PARP1 Trapping by PARP Inhibitors Drives Cytotoxicity Both in Cancer Cells and Healthy Bone Marrow

Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Title: PARP1 trapping by PARP inhibitors drives cytotoxicity both in cancer 2 cells and healthy bone marrow

4 Authors: Todd A. Hopkins, William B. Ainsworth, Paul A. Ellis, Cherrie K. 5 Donawho, Enrico L. DiGiammarino, Sanjay C. Panchal, Vivek C. 6 Abraham, Mikkel A. Algire, Yan Shi, Amanda M. Olson, Eric F. Johnson, 7 Julie L. Wilsbacher, David Maag*

9 Author affiliations: AbbVie, Inc., North Chicago, Illinois, USA

10 Running title: PARP trapping: impact on in vivo activity of PARP inhibitors

11 Keywords: PARP, DNA damage, veliparib,talazoparib, rucaparib, A-934935

12 Financial support: This work was financially supported by AbbVie, Inc.

13 Corresponding author: *David Maag, Oncology Development, AbbVie, Inc., 1 N. Waukegan 14 Road, North Chicago, Illinois, USA 60064. Phone: 847-937-3969; E- 15 mail: [email protected].

16 Disclosure: All authors are employees of AbbVie. The design, study conduct, and 17 financial support for this research were provided by AbbVie. AbbVie 18 participated in the interpretation of data, review, and approval of the 19 publication.

20 Word count: 5163/5000

21 Total number of figures and tables: 12 (6 plus 6 supplemental)

Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

24 Abstract

25 Poly (ADP-ribose) polymerase (PARP) inhibitors have recently been approved as 26 monotherapies for the treatment of recurrent ovarian cancer and metastatic BRCA-associated 27 breast cancer, and ongoing studies are exploring additional indications and combinations with 28 other agents. PARP inhibitors trap PARP onto damaged chromatin when combined with 29 temozolomide and methyl methanesulfonate, but the clinical relevance of these findings 30 remains unknown. PARP trapping has thus far been undetectable in cancer cells treated with 31 PARP inhibitors alone. Here we evaluate the contribution of PARP trapping to the tolerability 32 and efficacy of PARP inhibitors in the monotherapy setting. We developed a novel 33 implementation of the proximity ligation assay to detect chromatin-trapped PARP1 at single- 34 cell resolution with higher sensitivity and throughput than previously reported methods. We 35 further demonstrate that the PARP inhibitor-induced trapping appears to drive single-agent 36 cytotoxicity in healthy human bone marrow, indicating that the toxicity of trapped PARP 37 complexes is not restricted to cancer cells with homologous recombination deficiency (HRD). 38 Finally, we show that PARP inhibitors with dramatically different trapping potencies exhibit 39 comparable tumor growth inhibition at maximum tolerated doses in a xenograft model of 40 BRCA1-mutant triple negative breast cancer. These results are consistent with emerging clinical 41 data and suggest that the inverse relationship between trapping potency and tolerability may 42 limit the potential therapeutic advantage of potent trapping activity. 43 44 Implications: PARP trapping contributes to single-agent cytotoxicity of PARP inhibitors in both 45 cancer cells and healthy bone marrow, and the therapeutic advantage of potent trapping 46 activity appears to be limited. 47 48 49 50 51

52 INTRODUCTION 53 The recent approvals of the poly (ADP-ribose) polymerase (PARP) inhibitors olaparib 54 (Lynparza) and rucaparib (Rubraca) for the treatment of BRCA-mutated ovarian cancer (1) and 55 niraparib (Zejula) for the treatment of recurrent ovarian cancer with or without BRCA mutation 56 (2) are important benchmarks in over 50 years of research since the initial discovery of this 57 important class of enzymes. The enzymatic function of PARP1, known as PARylation, is the 58 synthesis of ADP-ribose polymers (PAR) that covalently modify host proteins or its own 59 automodification domain (3). When DNA is damaged, PARP1 binds the damage site and 60 catalyzes synthesis of PAR that recruits host DNA repair proteins. AutoPARylation of PARP1 61 induces an electrostatic destabilization and dissociation from the DNA damage site which 62 enables repair proteins to localize to the lesion (4,5). As a result, DNA damage is repaired and 63 cell viability is maintained. When DNA damage occurs in the presence of a PARP inhibitor, 64 PARP1 binds to damage sites and remains tightly bound or trapped onto the chromatin. 65 PARylation is inhibited and PARP1 remains bound to the lesion, which progresses to replication 66 fork collapse and DNA double strand breaks (DSB) (6). In homologous recombination (HR) 67 competent cells, DNA damage is repaired by the HR pathway and cells remain viable. In HR 68 defective (HRD) cells such as BRCA 1/2 mutants, DSB damage is not repaired with high fidelity, 69 resulting in cell death. The cytotoxicity induced by the combination of HRD and PARP inhibitors 70 is known as synthetic lethality (1).

