Elaborating the Mechanism of PARPthe Mechanism Elaborating Following Lethality Synthetic ATMDLD in Loss inhibitors BRCA could Our mediated This required inhibition mediated BRCA immunofluorescence assays, CRISPR/Cas aimed respond ATM carcinoma), harbor monotherapy these DNA patients PD benefit proven growth and Non such recognized and determined reported homologous genetic of deficient caused loss which PARP Abstract No. 1699, Session Title: Basic Mechanisms of Genome IntegrityofGenome Title:MechanismsBasicSession 1699, No. Abstract Background and Highlights of theFindings andHighlights of Background niraparib ATM treatment deficient a Method acquisition CellMaskTM (DMSO) 1450 Method longer set Method Methods GlaxoSmithKline, Synthetic Lethality Research Unit, Waltham, MA, US MA, Waltham, Unit, Research Lethality Synthetic Coleman G. Kevin GlaxoSmithKline, Muvaffak*, Asli high both - 1 of - of accounts findings Small - activates : ( as Damage data 605 functions PD - be patients, 1 1 orally, / clinically is (Poly BRCA content - from to / ) mutations to factor SSB III I or 2 prostate, II were by who DLD treated : . cells ) and - KO, to known for : and to L DNA regulated for Niraparib and was of & DNA be expression Colony identify 1 epigenetic provided . Cell inhibition germline, recently deep once which 18 IV - A ADP contribute blockade, these ATM/BAP incubated 1 highlight 9 1 (Single recombination ATM BRCA may through phosphorylation in effective immunofluorescence : treatment performed hrs for relevant receptor key Response cell characterize tumors repair or major HR in in we Lung NSCLC checkpoint to daily red ; - lung formation BRCA 80 Ribose both WB DLD single is in lines (homologous not DNA PARPi have targeted activation sought plasma 2 through - as . involved 85 Strand of 1 in . 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ATM cells HR HR HR loss loss in and low no cell the the we % on for be by - or . 2 is to of a 1 . . ) - . - - ATMmediatedphosphorylation of BRCA proteins is required for proper functioningof homology directed repair DSBs of In Results high antihigh In activity, Tumor(TGI%): vivo GrowthInhibition% {1 niraparib niraparib Fi Yoshida, K and Miki, Yoshida,KY Miki, and2017Sci etExpertInvestig Brandsma,al.CancerOpin I 2004;Drugs. Fi DLD ▪ B. ▪ g g A. ▪ u u Vitro Role of ATM DNAin ResponseStrand Break& DamageDouble Repair r r e e - compared In tumor activity tumorIn activity level; vitro activity (3D 2 tumor In 1 - . in . clonogenic Niraparib 1 in Dose TGI: 88% DDR DLD Vitro vivo Vivo BRCA2 response & - pathway 1 activity I BRCA . xenograft : In : Homozygous Niraparib curves Niraparib 2 overview, in Vivo to and DLD in ( for - assay the ATM / niraparib - - 1 ) vivo and hBRCA (+/ Sensitivity DLD clonogenic model - in ), the - showed BRCA (∆T/∆C)}*100; Response Criteria: to 42%i NCI standards,a T/C≤ Criteria: According (∆T/∆C)}*100; Response ( in - / 2 monotherapy - role Yoshida, K and Miki, Y Cancer Sci I Brandsma, Sci 2004; et Y Miki, Cancer Expert 2017 Kal. Investig Yoshida, and Opin Drugs ) activity ( - KO ), TGI%: ), {1 - DLD 1 / 2 - ), of Niraparib ActivitySummary(CFA, 14d) TGI: 60% ( - cell / parental - DLD ), key ATM ATM lines Loss - - checkpoint - (T 1 1 ( IC50 a - / hATM - (C) BRCA ), /C correlates ( of & 80 IC50 - . ATM / )}*100, where T where)}*100, - - ( ) - Tumor fold / regulators, PathwayOverview of (+/ - ) - inhibited KO ) 2 KO ( ATM & cell - difference / IC50 DLD - Presented at the Presented lines ), ATM, : niraparib IC : niraparib well - Cells ATM 1 and hATM ATR, niraparib Causes Niraparib Niraparib TGI: 20% IC50 IC50 (M) Inactive tumor 50 ATM and with onDLD (+/ (+/ sensitivity to - ) in DNA - KO ) - 1 +/ ATM & Niraparib - sensitivity PK growth cell its summary s the minimum level s of thelevel anti minimum - 6.33x10 6.33x10 ( KO or ) in - a lines / hBRCA2 - DSB ( in - ) BRCA2 / - Marked KO ) KO and (B) repair - 9 vitro , sensitivity KO, C (B4) in Comparison cell processes IC50 in DLD : niraparib IC niraparib : ranking - tumor activity tumor& aT/C<10% is considered activity activity lines C. In Vitro vsIn Vivo ATM Niraparib Activity 19.25x10 ( hATM - Increase of - . / using - 1 DLD ) KO AACR 2020 Virtual2020AACR Meeting, In 50 ATM 2 vitro onDLD ( ( D - - - - 9 1 1 / / colony - - ) HOMO KO HOMO ) vs ) BRCA2 BRCA2 from - 1 parental) 1 cell in formation ( vivo - 2.93x10 (+/ / hATM - ) - in ) KO & line activity 3 assay KO ATM D - 6 - the (A), of compared DLD Fi (A) Fi Inhibition Results g HOMO Results u g - r B. u signaling NBN, Preliminary 1 in e r ATM ATM e vitro 4 A. B. . 3 to C . Mean % RAD51expression is hange ( WB levels - Rad in / the - (%Rad51/beta-actin) ) In vitro KO analysis DLD In vivo 51 primary KO in III in in - cell foci II BRCA Dld of data . ATM *Rad51 levels normalized to Dld to normalized levels *Rad51 - . 1 line - & 1 DDR of Loss HR ATM Phosphorylation Cell parental  1 H tumor HR , loss 2 BRCA indicates target AX June 22 ▪ ▪ function ( Capacity - model, - foci / cell Rad51 foci formation is inhibited in ATM 2 that - and Lines ) of Niraparib & assay cell Vehicle line DLD RAD (3µM) phosphorylates : A. n=

