( :(#m[1:) 3"@P 319)9:#1:>):(:(#(@63:(#:) /9: )/)A:)313&:(#'m0;:1:

683:#)1=) ulmom_%#=FF`S&13:#P:(#m&3 )>#8#/>@98#9:8) :#":3 #//9)1:(# Xn[6(9#3&:(# #// @ /#_'#0)1)1[639):)=#`S 1""):)31P:(#")&#1:m)93&3809 >#8#"#:# :#" @>#9:#81 /3:::(#8#96# :)=#68#") :#"9)A#93&:(#:8;1 :#"683:#)19

_%#=FF`S3"#:# :)313&m1; /#8&3 )>99##11#):(#8)1:(#'mnolP:(: 9(3>#":3):8#:0#1:P138)1:(#:(8##8#0)1)1')[8#9)9:1:9P10#/@ ur

mnsPnol1"nqnP:3'#:(#8>):(ntl_%#=FF`S 19;008@P:(#9#": 9(3>#":(:/8'#683638:)313&)[8#9)9:1:'m03"#/9#?() ):8# 8;):0#1: 3&")&#1:(@630386() m)93&3809:3&3801; /#8&3 )S

2.0 p=0.6345 2.0 p=0.7203 1.5 1.5

1.0 1.0

0.5 0.5 (RPPA, NormLinear) (RPPA, PARP1 protein levels protein PARP1 (RPPA, NormLinear)

levels protein PARP1

0.0 0.0 gBRCA non-gBRCA SD/CR PD

8 6

Mean in 2 TNBC BRCA1 Normalized Ct

PDX230 PDX127 PDX179 STG316 PDX274 PDX124 PDX196 PDX221 PDX252 PDX236 PDX280 PDX230OR

N-terminus Exon 11 C-terminus

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Loss of 53BP1 and FAM35A in PARPi-resistant gBRCA1 PDX models

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2.5 PDX071 2.0

1.5 $0(((#*%&/ 1.0 0.5 53BP1 protein levels protein 53BP1 (RPPA, NormLinear) (RPPA, PDX230OR STG316 0.0

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PARPi-resistant gBRCA1 PDXs are able to form RAD51 nuclear foci

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nor`_%#=FG`S

mll

RAD51 nuclear foci formation can be assessed in untreated tumor samples and is associated with PARPi resistance in PDX cohort-1

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mlm

3 p=0.0047 p=0.0244 p=0.0345 2 1 RAD51/GAPDH (AU) RAD51/GAPDH 0 PDX230 PDX179 PDX124 PDX196

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mln

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mlo

100

Vehicle 80 Olaparib

20 RAD51 (+) / Geminin (+) cells (%)

0 STG316 PDX236 PDX274 PDX127 PDX280 PDX252 PDX221 PDX179 PDX196 PDX124 PDX071 PDX230 PDX230OR

p=0.0192 p<0.0001 p=0.0070 100 100 100 p=0.0070 p=0.0400 80 80 80 p=0.0008 p=0.0323

60 60 60

40 40 40 Geminin (+) (%) cells

20 20 20 H2AX (+) / Geminin (+) (%) cells γ RAD51 (+) / Geminin (+) cells (%) (+) cells Geminin (+) / RAD51 0 0 0

Olaparib Olaparib Vehicle Olaparib Vehicle Olaparib Vehicle Olaparib Vehicle Olaparib Vehicle Vehicle

SD/CR PD SD/CR PD SD/CR PD

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mlp

RAD51 foci formation in FFPE tumor samples from patients predicts response to PARPi

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γ n[639):)=# #//98#'8"/#993&>(#:(#83813:6:)#1:98# #)=#"68)38:8#:0#1:_%#=FI

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mlq

100 BRCA1 %, %/?62:? BRCA2 80

40 %, 20 '

0 RAD51 (+) / (+) cells Geminin (+) (%) RAD51

Treatment-naïve With prior treatment %,

100 BRCA1 BRCA2 80

40 %, 20 H2AX (+) / cells (+) Geminin (+) (%) H2AX

γ 0

Treatment-naïve With prior treatment %, %#30 FIYJF , γ G "-% %, .2% ,2>1 23+-0 ' 1+.* 1=B;1:)&) :)313&qm_11\():1#@[ :#9:` 1" B γ n &3 ) &380:)31 _16)8#" .;:#9:` )1

