Supplementary Figure 1: BMN 673 induces DNA damage at lower doses and with higher intensity than olaparib. HeLa cells were treated with a dose range of olaparib or BMN 673 and stained for γH2Ax foci after 24 hours. A) Confocal image 24 hours of 10 nM BMN 673 treatment. Blue = DAPI, red = γH2Ax. B) 24 hours of 10nM olaparib treatment, showing fewer γH2Ax foci than the same concentration of BMN 673. C) Percentage of cells with >5 nuclear foci, 24 hours after PARP inhibitor treatment in different concentrations, as determined by confocal microscopy. A minimum of 100 cells were counted per coverslip. Error bars represent SD in three replicates.

Supplementary Figure 2: BMN 673 is active in vivo against xenograft models with PTEN deficiency. Nude mice carrying xenografted tumors from A) LNCAP and B) MDA-MB-468 cell lines were treated once-a-day for 28 consecutive days with oral dosing of BMN 673 at 0.33 mg/kg/day or its vehicle. Tumor growth delay was observed in both PTEN-null xenografts. Median tumor volume was plotted against days of treatment (first day of treatment is defined at Day1).

Supplementary Figure 3: Balb/c nude mice carrying sc MDA-MB-468 xenograft tumors were treated with BMN673 at 0.33 mg/kg, QD or BMN673 at 0.165 mg/kg, BID for 28 consecutive days (Days 15-42 post tumor implantation) and the animals were terminated at Day 64 post tumor implantation.

Supplementary Table 1. Inhibition of PARP1 enzymatic activity by chiral isomers of BMN 673. LT-00628 is a racemic mixture of trans isomers that include BMN 673 and LT-00674. LT-00878 is the racemic mixture of cis isomers.

PARP1 IC50 was determined for BMN 673 (0.57 nM), LT-00674 (144 nM), LT- 00628 (1.82 nM) and LT-00878 (>100nM). Lack of potent inhibition of PARP1 suggesting both cis isomers have weak or no activity of PARP1 inhibition.

Racemate Enantiomers PARP1 IC50 (nM)

LT-00878 >100 (cis) Not isolated N/A LT-00628 1.82 (trans) BMN 673 0.57

LT-00674 144

Supplementary Table 2. List of genes that are targeted by the siRNA library. siRNA library targeting 960 genes, encompassing kinases and kinase related genes as well as a series of tumor suppressors and DNA repair proteins.