71 There are now more than 150 completed, running or planned registered trials for the 72 PARP inhibitors niraparib, olaparib, rucaparib, talazoparib and veliparib (www.clinicaltrials.gov). 73 The majority of these trials are monotherapy studies in patients with tumors harboring DNA 74 repair defects while the remaining trials are combinations with chemotherapies including 75 platinums, taxanes, ATM inhibitors, ATR inhibitors, Wee1 inhibitors and PI3K inhibitors as well 76 as with immune-oncology therapies. The role of PARP trapping in the efficacy of clinical PARP 77 inhibitors is under intensive investigation. In vitro studies in DT40 cells showed that all five 78 clinical PARP inhibitors were able to potentiate DNA damage induced by alkylating agents and 79 knockout of PARP1 eliminated this effect (7,8). Although there is only a 40-fold difference in 80 IC50s between the most potent (talazoparib) and least potent (veliparib) compound in catalytic

81 inhibition studies, the difference in PARP trapping activity between the inhibitors was greater 82 than 10000-fold (9). This differential trapping potency was theorized to be the result of an 83 allosteric change in protein conformation induced by the more potent trapping agents (7,8); 84 however, we found no evidence of allostery after rigorous biochemical testing (9). Thus, the 85 underlying mechanism behind the high trapping potency of talazoparib relative to other PARP 86 inhibitors remains to be determined.

87 The importance of PARP trapping to the cytotoxic mechanism of PARP inhibitors 88 appears to be context dependent. While the activity of PARP inhibitor combinations with 89 temozolomide appear to be mediated largely by PARP trapping, in the context of combinations 90 with other DNA damaging agents like platinums and irinotecan, efficacy appears to be less 91 dependent on PARP1 trapping (10). The importance of PARP1 trapping in the monotherapy 92 setting remains equivocal. Genetic evidence and correlations with preclinical and clinical 93 maximum tolerated doses (MTDs) suggest that trapping occurs in this context (7-9), but it has 94 yet to be demonstrated directly.

95 In this study, we sought to better understand the effects of PARP1 trapping on PARP 96 inhibitor tolerability and efficacy in the monotherapy setting. To assess the properties of PARP 97 inhibitors that contribute to efficient trapping, we characterized a panel of compounds with 98 similar catalytic potencies and PARP binding residence times to the potent PARP trapping agent 99 talazoparib. This includes the first reported characterization of a novel PARP inhibitor, A- 100 934935. Utilizing a highly sensitive proximity ligation assay (PLA), we were able to detect 101 chromatin-trapped PARP at single cell resolution following single agent PARP inhibitor 102 treatment. Kinetics of PARP trapping relative to PAR inhibition, DNA damage induction, and 103 apoptosis were also analyzed. In addition, PARP inhibitor cytotoxicity was determined in bone 104 marrow progenitors to evaluate contributions of trapping to bone marrow toxicity observed in 105 the clinic. Our data suggest an inverse relationship between PARP inhibitor trapping potency 106 and tolerability due to cytotoxicity within normal cells, and provide important context for 107 translation of reported in vitro trapping activity differences to clinical outcomes. 108

109

110 MATERIALS AND METHODS

111 Cell culture

112 HeyA8 cells purchased from M.D. Anderson were maintained in RPMI 1640 with 10% -/- 113 FBS at 5% CO2 and 37°C. Isogenic DLD1 and DLD1-BRCA2 cells with homozygous deletion of

114 exon 11 of BRCA2 were maintained in RPMI 1640 with 10% FBS at 5% CO2 and 37°C. Isogenic 115 haploid cell line HAP1 and HAP1 PARP1 knockout cells were maintained in Iscove’s Modified