:8#:0#1:[1+=# 6:)#1: :;038 906/#9P 1" )1 :;038 906/#9 >):( 68)38 :8#:0#1:S #1 :8#:0#1:[1+=#

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1; /#8&3 )S; /#)>#8#=)9;/)A#">):( _ /;#`S// %, 6) :;8#9>#8#:.#1:rll?0'1)&) :)31S

' 296:6: %

mlr

100 BRCA1 BRCA2 80 p=0.0005 60 p=0.0003 Pt310post 40 Pt124post Pt124pre 20 Pt310pre 0 RAD51 (+) / (+) cells Geminin (+) (%) RAD51

Sensitive

Primary-resistant Acquired-resistant

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mls

PART 2 The RAD51 assay identifies homologous recombination repair deficient sporadic breast tumors with PARP inhibitor sensitivity.

The establishment of a PDX panel from non-gBRCA patient’s tumors

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Olaparib antitumor activity in the non-gBRCA PDX panel distinguishes a subset of tumor highly sensitive to PARPi

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NO YES # ;9#n6:)#1:9 906/#>9 3//# :#" Y106X Y106X - - Result Result TP53 Negative Negative Positive % n - - - - - 1 12 tor - - 2 11 - - - p tor - - 4 9 - - - py p ies - - ies - - - - 4 9 p e I 2 15.39 - - - - - e III 3 23.08 - - - - - g g en rece en Levels Levels g #")1:)0# #:>##1")'139)91"&)89:8#/69#_)1@#89`zmm esterone rece esterone g 31:(9`1zt # ;9#n6:)#1:9("138#/69#1")1o 9#9 Median (Min-Max) (Min-Max) Median Mean ------II - - 53.85 IV Stage 7 - - - Sta Pro 7.69 1 Stage - - - - status - - - Her2 - - B - - - 0 - 4 - Luminal - - - - 13 assessed - - TNBC - - 11 - - - - - Not - - - - - 8 WT ------Taxanes - - thera - 1 Platinum Capecitabine - - - NA - Gemcitabine - Hormone - - - thera ------Other - Eribuline ------NA ------9 - 3 4 4 10 3 9 10 - 1 12 - - Estro - - Mutation 1 - - - - - Anthracyclines - - - - - 8 5 - - A 1 Luminal ------Sta 8 6.88 (0-15) 6 ------N 13 57.85 58.19 (31.59-93.53) 13 57.85 58.19 ------11 20.27 (5-96) 15 13 18.92 (1-97) 11 ------py p Variable to PDX (n=13) to PDX (n=13)

10%.2%4 ,*71%1"0-+4%** .2% ,21-"-$-02@G= )

Y ) Cancer subtype (n=13) subtype Cancer Stage at diagnosis (n=13)at Stage diagnosis IHC characterization (n=13) characterization IHC ears y Mutational panel assessed (n=13) assessed panel Mutational Patient's therapy exposure previous previous therapyPatient's exposure * I ("138#/69#S#")1:)0# #:>##11"&)89:8#/69#_)10 in in months in Median age at cancer diagnosis age diagnosis atMedian cancer ( and time diagnosis between Median relapse first (in months) time PDX and between Median diagnosis (in months) time first PDX and between Median relapse (

mlu

600 600

500 500 STG201 400 400

300 300 PDX302 200 200 PDX270 100 100 PDX093 Tumor Volume Change (%) Tumor Volume Change (%) 0 0 PDX197 -100 0 20 40 60 80 100 120 Day of treatment PDX098 STG139 PDX137 PDX102 PDX044 PDX156 PDX094 PDX291 PDX060 PDX288 PDX270 PDX302 STG201 PDX197 PDX093 PDX302OR STG201OR PDX093OR

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PARPi-sensitive PDX models harbor epigenetic silencing of BRCA1

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Somatic alterations in HRR-related genes within PDX cohort-2

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mmm

3 60 BRCA1

2 40 mRNA (normalized Ct)

1 20 promoter methylation (ratio) BRCA1

0 0 T127 T162 PDX098 STG139 PDX137 PDX102 PDX044 PDX156 PDX094 PDX291 PDX060 PDX288 PDX270 PDX302 STG201 PDX197 PDX093 PDX302OR STG201OR PDX093OR

! !