AAK1 CKMT2 FRAP1 MAPKAPK2 PIK3C3 RPS6KA4 AATK CKS1B FRDA MAPKAPK3 PIK3CA RPS6KA5 ABL1 CKS2 FRK MAPKAPK5 PIK3CB RPS6KA6 ABL2 CLK1 FUK MARK1 PIK3CD RPS6KB1 ACVR1 CLK2 FYN MARK2 PIK3CG RPS6KB2 ACVR1B CLK2 GAK MARK3 PIK3R1 RPS6KC1 ACVR1C CLK3 GALK1 MARK4 PIK3R1 RPS6KL1 ACVR2 CLK4 GALK2 MAST2 PIK3R2 RRM2B ACVR2B COASY GCK MAST3 PIK3R3 RYK ACVRL1 COL4A3BP GEN1 MAST4 PIK3R4 SAST ADCK1 COMMD3 GIYD1 MASTL PIK4CA SBDS ADCK2 COPEB GK MATK PIK4CB SBK1 ADCK4 CPNE3 GK2 MBD4 PIM1 SCAP1 ADCK5 CRIM1 GNE MDC1 PIM2 SCYL1 ADK CRK7 GOLGA5 MELK PIM3 SDHB ADP-GK CRKL GPC3 MEN1 PINK1 SDHC ADRBK1 CSF1R GRK1 MERTK PIP5K1A SDHD ADRBK2 CSK GRK4 MET PIP5K1B SETMAR AIP1 CSNK1A1 GRK5 MGC16169 PIP5K1C SGK AK1 CSNK1A1L GRK6 MGC42105 PIP5K2A SGK2 AK2 CSNK1D GRK7 MGC45428 PIP5K2B SGKL AK3 CSNK1E GSG2 MGC4796 PIP5K2C SHFM1 AK3L1 CSNK1G1 GSK3A MGC4796 PIP5K3 SIK2 AK5 CSNK1G2 GSK3B MGC8407 PIP5KL1 SLK AK7 CSNK1G3 GTF2H1 MGMT PKIA SMAD4 AKT1 CSNK2A1 GTF2H1 MIDORI PKIB SMARCB1 AKT2 CSNK2A2 GTF2H2 MINK PKLR SMG1 AKT3 CSNK2B GTF2H3 MKNK1 PKM2 SMUG1 ALK CYLD GTF2H4 MKNK2 PKMYT1 SNARK ALKBH2 DAPK1 GTF2H5 MLCK PKN3 SNF1LK ALKBH3 DAPK2 GUCY2C MLH1 PLK1 SNRK ALS2CR2 DAPK3 GUCY2D MLH1 PLK2 SOCS1 ALS2CR7 DCAMKL1 GUCY2F MLH3 PLK3 SPEG AMHR2 DCK GUK1 MMS19 PLK4 SPHK1 ANKK1 DCLRE1A H2AFX MNAT1 PMS1 SPHK2 ANKRD3 DCLRE1B HAK MOS PMS1 SPO11 APC DCLRE1C HCK MPG PMS2 SRC APEX1 DDB1 HEL308 MPP1 PMS2 SRMS APEX2 DDB2 HIPK1 MPP2 PMVK SRP72 APTX DDB2 HIPK2 MPP3 PNCK SRPK1 ARAF1 DDR1 HIPK3 MRE11A PNKP SRPK2 ARK5 DDR2 HIPK4 MSH2 PNKP SSTK ASK DGKA HK1 MSH2 POLB SSTK ATM DGKB HK2 MSH3 POLD1 STK10 ATM DGKD HK3 MSH4 POLE STK11 ATM DGKG HRI MSH5 POLG STK11 ATR DGKH HRPT2 MSH6 POLH STK16 ATR DGKI HSMDPKIN MSH6 POLI STK17A ATRIP DGKK HSPB8 MST1R POLK STK17B AURKA DGKQ HUNK MTMR15 POLL STK19 AURKB DGUOK HUS1 MULK POLM STK22B AURKC DKFZP434C131 HUS1 MUS81 POLN STK22C AXL DKFZP761P0423 ICK MUSK POLQ STK22D BAIAP1 DLG1 IGF1R MUTYH PRKAA1 STK22D BCKDK DLG2 IGF2R MUTYH PRKAA2 STK23 BCR DLG3 IHPK1 MVK PRKAB1 STK24 BHD DLG4 IHPK2 MYLK PRKAB2 STK25 BLK DMC1 IHPK3 MYLK2 PRKACA STK29 BLM DMPK IKBKAP MYO3A PRKACB STK3 BLM DTYMK IKBKB MYO3B PRKACG STK31 BMP2K DUSP21 IKBKE N4BP2 PRKAG1 STK32A BMPR1A DUSTYPK IKBKG NAGK PRKAG2 STK32B BMPR1A DUT ILK NBN PRKAG3 STK32C BMPR1B DYRK1A ILK-2 NBS1 PRKAR1A STK33 BMPR2 DYRK1B INSR NEIL1 PRKAR1A STK35 BMX DYRK2 INSRR NEIL2 PRKAR1B STK36 BRAF DYRK3 IPMK NEIL3 PRKAR2A STK38 BRCA1 DYRK4 IRAK1 NEK1 PRKAR2B STK38L BRCA1 EEF2K IRAK2 NEK11 PRKCA STK39 BRCA2 EFNA3 IRAK3 NEK2 PRKCB1 STK4 BRCA2 EFNA4 IRAK4 NEK3 PRKCD STYK1 BRD2 EFNA5 ITK NEK4 PRKCE SUFU BRD3 EFNB3 ITPK1 NEK5 PRKCG SYK BRD4 EGFR ITPKA NEK6 PRKCH TAF1 BRDT EIF2AK3 ITPKB NEK7 PRKCI TAF1L BRIP1 EIF2AK4 ITPKC NEK8 PRKCL1 TAO1 BRIP1 EME1 JAK1 NEK9 PRKCL2 TBK1 BTBD12 EME2 JAK2 NF1 PRKCM TCF1 BTK EP300 JAK3 NF2 PRKCN TDG BUB1 EPHA1 JIK NHEJ1 PRKCQ TDP1 BUB1B EPHA10 KALRN NLK PRKCSH TDP2 BUB1B EPHA2 KCNH2 NME1 PRKCZ TEC C10ORF89 EPHA3 KCNH8 NME2 PRKD2 TEK C14ORF20 EPHA4 KDR NME3 PRKDC TESK1 C19orf40 EPHA5 KHK NME4 PRKDC TESK2 C7ORF11 EPHA6 KIAA0999 NME5 PRKG1 TEX14 C7ORF2 EPHA7 KIAA1361 NME6 PRKG2 TGFBR1 C9ORF12 EPHA8 KIAA1639 NME7 PRKR TGFBR2 C9ORF96 EPHB1 KIAA1765 NPR2 PRKWNK1 TGFBR3 CALM1 EPHB2 KIAA1804 NRBP PRKWNK2 THNSL1 CALM2 EPHB3 KIAA1811 NRBP2 PRKWNK3 TJP2 CALM3 EPHB4 KIAA1811 NRK PRKX TK2 CAMK1 EPHB6 KIAA2002 NTHL1 PRKY TLK1 CAMK1D ERBB2 KIT NTRK1 PRPF4B TLK2 CAMK1G ERBB3 KSR NTRK2 PRPS1 TNIK CAMK2A ERBB4 KSR2 NTRK3 PRPS1L1 TNK1 CAMK2B ERCC1 KUB3 NUCKS PRPS2 TNK2 CAMK2D ERCC2 LAK NUDT1 PSKH1 TNNI3K CAMK2G ERCC2 LATS1 NUP62 PSKH2 TOPBP1 CAMK4 ERCC3 LATS2 NYD-SP25 PTCH TOPK CAMKIINALPHA ERCC3 LCK OBFC2B PTEN TP53 CAMKK1 ERCC4 LIG1 OGG1 PTEN TP53 CAMKK1 ERCC4 LIG3 OSR1 PTK2 TP53BP1 CAMKK2 ERCC5 LIG4 P101-PI3K PTK2B TP53RK CARKL ERCC5 LIMK1 PACE-1 PTK6 TPK1 CASK ERCC6 LIMK2 PACSIN1 PTK7 TREX1 CCNH ERCC8 LMTK2 PAK1 PTK9 TREX2 CCRK ERK8 LMTK3 PAK2 PTK9L TRIB1 CDADC1 ERN1 LOC340156 PAK3 PXK TRIB2 CDC2 ERN2 LOC390226 PAK4 PYCS TRIB3 CDC2L1 ETNK1 LOC91461 PAK6 RAD1 TRIO CDC2L2 EXO1 LRRK1 PAK7 RAD17 TRPM6 CDC2L5 EXOSC10 LRRK2 PALB2 RAD18 TRPM7 CDC42BPA EXT1 LTK PANK1 RAD23A TSC1 CDC42BPB EXT2 LYK5 PANK2 RAD23B TSC2 CDC7 FANCA LYN PANK3 RAD50 TSKS CDH1 FANCA MAD2L2 PANK4 RAD51 TTBK1 CDK10 FANCB MAGI-3 PAPSS1 RAD51C TTBK2 CDK11 FANCC MAK PAPSS2 RAD51L1 TTK CDK2 FANCC MAP2K1 PARP1 RAD51L3 TYK2 CDK3 FANCD2 MAP2K2 PARP2 RAD52 TYRO3 CDK4 FANCD2 MAP2K3 PASK RAD54B UBE2A CDK5 FANCE MAP2K4 PCK1 RAD54L UBE2B CDK5 FANCE MAP2K4 PCK2 RAD9A UBE2N CDK5R1 FANCF MAP2K5 PCNA RAF1 UBE2V2 CDK5R2 FANCF MAP2K6 PCTK1 RAGE UCK1 CDK6 FANCG MAP2K7 PCTK2 RB1 UHMK1 CDK7 FANCG MAP3K1 PCTK3 RBBP8 ULK1 CDK7 FANCI MAP3K10 PDGFRA RBKS ULK2 CDK8 FANCL MAP3K11 PDGFRB RDM1 ULK4 UMP- CDK9 FANCM MAP3K12 PDGFRL RECQL CMPK CDKL1 FAS MAP3K13 PDIK1L RECQL4 UMPK CDKL2 FASTK MAP3K14 PDK1 RECQL4 UNG CDKL3 FBXW7 MAP3K15 PDK2 RECQL5 URKL1 CDKL4 FEN1 MAP3K2 PDK3 RELA VHL CDKL5 FER MAP3K3 PDK4 RET VRK1 CDKN1A FES MAP3K4 PDPK1 REV1 VRK2 CDKN1B FGFR1 MAP3K5 PDXK REV3L VRK3 CDKN1C FGFR2 MAP3K6 PER1 RFK WAS CDKN2A FGFR3 MAP3K7 PFKFB1 RFP WEE1 CDKN2A FGFR4 MAP3K7IP1 PFKFB2 RIOK1 WNK4 CDKN2B FGFRL1 MAP3K8 PFKFB3 RIOK2 WRN CDKN2C FGR MAP3K9 PFKFB4 RIOK3 WRN CDKN2D FH MAP4K1 PFKL RIPK1 WT1 CERK FLJ10761 MAP4K2 PFKM RIPK2 XAB2 CETN2 FLJ13052 MAP4K3 PFKP RIPK3 XPA CHAF1A FLJ21816 MAP4K4 PFTK1 RNASEL XPA CHEK1 FLJ23074 MAP4K5 PGK1 ROCK1 XPC CHEK1 FLJ23356 MAPK1 PGK2 ROCK2 XPC CHEK2 FLJ23356 MAPK10 PHKA1 ROR1 XRCC1 CHEK2 FLJ25006 MAPK11 PHKA2 ROR2 XRCC2 CHEK2 FLJ32685 MAPK12 PHKB ROS1 XRCC3 RP6- CHKA FLJ34389 MAPK13 PHKG1 213H19.1 XRCC4 CHKB FLJ35220 MAPK14 PHKG2 RPA1 XRCC5 CHUK FLJ39827 MAPK3 PHOX2B RPA2 XRCC6 CIB2 FLT1 MAPK4 PI4K2B RPA3 XYLB CIT FLT3 MAPK6 PI4KII RPA4 YES1 CKB FLT4 MAPK7 PIK3C2A RPS6KA1 ZAK CKM FN3K MAPK8 PIK3C2B RPS6KA2 ZAP70 CKMT1B FN3KRP MAPK9 PIK3C2G RPS6KA3 ZNFN1A1