116 Dulbecco’s Medium (IMDM) with 10% FBS at 5% CO2 and 37°C. DLD1 and HAP1 lines were 117 licensed from Horizon Discovery, Ltd. (Cambridge, UK). Cell lines H69, H1048, H1930, H2081, 118 T47D, UACC812 and MDA-MB-436 were purchased from ATCC. SUM149PT was purchased from 119 Asterand Bioscience. All cell lines were authenticated immediately prior to freezing aliquots 120 between October of 2013 and January of 2015 using the Promega GenePrint 10 system. Cells 121 were used within 40 passages after thawing. SUM149PT cells for in vivo studies were grown to 122 passage 3 in vitro in RPMI (Life Technologies Corp) containing 10% FBS (Hyclone) and harvested 123 while in log phase.

124 High-content imaging of H2A.X, Caspase 3/7 and cellular proximity ligation assays:

125 Cells were plated in 96-well collagen I coated Costar plates (Corning; cat# 356649) at 126 densities to achieve 90% confluence at time of fixation. Veliparib, olaparib, and talazoparib 127 were synthesized as previously described (9). Rucaparib was synthesized using published 128 methods (11) or purchased from SelleckChem (Houston, TX). A-934935 was synthesized using 129 methods disclosed in (12). Compounds were diluted in DMSO, serial diluted in DMSO and 130 added to cell plates at indicated concentrations with final DMSO concentrations <0.1%. Cells 131 were fixed with 2% formaldehyde (Polysciences #04018) for 10 minutes, permeablized with 132 0.1% Triton X-100 in 1xPBS for 10 minutes, and blocked in 2% BSA for 1 hour with washes in 133 1xPBS between each step. Cells were treated with indicated antibodies at a 1:400 dilution in 134 antibody dilution buffer (0.3% BSA in 1xPBS) at 4oC overnight. Antibodies used for H2A.X and 135 PLA include: PARP-1 mAb (Sigma #WH0000142M1), PARP1, Histone H2B, total H2A.X and

136 үH2A.X (Cell Signaling Technologies #9542, #12364, #7631 and #9718 ), NPM1, KAP1, Histone 137 H3, and Histone H4 (Bethyl #A302-404A, #A300-274A, #A300-823A and #A300-646A), Lamin 138 A/C (Invitrogen #MA3-1000) and BRCA1 (Gentex #GTX70111).

139 Cell Imaging

140 For H2A.X, cells were washed 3x in PBS and incubated with Alexa Fluor 488-conjugated 141 goat anti-rabbit Ab (Life Technologies, #A11034) and Hoechst 33342 (Life Technologies, # 142 H2570) diluted 1:400 and 1:8000, respectively, in antibody dilution buffer for 1 hour at 143 RT. Plates were washed 3x with PBS and scanned using 10x objective within 24 hours. For 144 caspase 3/7, samples were treated with CellEvent Green Caspase 3/7 Detection Reagent

145 (Molecular Probes, #C10423) in complete media at a final concentration of 2M for 24 hours 146 prior to fixation. Cells were fixed, permeablized and treated with Hoechst as previously 147 described. Plates were washed and scanned immediately using 10x objective on a CellInsight.

148 Duolink PLA

149 The Duolink Proximity Ligation Assay was performed per the manufacturer’s protocol 150 (Sigma DUO92008-100RXN, DUO92004-100RXN, and DUO92002-100RXN). Labeling of nuclear

151 DNA was performed in antibody dilution buffer with Hoechst 33342 at 2 M. Final reaction 152 plates were washed 3X with PBS and scanned within 24 hours by automated image acquisition 153 using a 20x objective on a CellInsight. Image analysis was performed on the CellInsight HCS 154 Reader and data analysis was performed using Cellomics View Software. Graphs are the mean 155 and standard errors of duplicate samples tested in at least three independent experiments.

156 Cellular and biochemical assays

157 Cellular trapping, cell viability, PAR ELISA and TR-FRET binding assays were performed as 158 previously described (9). Biochemical assays for the specified PARP family of enzymes were 159 conducted at BPS Bioscience Inc.

160 Equilibrium binding and kinetic studies of PARP1-PARP inhibitor complexes

161 Biacore assays were performed as previously described (9) with the following 162 modification: Analysis used single-cycle kinetics mode in 5-point and 3-fold concentration 163 series from 0.62 nM to 50 nM (talazoparib) or 1.1 nM to 90 nM (A-934935 and rucaparib), 164 respectively.

165 Bone marrow PAR assay

166 Frozen bone marrow mononuclear cells were purchased from Lonza (cat# 2M-125C). 167 Cultures were thawed per manufacturer protocol. Cells were maintained in IMDM + 2%FBS + 168 supplemented with StemSpan CC100 bullet (StemCell Technologies, cat# 02690). Cells were 169 plated in complete media at a density of 7x105 cells per well and treated with compounds for 5 170 days. Cellular PAR levels were measured by ELISA as described (13-15).

171 Bone marrow colony formation assays

172 All bone marrow colony formation assays were conducted at StemCell Technologies 173 (Vancouver, BC, Canada). Normal human bone marrow (BM) lot BM070525B, lot BM10A33225, 174 and lot BM070973A (Lonza: Walkersville, Maryland) were thawed rapidly at 37oC into DNaseI, 175 then diluted into 10 mL of IMDM + 2%FBS and washed by centrifugation. The resulting cell 176 pellet was re-suspended into IMDM+2%FBS. Hematopoietic cultures were plated in triplicate at 177 a density of 1x104 cells per dish in MethoCult TM media (StemCell Technologies, MethoCult TM 178 cat# GF H84434). Compounds were diluted to make 1000X stocks in DMSO followed by further 179 dilution into MethoCult TM to achieve final concentrations. Cultures were incubated for 13-16 180 days at 37°C at 5% CO2. Mean colony numbers following compound treatment were used to

181 calculate IC20, IC50 and IC90 values using GraphPad Prism 5 software.

182 In vivo pharmacology

183 Determination of maximum tolerated doses (MTDs)

184 Animal research was approved and overseen by the AbbVie Institutional Animal Care 185 and Use Committee (AbbVie IACUC, in accordance with all ALAAC guidelines). Veliparib, 186 olaparib and talazoparib formulations were prepared as previously described (6). Tolerability

187 studies were conducted in naïve C.B-17 SCID female mice. Talazoparib was dosed orally (PO) 188 once a day (QD) and other PARP inhibitors were administered PO, twice a day (BID) for 21 days. 189 Doses resulting in <15% weight loss were considered tolerable.

190 SUM149PT xenograft models

191 2 x 106 SUM149PT cells in 0.1 mL of a 1:1 mixture of S-MEM (Life Technologies Corp) 192 and Matrigel (Corning) were inoculated subcutaneously into the right flank of female C.B-17 193 SCID mice (Charles Rivers Laboratories) on Day 0.

194 Efficacy studies

195 SUM149PT xenografts were size matched on day 29 with a mean tumor volume of 235 + 196 14mm3 and dosed starting on day 30. Ten mice per group were treated with the indicated oral 197 doses of PARP inhibitors administered BID for vehicle, veliparib, olaparib, and QD for 198 talazoparib, for 42 days. Mice were observed daily and tumors were measured two times per 199 week. When tumor volumes reached a maximum of 2500 mm3 or when skin ulcerations 200 occurred, mice were euthanized. Electronic calipers were used to measure tumor length (L) 201 and width (W) and tumor volume was calculated according to the following equation: V = L x 202 W2/2 using Study Director version 3.1 (Studylog Systems, Inc., South San Francisco).

203 Pharmacokinetics (PK) and pharmacodynamics (PD)

204 SUM149PT tumor-bearing mice were size matched and treated with the same doses and 205 schedules as for the efficacy study but only for 5 days. Plasma and tumor samples were 206 collected at 1, 4, 24, 32 and 48 hrs following the final dose. Blood samples were collected in 207 microtainer tubes (BD bioscience, San Jose, CA) containing K+-EDTA and centrifuged. The upper 208 layer, consisting of the plasma was collected and frozen. Tumor samples were flash frozen in 209 liquid nitrogen. Inhibitor (16) and PAR levels (13-15) were measured as described.