" "! () () $' %, %, $'

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mmo

! ! p<0.0001 p<0.0001 20 p<0.0001 p=0.0009

15 p=0.0010

10 !

5 !

Clover-positive (%) cells

0 ""

siCTL + pcDNA3siPALB2 + siPALB2 + siPALB2 + pcDNA3 PALB2 WT PALB2 p.M296Nfs

! ! 30 p=0.0011 p=0.0159

200 !

p<0.0001 10 150 ! Clover-positive cells (%) 100 0

YFP intensity (%) 50 PALB2 wt + pcDNA3 PALB2 p.M296Nfs + PALB2 WT 0 0246810121416 + PALB2 p.M296Nfs Time (min)

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mmp

RAD51 score is a robust biomarker to predict PARPi response in non-gBRCA PDX models

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906/#9P>#9: /)9(#"683:3 3/9(3>1)1%#=GF:(:)1 /;"#99#=#8/7;/):@ 31:83/9 _`S)89:/@P>#;9#"639):)=#1"1#':)=##?:#81/ 31:83/9>):(#=#8@8;13&9:)1)1'S # 31"/@P>#;9#":>3)1:#81/ 31:83/9R_)`:(#1;0 #83&'#0)1)1[639):)=# #//9(":3 # :/#9:plP:3#19;8#:(::(#8#8#9;&&) )#1: #//9)1Xn[6(9#3&:(# #// @ /#Q1"_))` :(#x3&γ nX'#0)1)1[639):)=# #//9(":3 #:/#9:3&nqxP:3#19;8#:(::(#:;038 (99;&&) )#1:#1"3'#13;9"0'#S .3&#):(#8'#0)1)1[38γ n[639):)=# #//9 _ #/3>:(39#:(8#9(3/"9`>3;/"68# /;"#9 38)1'3&qmS

mmq

50 40 30 20 20

5 Number of RAD51 foci-per-nucleus of RAD51 Number

0 PDX098 PDX060 PDX094 PDX044 PDX102 PDX270 PDX197 STG201 PDX302

50 40 30 20 20

5 Number of RAD51 foci-per-nucleus of RAD51 Number

0 PDX098 PDX060 PDX094 PDX044 PDX102 PDX270 PDX197 STG201 PDX302

100 PDX197 100 %, PDX98 STG201 %, PDX60 80 PDX302 80 %, PDX94 %, PDX44 60 60 %, PDX102 %, PDX270 40 40 Number of cells Number of cells 20 20

0 0 0 5 0 5 10 15 20 25 30 35 40 45 50 10 15 20 25 30 35 40 45 50 Number of RAD51 foci-per-nucleus Number of RAD51 foci-per-nucleus

%#30 FNY$ JJF"-%@. 0@,3* 3132-""%"" 0 ,2%2 1%@1 ,1%2%4 ,%@0 1%12,2 1=@B ::#86/3:99(3>)1':(#1;0 #83&qm&3 ))1# ()1")=)";/'#0)1)1[639):)=# #//_mll #//9&38# (03"#/`)1B =#() /#[ 38B 3/68) [:8#:#"906/#9&8309)?)[8#9)9:1:1":(8## )[9#19):)=#903"#/9&830 3(38:[nS(#"9(#"/)1#)1") :#9:(# ;:[3&&3&qqm&3 )[6#8[ 1; /#;9SB )9:3'80")9:8) ;:)313&:(#1;0 #83&'#0)1)1[639):)=# #//9>):()1 8#9)1'1;0 #83& qm&3 )[6#8[1; /#;9_l:3ql`)1:(8##)[9#19):)=#_/#&:61#/`1"9)?)[8#9)9:1:_8)'(:61#/` 9&830 3(38:[nS ( 3/388#68#9#1:931#03"#/S

mmr

OR 95% CI p value

> 10 foci-per-nucleus 3.889 0.1374 to 110.1 0.5000 > 9 foci-per-nucleus 3.889 0.1374 to 110.1 0.5000