Supplementary Table 3: Drug effect (DE) scores for significant siRNA sensitization genes from siRNA PARPi drug sensitization screens. CAL51 cells were transfected with a library of siRNAs and treated with BMN 673, olaparib, veliparib or rucaparib. The sensitization effect of each siRNA on PARP inhibitor was quantified by the calculation of a Drug Effect (DE) Z score, with genes returning DE Z scores of <-2 being considered significant sensitization effects.

Gene (HR genes in bold) BMN 673 Olaparib Veliparib Rucaparib RAD51 -10.88 -15.44 -9.57 -10.28 RBBP8 -8.47 -3.09 -9.53 -6.36 SHFM1 -6.92 -7.60 -5.68 -6.76 RPA2 -6.60 -3.48 1.39 -4.08 CSNK1G1 -5.97 -2.08 -9.71 -3.54 BRCA2 -5.62 -2.24 -3.41 -2.18 TOPBP1 -5.25 -2.06 -6.35 -3.82 TTBK1 -4.92 -5.60 -4.06 -3.43 PIK3C2A -4.82 -6.05 -5.42 -2.77 GUCY2D -4.79 -2.36 -3.83 -9.16 FN3KRP -4.75 -1.48 -4.75 -2.26 PRKCL2 -4.45 -2.48 -3.09 -3.32 PALB2 -4.44 -3.03 -4.65 -3.38 PNKP -4.38 -1.39 -2.50 -1.64 RAD17 -4.29 -0.73 -2.70 -0.66 CHEK1 -4.01 -1.26 -4.70 -2.05 STK39 -3.99 -1.11 -4.06 -2.97 NEK3 -3.92 -4.35 -5.36 -5.38 ATRIP -3.81 -1.95 -3.34 -2.43 WEE1 -3.60 -3.85 1.27 -3.70 DDB1 -3.38 -3.24 -3.26 -5.14 BCKDK -3.34 -1.94 -2.89 -3.29 RELA -3.34 -1.89 -2.57 -2.78 BRCA1 -3.30 -0.96 -0.99 -1.59 CAMK1 -3.28 0.20 -6.22 -4.60 PANK4 -3.07 -0.89 -1.40 0.09 PCNA -3.06 -1.37 -0.20 -3.41 NUP62 -3.00 -3.21 -15.70 -4.66 CHAF1A -2.91 -1.88 -2.23 -2.33 PLK4 -2.91 -2.90 -10.63 -2.50 MNAT1 -2.90 -2.97 -5.66 -0.11 ATR -2.86 -0.50 -1.58 -0.95 PFKFB1 -2.85 -2.16 -6.06 -2.63 LOC340156 -2.80 -1.11 -2.91 -0.51 ERK8 -2.78 -1.33 -2.95 -2.86 C9ORF96 -2.76 -2.23 -2.49 -2.00 FANCM -2.72 -4.15 -2.98 -3.07 GNE -2.69 -1.56 -1.35 0.22 PCTK3 -2.69 -1.51 -4.19 -2.09 STK11 -2.68 -2.52 -2.40 -4.08 TESK1 -2.65 -3.64 -3.62 -0.58 ATM -2.61 -2.53 -2.05 -2.86 DGKQ -2.60 -2.29 -2.59 -6.45 TRIB1 -2.56 -1.51 -3.89 -1.71 PMVK -2.56 -1.37 -1.55 -2.12 CRKL -2.55 -4.65 -1.44 -5.62 CSF1R -2.53 -2.34 -1.10 -1.08 HUS1 -2.51 -2.26 -1.98 -4.55 SRMS -2.44 -1.33 -0.72 0.03 MSH4 -2.42 -0.41 -1.08 -0.27 BTK -2.39 0.65 -0.67 1.24 TDP1 -2.33 0.40 -0.80 -1.95 GRK4 -2.32 -1.69 -3.43 -3.13 PRKX -2.32 -1.77 -1.28 -0.80 RPS6KA4 -2.30 -0.84 -0.63 -0.10 AURKB -2.25 -4.63 -4.26 -2.30 SDHD -2.23 -4.58 -3.11 -3.09 CDK5R2 -2.23 -3.40 -2.66 -4.80 FUK -2.18 -6.23 -1.59 -3.31 SYK -2.18 -0.27 -2.08 -0.87 FANCA -2.18 -1.90 -3.57 -0.02 CDC2L2 -2.17 -0.07 -15.33 -5.46 GRK6 -2.16 -1.62 -0.78 -3.28 PTK7 -2.14 -4.75 -1.47 -9.14 RPS6KB1 -2.13 -0.22 -2.72 -0.53 FGR -2.12 -3.81 -1.86 -6.21 XAB2 -2.11 -4.85 -11.94 -5.24 EGFR -2.07 -1.48 -0.58 -1.21 TDP2 -2.06 -3.39 -0.20 -2.89 SBK1 -2.06 -0.83 -0.29 -0.63 PRKACG -2.02 -1.53 -1.33 -0.72 HK3 -2.02 -1.15 -2.54 -1.89 IPMK -2.01 -0.16 -0.81 -0.13