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and 50 DLD abrogates **** (Rad ATM Rad is is levels H (A) - 2 - provides 1 AX 51 50 24 ; BRCA % Impaired of Associated )/beta in WT leads (by protein pRad of Merged Merged of Images Rad51 & response MRN WB th regulation 2 - 50 actin), **** p<0.0001, *** p:0.0009,** p:0.0024,* p:0.0467 pairedt ( - (Ser / levels analysis) 4.4 to 1.6 , 2020 , - further ) DSB (n=2) & to inhibition 638 (n= - - DLD fold DSBs, fold Complex in 1 )/Rad hATM

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- in - test ) DLD - 1 **** ) & KO, **** p<0.0001, ** p<0.0096, * t p:0.036,unpaired ATM - ** 2 ) MRN ( HR deficiency Rad51 foci & 50 (C), Hoechst  genes in H2AX foci treated mg/kg from - - / Dld - hBRCA2 ( mediated Rad Niraparib - cell complex 1 - 1 niraparib ( PO, line hATM - samples ATM in / (n=3) - 51 (Burma, QD this ) at HOMO & 3 28 treated µM in DNA (B) proteins, Expression, ( cell days S - ( . hBRCA - & . - DLD / KO et - / Etoposide - ) repair in line al, or ) cell lines 1 Cell Line 1 Cell cell KO vivo J longer) - Biol line 1 i (n=2) tumors . at ATM e Chem checkpoint Cell by . in 40 RAD 2 - WB test tumors µM) 21 2.7 2001 at ( analysis, . ( - termination - Line fold 50 - - ) / / fold and - from - ) ) & ● ● ● ● ● % { by Fi 5. 4. 3. 2. 1. 1 References Conclusions Acknowledgements Rad - g (∆T/∆C)}* WB u is MRN mutant Loss models extends ATM downregulation ATM Biallelic Results ▪ Gawden Deigner, generation We The r Birkelbach Burma Res Schmitt Lord, Lord, e 51 regulated analysis 5 expression C. . would . presenting in ATM Niraparib 2017 biallelic C C A. ( of 100 complex - S . . / both . A, NSLC - J J - . , sensitivityin Aninverse relationship is observed between BRCA1/2expression levels and niraparib (B), ATM , Bone ) beyond . . Konstantin ; following , Chen Knittel & & like Jun deficiency Niraparib Niraparib In Vivo M KO of monotherapy Correlation Ashworth, Ashworth, in . IV through Mutant et p PDX the to 1 Non and is - B author, 53 PDX / ; in al - . 77 proteins G . niraparib express P of . associated . samples BRCA DLD ( Differing . , J DLD , 11 deficient Simon - Welcker - ATM Rad Murphy Thorac Lashuk / analysis ) - models A Nat : in 3040 BRCA - Asli . * 1 monotherapy - Nature vivo is are email for more information: [email protected] information: foremail more by 1 . 51 ATM 1 NSCLC special BRCA Rev HRR_Bi in Scrace WT WT del( / WB - Oncol Muvaffak cell 2 M of associated D , activity 3056 NSCLC PDX Models from DLD particularly . indicative . tumor . , 1 . genes , analysis, and Cancer with Yang 2012 Kurimasa Tumor Growth Inhibition, TGI (%) Preliminary biallelic mutant NSCLC PDXmodels and 2 Activiity . line PDX #6 #5 #4 #3 #2 #3 #4 #5 PDX#6 2013 thanks summary Degrees - from Oncotest and at growth 1 ; - proficient T decreased 50 /0) WT 481 & BRCA ATM PDX ID: compared . caused ; and 2016 P PDX mg/kg 8 co ATM Horizon . 30% ND ND 30% : , inhibition A ( of to 287 3 in George - . , with ) author sensitive ; mainly : HRR ( in HRR Olivia Chen /CRL PO, impairment 16 - 279 2 – / data ( - to 294 - : Models #1 #1 #2 #4 #3 #5 #6 genes ) / QD 110 a - mutant - (TGI levels Discovery ), KO 286 markedly J D of lung . Kevin for BRCA . marked Li **** p<0.0001, *** p:0.0009,** p:0.0024,* p:0.0467 pairedt for , (+/ . – indicates J % driven Nowak 120 . . & - ) in 28 cell Bialleic supporting - / J NSCLC to - and Niraparib ) in Dld Cindy Biol days . ATM adeno CRISPR/Cas G niraparib del( 1 DLD - downregulation of line, 1 Coleman / M for Chem 2 ND by parental or Del( PDX increase . HR - _Bi , Liu /0) increased expression longer, Leeser performing ATM - - ATM, /0): deleted, hemizygousdeleted, /0): Mutant Mutant suggestive 1 - models - the - from capacity 2001 & ATM / ND: Not Determined Not ND: - cell and in squamous are ( ND in U - 9 line of ; BAP / (A), Applied . Sensitivity NSCLC - 276 in baseline , WT WT ( vitro KO ) BRCA n=6 * both Büttner - ( % / loss & the - niraparib Rad ( ) 1 sensitivity in ATM in 45 cell , KO 1 of & employees B. / - * 37% Rad 51 ) BRCA 2 & DLD both / : leads Stem - in expression expression downstream R /beta 42462 lines, PDX MRE cell . , vivo 51 cell ATM Expression is Lost in 1 Perner

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