> 8 foci-per-nucleus 25.67 0.7981 to 825.4 0.0476 > 7 foci-per-nucleus 25.67 0.7981 to 825.4 0.0476 > 6 foci-per-nucleus 25.67 0.7981 to 825.4 0.0476 > 5 foci-per-nucleus 91.00 1.462 to 5662 0.0119 > 4 foci-per-nucleus 91.00 1.462 to 5662 0.0119 > 3 foci-per-nucleus 91.00 1.462 to 5662 0.0119 > 2 foci-per-nucleus 91.00 1.462 to 5662 0.0119 > 1 foci-per-nucleus 91.00 1.462 to 5662 0.0119

10-1 100 101 102 103 104 Odds ratio

100 1.0 Vehicle Olaparib 80

0.5 40

ab-133534 20 Accuracy Specificity

RAD51 (+) / Geminin (+) cells (%) (+) cells Geminin (+) / RAD51 0 0.0 12345678910 0 102030405060708090100 Number of RAD51 foci-per-nucleous cut off RAD51 (+) / Geminin (+) cells (%) sc-8349

%#30 GEY,*71%1-"2$ JF"-%@. 0@,3* 3132-"",4*%2%-,-"JF1-0%,#5%2$25- %"" 0 ,2,2%-% 1=B38#9:6/3:91"""U98:)31/@9)91" B ;8 @1"96# )&) ):@3&:(# qm 9 38# ;9)1' ")&#1: ;: 3&&9 &830 m :3 ml &3 ) 6#8 1; /#;9SB 6#801 388#/:)31 3& :(# 6#8 #1:'# 3& qm _y`X#0)1)1 _y` #//9 )1 3(38:[n 99#99#">):(:>3")&#1:1:) 3")#9 ')19:qm 3:()1=#() /#[1")[:8#:#"906/#9S

mms

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&380:)31 )1 nlm 1" oln _%#=FL , .. ,%6 %#=J`S 1 uo 8# 3=#8@)9 319)9:#1:>):(:(#/3993&:(# A0;::)3168#9#1:)1:(#3/68) [9#19):)=#

3;1:#868: luo _%#=FL , .. ,%6 * F`S (#9# ": 9;''#9:#" :(: )1 :(# (#0) )'39)9 31:#?: 3& luoP :(# "30)11: 1#':)=# #&&# : 3& n 6Snur&9 ")966#8#"P//3>)1':3 683&) )#1 @1") 7;)8#"8#9)9:1 #S/:3'#:(#8P:(#9# ":)1") :#:(:/3993& 6 ):@)9&8#7;#1:#=#1:993 ):#">):()9#19):)=):@ 1""#0319:8:#":(::(#qm9 38#)983 ;9:1"()'(/@")9 8)0)1:)=# )308.#83& )9#19):)=):@=9)8#9)9:1 #)1:(# 3(38:[nS

mmt

!5.02%-0&,3

544+335(3(&4+0/3 μ.

γ " (.+/+/ (.+/+/

+/'2(12(3(/4$4+6(45.02$2($3 (.+/+/&(--3 5$/4+)8(.+/+/&(--3 02 0/(6$-5$%-( 5$/4+)8γ " (.+/+/&(--3 γ " (.+/+/&(--3

*(&,+/'+6+'5$--80/((.+/+/&(-- 2(4*(2( /5&-($2)0&+

(1($45/4+- # 2(4*(2(9 )0&+ (.+/+/&(--3 7*(/1033+%-(

&(-- # &(--

p=0.0213 p<0.0001 100 100

80 80

60 60

40 40 Geminin (+) (%) cells 20

H2AX (+) / Geminin (+) (%) cells 20 γ

0 0

Vehicle Olaparib Vehicle Olaparib Vehicle Olaparib Vehicle Olaparib

CR/PR PD CR/PR PD

%#30 GFY1 *%, * 4 *1-"+# -0*-5,3+ 0-" **1%,?G@.$1 -"2$ **7* % ,-2%+.%01JF1-0%,#%,-$-02@G=B/3>")'803&:(#qm9 38)1' 8):#8)1"7;/):@ 31:83/9S@B;1:)&) :)313&B #//9)1Xn[6(9#3&:(# #// @ /#_'#0)1)1[639):)=#`:(:#?() ): H n&3 )&380:)311"B #//9)1Xn[6(9#3&:(# #// @ /#&3//3>)1':8#:0#1:>):(=#() /#1" 3/68) )1)[9#19):)=#_X`1")[8#9)9:1:_`9_)8#".;:#9:)1=#() /#[=9)[ :8#:#":;0389Q16)8#".;:#9:)1X=9:;0389`S

mmu

100

Vehicle 80 Olaparib

20 RAD51 (+) / Geminin (+) cells (%)

0 PDX098 PDX060 PDX094 PDX044 PDX102 PDX291 PDX288 PDX156 STG139 PDX137 PDX270 PDX197 STG201 PDX093 PDX302 STG201OR PDX302OR PDX093OR