210

211

212 RESULTS

213 Evaluation of trapping vs catalytic inhibition for compounds that are equipotent to 214 talazoparib

215 Studies have shown that PARP inhibitors with similar catalytic potency can differ in their 216 ability to induce PARP1 trapping (6,8); however, the unique potency and binding residence time 217 exhibited by the PARP inhibitor talazoparib confound interpretation of the relationship 218 between trapping and cytotoxic potency. Given the differential resolution between catalytic 219 inhibition and trapping of PARP inhibitors, we sought to identify compounds that inhibit PARP1 220 equipotently to talazoparib while exhibiting a much greater resolution between catalytic 221 inhibition and trapping activities. Rucaparib and a proprietary PARP inhibitor, A-934935, were 222 identified as compounds with similar binding properties as talazoparib, as determined by 223 surface plasmon resonance (Figure 1A). Talazoparib, rucaparib and A-934935 also inhibit 224 PARP1 activity in cells equipotently, with IC50 values of 3, 9 and 4 nM, respectively (Figure 1B)

225 and trap full length PARP1 in a biochemical system identically with EC50 values of 2, 2 and 4 nM, 226 respectively (Figure 1C). Despite these similarities, talazoparib produces substantially more 227 PARP1 trapping and DNA damage potentiation activities in cells treated with MMS (Figure 1D 228 and 1E). These results indicate that the potent trapping and DNA damaging activities of 229 talazoparib are not simply attributable to PARP1 binding properties. Although these data 230 would be consistent with the proposed allosteric trapping hypothesis (7), a rigorous 231 biochemical investigation revealed no evidence of allosteric trapping for talazoparib (9). 232 Moreover, the unique trapping efficiency of talazoparib relative to rucaparib and A-934935 is 233 only evident in intact cells, while any potential unique allosteric trapping activity would be 234 expected to manifest in biochemical trapping assays (Figure 1C).

235 To investigate whether these differences in PARP inhibitor trapping activity and DNA 236 damage potentiation were due to variability in polypharmacology, the compounds were 237 characterized for inhibition of other PARP family members using biochemical activity assays for 238 12 of the 17 PARP family members (Supplementary table S1). In comparison to PARP1 and 239 PARP2, the role of other PARP family members in DNA damage repair and the cytotoxicity of

240 different PARP inhibitors have not yet been well established. Talazoparib, rucaparib and A- 241 934935 were equipotent inhibitors of PARP1/2. Relative to their PARP1/2 IC50s, rucaparib was 242 approximately 100 fold less potent against PARP3, talazoparib was approximately 100 fold less 243 potent against PARP3, TNKS1 and TNKS2, and A-934935 was approximately 100 fold less potent 244 against TNKS2. All three compounds were inactive against PARP6, 7, 8, 10, 11, 12 and 15. 245 Although talazoparib displayed somewhat unique potency at inhibiting TNKS1 and TNKS2, this 246 did not appear sufficient to account for the potency with which the compound potentiates DNA 247 damage or traps PARP1.

248 Cytotoxicity of talazoparib is driven by PARP1

249 Avian cells such as DT40 are naturally PARP2 deficient, and knockout of PARP1 250 expression results in loss of cytotoxicity to single agent PARP inhibitor treatments (7,8). We 251 performed analogous experiments utilizing a PARP2 proficient HAP1 cell line with CRISPR/Cas9- 252 generated PARP1 knockout (HAP1 PARP1 KO). Talazoparib, rucaparib and A-934935 showed 253 decreased cytotoxicity in the HAP1 PARP1 KO cells relative to the isogenic HAP1 wild-type 254 (HAP1 WT) cells. If inhibition of PARP1 catalytic activity is the exclusive driver of cytotoxicity in 255 a particular cellular context, then all three compounds should be equipotent in cytotoxicity 256 assays, yet talazoparib displayed significantly greater cytotoxic potency than either rucaparib or 257 A-934935 in HAP1 WT cells (Figure 2). Knock out of PARP1 in HAP1 cells resulted in modest 4- 258 fold and 11-fold increases in IC50 for rucaparib and A-934935, suggesting that the PARP1 259 trapping activities of these compounds are sufficient to contribute to single-agent cytotoxicity 260 (Figure 2). In contrast, the cytotoxic potency of talazoparib is reduced by over 300-fold in HAP1 261 PARP1 KO cells, suggesting that PARP1 trapping is the primary driver of the cytotoxicity of this 262 compound (Figure 2). Collectively, these data suggest that PARP1 trapping is an important 263 driver of PARP inhibitor cytotoxicity in the monotherapy setting, and that talazoparib is unique 264 in the extent to which trapping drives cytotoxic potency.