Supplementary Table 4: siRNA drug sensitization profiles for all PARP inhibitors tested. The siRNA drug sensitization profiles of CAL51 cells transfected with a library of siRNAs and treated with one of olaparib, BMN 673, veliparib or rucaparib were compared by a two-sided t-test. No statistically significant differences (at p<0.05) were found between any of the drug pairs.

BMN 673 Olaparib Veliparib Rucaparib

BMN 673 1.00 0.22 0.57 0.07

Olaparib 0.22 1.00 0.60 0.54

Veliparib 0.57 0.60 1.00 0.30

Rucaparib 0.07 0.54 0.30 1.00 Supplementary Table 5. Chiral selectivity of anti-tumor activities and PARP inhibition potency. Enantiomer pair BMN 673 and LT-00674 were obtained using chiral separation, and tested in the PARP activity and cellular assays. One enantiomer of each pair was much more active than the other (such as BMN 673 and its trans-isomer LT-00674).

PARP1 Cellular TMZ Chemo- Enzyme PAR Capan-1 Racemate Enantiomers sensitization IC50 Inhibition IC50 (nM) GI50 (nM) (nM) IC50 (nM) BMN 673 0.57 2.51 3 5 LT-00628 LT-00674 144 864 1807 1135

Supplementary Table 6. Metabolic stability of BMN 673 in human, dog and rat liver microsomes. Metabolic stability was determined by incubating 1 μM test compounds with rat, dog or human liver microsomes, and the remaining concentrations of the test compounds at 0, 15, 30, 60 and 120 min were determined by liquid chromatography-tandem mass spectrometry. Half-life of greater than 120 minutes in liver microsomes suggests minimal metabolism of BMN 673.

Test Article Species T1/2 (minute) Human 52.9 Olaparib Dog 47.5 Rat 17.0 Human >120 BMN 673 Dog >120 Rat >120