$ *"+)('+"

"&$'$' %)*$ ,*","! "&$'$' "#$ %",*","! *"%,"! %,"*,$('+ # # # #

p<0.0001 p=0.0007 - p<0.0001 600 100

80 400

60 200 40 PDX270

20 0 10 20 30 40 50 60 70 Tumor Volume Change (%) Change Volume Tumor RAD51 (+) / Geminin (+) cells (%) (+) cells Geminin (+) / RAD51 0 RAD51 (+) / Geminin (+) cells (%) -200

Vehicle Olaparib Vehicle Olaparib

CR/PR PD %#30 GGY ) -" JF ,3* 0 "-% % ,2%"% 1 %@1 ,1%2%4 23+-01= B #8 #1:'# 3& '#0)1)1[639):)=#Pqm1; /#8&3 )[ 31:)1)1' #//9"#:# :#" @)00;13&/;38#9 #1 #)1906/#9 &830:;0389:8#:#">):()_'8##1 89`38=#() /#_ / . 89`S8838 89)1") :#9&830 )1"#6#1"#1::;0389S(# 388#/:)31>):()[8#96319#)99(3>1)1:(#9;008@;1"#81#:(R>():# 3?RQ / . 3?RXS 00;13&/;38#9 #1 #9:)1)1'3&qm&3 ))1)[1"=#() /#[:8#:#" :;0389)99(3>1Q31# #//)1:(#Xn[6(9#_'#0)1)1[639):)=#P'8##1`>):(38>):(3;:qm&3 ) _8#"`)99(3>1&38# (03"#/S/:#8:)319)1 [8#/:#"'#1#98#/939;008)A#"R(mR@ 68303:#8 (@6#80#:(@/:)31 1" / . 3& @ #?68#99)31 1" m 1; /#8 &3 ) &380:)31S B ;1:)&) :)31 3& #//9 )1 Xn[6(9# 3& :(# #// @ /# _'#0)1)1[639):)=#` :(: #?() ): qm &3 ) &380:)31&3//3>)1':8#:0#1:>):(=#() /#1"3/68) )1)[9#19):)=#_X`1")[8#9)9:1: _`9_11\():1#@[:#9:`SB#8931 388#/:)31 #:>##1:(#qm9 38#_6#8 #1:'#3& qm_y`X#0)1)1_y` #//9P99#99#")1;1:8#:#":;038906/#9`1":(#6#8 #1:'#3&:;038 =3/;0# (1'#)13/68) [:8#:#":;0389&830 3(38:[nS ("3:8#68#9#1:931#03"#/S8838 89)1") :#&830:/#9:o)1"#6#1"#1::;0389:8#:#">):(3/68) S mnl

RAD51 score predicts response to PARPi in an independent PDX cohort

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mnm

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03"#/9_%#=GI`S

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mnn

100

BRCA1 80 BRCA2 PALB2 non-BRCA

20 RAD51 (+) / (+) cells Geminin (+) (%) RAD51 10% cutoff

0 T174 T381 T330 T298 T311R T180R TC168 HBCx2 HBCx7 HBCx8 HBCx9 HBCx1 HBCx6 HBCx31 HBCx30 HBCx33 HBCx27 HBCx39 HBCx16 HBCx24 HBCx11 HBCx28 HBCx23 HBCx17 HBCx15 HBCx10 HBCx14 HBCx12b

Olaparib 100mg/kg Olaparib 100mg/kg & 50mg/kg Niraparib 75mg/kg Niraparib 50mg/kg Veliparib 100mg/kg

p=0.1107 1.0 100

60 0.5

Sensitivity 40

20 H2AX (+) / Geminin (+) (%) cells γ 0.0 0 0.0 0.5 1.0 High RAD51 score Low RAD51 score 1-Specificity (>10%) (≤10%)

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mnp

Scoring RAD51 in clinical samples identifies HRR-deficient tumors among patients with hereditary breast and ovarian cancer (HBOC) syndrome, including PALB2-related tumors

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PART 3 Inhibiting WEE1 and ATM induces replication stress and enhances the antitumor response to PARPi in BRCA1-altered tumors.

AZD1775 antitumor activity in the PDX panel distinguishes a subset of tumors highly sensitive to WEE1 inhibition

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mnt

900 600

300 300

200

100 % Change from Baseline % Change

-100 PDX179 PDX127 PDX044 PDX137 STG201 PDX102 PDX274 PDX093 PDX071 PDX196 PDX124 PDX221 PDX252 PDX288 PDX197 STG316 PDX270 STG139 PDX060 PDX156 PDX094 PDX280 PDX236 PDX098 PDX93OR STG201OR

PDX098 PD SD AZD1775 120mg/kg 5/9 200 PR AZD1775 60mg/kg 5/2 CR 7 AZD1775 30mg/kg 5/2 150 Vehicle n=5 PDX094 6 100 5 STG139

4 50 PDX098

3 0 PDX156 PDX280 2 -50 Tumor Volume Change (%)

Relative Tumor Volume 1 PDX236 0 0 5 10 15 20 25 30 0 102030405060708090 Day of treatment Day of treatment

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mnu

Response biomarkers of WEE1 inhibition

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WEE1 inhibition induces accelerated mitotic entry and replication stress in vivo

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mom

p=0.0065 p=0.0014 p=0.0199 p=0.0006 20 60 50 p=0.0076 p<0.0001 50 p=0.0147 40 p<0.0001 p=0.0001 40 30 10 30 H2AX (+) (%) cells

γ 20 20 pHH3 (+) (%) cells pan- 10 10

0 0 (%) (+) cells geminin (+) / S4/S8 pRPA32 0

Vehicle Olaparib Vehicle Olaparib Vehicle AZD1775 Vehicle AZD1775 Vehicle Olaparib Vehicle Olaparib

PR/CR SD/PD PR/CR SD/PD ' %' ( % ' %' ( % ' %'PR/CR( % SD/PD

γ %#30 GL Y0 2+ ,25%2$FLLJ%,3 1+%2-2% ,207,0 .*%2%-,120 11= B ;1:)&) :)313& #//9)1nX[6(9#3&:(# #// @ /#_6 o[639):)=#`&3//3>)1':8#:0#1:>):(=#() /#1"mssq)1 m)[9#19):)=#_X`1"m)[8#9)9:1:_X`9_)8#".;:#9:)1=#() /#[=9m)[:8#:#" :;0389Q16)8#".;:#9:)1X=9X:;0389`SB 00;13&/;38#9 #1 #9:)1)1'3&6 o)1:;038 906/#9:8#:#">):(=#() /#38mssq3&:>3m)[9#19):)=#9SB;1:)&) :)313& #//9>):( 61[1; /#8γ n9:)1)1'&3//3>)1':8#:0#1:>):(=#() /#1"mssq)1m)[9#19):)=#_X` 1"m)[8#9)9:1:_X`9 _11\():1#@[:#9:`SB 00;13&/;38#9 #1 #9:)1)1'3&γ n )1:;038906/#9:8#:#">):(=#() /#38mssq3&:>3m)[9#19):)=#9SB;1:)&) :)313& '#0)1)1[639):)=# #//9:(:#?() ):6onpXt&3 )&380:)31&3//3>)1':8#:0#1:>):(=#() /#1" mssq )1 m)[9#19):)=# _X` 1" m)[8#9)9:1: _X` 9 _11\():1#@ [:#9:`S B 00;13&/;38#9 #1 #9:)1)1'3&6onpXt1"'#0)1)1)1:;038906/#9:8#:#">):(=#() /#38 mssq3&:>3m)[9#19):)=#9S; /#)>#8#=)9;/)A#">):( _ /;#`S//6) :;8#9>#8#:.#1 :rll?0'1)&) :)31S

mon

γ γ

60 60

50 50

40 40

30 30 PDX156 PDX098 20 20 H2AX (+) (%) cells γ PDX098 PDX156 10 PDX094 pan- 10 PDX280 PDX280 PDX094 0 0 pRPA32 S4/S8 (+)pRPA32 S4/S8 / geminin (+) (%) cells

Vehicle Vehicle

AZD1775-sensitive AZD1775-sensitive AZD1775-progression AZD1775-progression

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moo

WEE1 blockade in combination with PARP inhibition sensitizes BRCA1-altered tumors

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3&61[1; /#8γ n9:)1)1'1"6onpXt1; /#8&3 ) 31:)1)1' #//9_%#=HF`S mop

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Olaparib

800 600 400

300

200

100 % Change from Baseline % Change 0

-100 PDX127 PDX274 PDX221 PDX098 STG139 PDX137 STG316 PDX179 PDX102 PDX280 PDX044 PDX156 PDX094 PDX252 PDX060 PDX288 PDX196 PDX270 PDX236 PDX124 PDX093 PDX071 STG201 PDX197 PDX93OR STG201OR

300 Olaparib+AZD1775

200

100

% Change from Baseline 0

-100 PDX102 PDX044 PDX127 PDX137 PDX288 PDX274 PDX179 STG316 PDX156 PDX196 PDX094 PDX071 PDX221 STG139 PDX270 PDX060 PDX093 PDX252 STG201 PDX124 PDX236 PDX197 PDX280 PDX098 PDX93OR STG201OR

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PDX270

6 PDX124 7 Vehicle n=3 Vehicle n=4 Olaparib n=4 Olaparib n=7 6 5 AZD1775 n=6 AZD1775 n=6 AZD1775 n=9 Olaparib + AZD1775 n=5 5 4 4 3 3 2 p<0.0001 2 Relative Tumor Volume Tumor Relative 1 Relative Tumor Volume Relative Tumor 1 p<0.0035

0 0 0 102030405060708090100110120 0 10203040 Day of treatment Day of treatment

PDX196 PDX252

6 Vehicle n=5 7 Olaparib n=5 Vehicle n=7 AZD1775 n=6 6 Olaparib n=7 5 Olaparib+AZD1775 n=6 AZD1775 n=5 Olaparib + AZD1775 n=5 5 4 4 3 3 2 2

Relative Tumor Volume p<0.0001 p<0.0001 Volume Relative Tumor 1 1

0 0 0 102030405060 0 1020304050 Day of treatment Day of treatment

Vehicle n=4 STG201 6 Olaparib n=7 AZD1775 n=6 5 Olaparib + AZD1775 n=4

2 p<0.0001

Relative Tumor1 Volume

0 0 102030405060708090100110 Day of treatment

%#30 HEYF %,$%%2%-, ,$, 1 %@1 ,1%2%4%27 %, F@*2 0 23+-01 , .0 4 ,2 2$ /3%1%2%-,-"%0 1%12, =#8 #1:'#3&:;038=3/;0# (1'#";8)1':8#:0#1:>):(=#() /#P 3/68) Pmssq38 30 )1:)31_3/68) ymssq`)131#)[ _mnp`P:(8## )[ _murPnsl1"nqn`1"31#)[_nlm`9_>3[>@`S(#0#1yX[ )98#68#9#1:#"&38# (803&:8#:0#1:S

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p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001 40 40 p<0.0001 p<0.0001

p=0.0068 30 30 p=0.0157

20 20 H2AX (+) (%) cells γ

pan- 10 10 pRPA32 S4/S8 (+)pRPA32 S4/S8 / geminin (+) (%) cells 0 0

Combination CR/PR Combination SD/PD Combination CR/PR Combination SD/PD

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ATM blockade in combination with PARP inhibition sensitizes other BRCA1- altered tumors

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p<0.0001 p=0.0001 300 100 100 p<0.0001 p=0.0001 p=0.0003 ATM VUS 80 80 200 p=0.0027 p=0.0034 60 60

100 40 ATM H-score ATM 40

20 20 0 0

H2AX (+) / cells (+) Geminin (+) (%) H2AX 0 γ RAD51 (+) / Geminin (+) cells (%) STG316 PDX221 PDX230 PDX252 PDX236 PDX124 PDX280 PDX071 PDX179 PDX127 PDX274 PDX196 Vehicle Olaparib Vehicle AZD0156 PDX230OR Olaparib + Olaparib AZD0156 AZD0156 Olaparib + AZD0156

Vehicle n=13 Vehicle n=9 Vehicle n=6 Olaparib n=6 7 Olaparib n=11 7 Olaparib n=13 7 AZD0156 n=11 AZD0156 n=15 AZD0156 n=4 6 Olaparib+AZD0156 n=10 6 Olaparib+AZD0156 n=9 6 Olaparib+AZD0156 n=6

5 5 5

4 4 4

3 3 p=0.0014 3 p=0.0002 2 2 2

1 Volume Relative Tumor 1 1 Relative Tumor Volume Relative Tumor Relative Tumor Volume 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 Day of treatment Day of treatment Day of treatment

p=0.0031 20 20 20 p=0.0107 p=0.0082 p=0.0107 15 15 p=0.0281 15

10 10 10 H2AX (+) (%) cells γ H2AX (+) (%) cells H2AX (+) (%) cells γ γ

pan- 5 5 5 pan- pan-

0 0 0

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TARGETING DDR AND CELL CYCLE A Drug Anti-tumoral Immune Evasion

Cell cycle checkpoint

Effect is manifest

M G1 G2 S Maximize DNA damage C TARGETING IMMUNE RESPONSE Prevent repair

CD4

Anti-tumor Immunity CD8 T naive DNA damage CytosolicC t li DNANAA α PD-L1 cGAS B TARGETING HRR CAPACITY PD-L1 STING IRF3IRF3 PD-L1 EGFR DC MET

VEGFR Activation DC + cGAS Maduration IRF3 RAS STING Type I IFN CXCL10 RAF PI3K CCL5 Anti-tumor MEK AKT Immunity Angiogenesis Activation AR ERK mTOR

NK MØ

Promotes HRR

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CONCLUSIONS

mqr

PART 1

• :)#1:"#8)=#"?#13'8&:9_`&830'#80/)1#@:A[0;::#"_'` 8#9: 1" 3=8)1 1 #8 6:)#1:9 8# 6):;/:# :(# 6:)#1:U9 8#96319# :3 ) 1" 8#68#9#1:9;): /#6/:&380:39:;"@!(0!0)1:):;038 :)=):@3&) • #9:38:)313& &;1 :)31/):@)9&8#7;#1:031''9:(:"313:8#9631" :3):8#:0#1: • 8# 3=#8@)1:(#'9)9 ()#=#" @9#=#8/0# (1)909)1 /;")1':(# #?68#99)313&(@630386() m683:#)191"/3993&1#1"8#9# :)31 88)#8P )1 /;")1'qom1"oq • #:# :)313&qm&3 ))1':;0389&83091"6:)#1:9 388#/:#9>):( )8#9)9:1 #8#'8"/#993&:(#;1"#8/@)1'0# (1)9093& 8#9:38:)31 • (#qm99@)96830)9)1' )308.#8:3 #;9#")1:(# /)1) &38 #::#89#/# : '6:)#1:9:(:>)// #1#&):&830)0313:(#86@

PART 2

• (#8#8#9&830@:A>)/":@6#_131['` 8#9: 1 #8_`6:)#1:9 :(:9(3>#"1:):;038 :)=):@;631)1() ):)31 • @68303:#8(@6#80#:(@/:)311"'#1#:) /:#8:)319)1 [8#/:#"'#1#9 8#993 ):#">):()9#19):)=):@)1131['9 • (#qm9 38#)9&;1 :)31/ )308.#8>):( 306/#:#")9 8)0)1:)=# 6 ):@)1 68#") :)1' ) 1:):;038 8#96319# )1 9 1" 3;:6#8&380#" :(# '#130) :#9: • (#qm99@)9&#9) /#)183;:)1# /)1) /906/#9>):(3;:68)38#?639;8#:3 "0')1''#1:9 • 1 /)1) /906/#9&8306:)#1:9>):( 9@1"830#P 88@)1' A0;::)31 )9993 ):#">):(/3>qm9 38# • (#qm9 38#)9&;1 :)31/ )308.#8:(:#1 /#9:(#)"#1:)&) :)313&)[ 9#19):)=# 1" 83"#19 :(# 636;/:)31 >(3 0@ #1#&): &830 :()9 :8'#:#" :(#86@ #@31"[8#/:#" 1 #89

mqs

PART 3

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mqt

REFERENCES

mrl

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APPENDIX

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