265 Direct evidence of PARP1 trapping after single-agent treatment of cancer cells with PARP 266 inhibitors

267 It has been well documented that alkylating agent combinations with PARP inhibitors 268 induce PARP1 trapping to chromatin (7-10); however, detection of single agent PARP inhibitor 269 induced trapping has not been shown. Established methods to detect PARP1 trapping, such as 270 subcellular fractionation followed by Western detection, have several limitations including low 271 throughput, potential loss of trapped complexes during processing, non-native conditions, and 272 insufficient sensitivity for detecting trapping induced by PARP inhibitor monotherapy. To 273 address these pitfalls, we utilized a highly sensitive, fluorescence-based proximity ligation assay 274 (PLA) to detect trapped PARP1 at the single cell level. Histone H2A.X is an abundantly 275 expressed, chromatin-associated protein that is phosphorylated at Ser139 (also known as

276 H2A.X) upon DNA damage. Previous combination studies with alkylating agents and PARP

277 inhibitors have shown enhancement of H2A.X signal (9); we therefore theorized that under 278 DNA damaging conditions PARP1 and total H2A.X (tH2A.X) would associate within the 40 279 angstrom distance required for PLA ligation and amplification reactions. Treatment of DLD-1 280 cells with 1mM methyl methanesulfonate (MMS) and 0.1 µM talazoparib followed by PLA

281 staining with antibody pairs PARP1/tH2A.X or PARP1/H2A.X, resulted in robust PLA signal 282 (Supplementary figures 1A and 1B). This signal strongly correlates with data generated with 283 previously established fractionation and Western blot methods, and recapitulate the reported 284 rank order of weaker trapping by veliparib relative to stronger trapping by talazoparib (7-9)

285 (Supplementary figures 1C and 1D). The PLA signal for H2A.X was higher than that observed 286 for total H2A.X (Supplementary figures 1A and 1B). Total loss of PLA signal was observed if

287 PARP1, tH2A.X or H2A. X antibodies were removed individually from the PLA reaction. The PLA 288 signal appears limited to specific partner proteins, as much weaker signal was observed with 289 Histone H2B and Histone H3 (Supplementary Figure 2), and no signal was observed with 290 Histone H4, NPM1, KAP1, Lamin A/C, or BRCA1 (data not shown), although it cannot be ruled 291 out that some of these antibodies are simply inadequate for the method. The total H2A.X 292 antibody was chosen for further experiments because it produces a robust signal when paired 293 with PARP1 and its abundance is not dependent on DNA damage (which may confound 294 interpretation of results).

295 To investigate the cytotoxic role of PARP trapping following PARP inhibitor 296 monotherapy, we treated the HR-proficient cell line DLD wild-type (DLDwt) and two HR 297 deficient cell lines DLD BRCA2 (-/-) and SUM149PT (a BRCA1-mutant triple negative breast 298 cancer line) with increasing concentrations of veliparib in a time course study. In all three cell 299 lines, there was a dose dependent inhibition of PAR formation, indicating inhibition of PARP1 300 catalytic activity (Figure 3A-C). In DLD BRCA2 (-/-) and SUM149PT cells, a time-dependent

301 synthetic lethality was observed (Figure 3E-F) with concordant increases in H2A.X associated 302 DNA damage, caspase 3/7 activity and PLA signal (Figure 3B-C). These data are consistent with 303 previously reported PARP inhibitor concentrations that inhibit greater than 95% of PAR 304 formation and induce PARP1 trapping in combination with alkylating agents (9). In the DLDwt 305 cell line, PAR was depleted to similar levels as in DLD BRCA2-/- cells; however, no dose-

306 dependent increases in H2AX associated DNA damage, caspase 3/7 activity or PLA signal were 307 observed (Figure 3A). Likewise, veliparib monotherapy did not induce cytotoxicity in the DLDwt 308 lines (Figure 3D).

309 PARP1 trapping drives cytotoxicity in bone marrow

310 Using the set of PARP inhibitors with identical PAR inhibition potencies but dramatically 311 different trapping potencies, we sought to understand the significance of potent trapping 312 activity for the tolerability and efficacy of PARP inhibitors in the monotherapy setting. Common 313 adverse events reported in PARP inhibitor monotherapy clinical trials include cytopenias related 314 to bone marrow toxicity such as anemia, thrombocytopenia and neutropenia (17-22). A well- 315 validated method to model such toxicity is ex vivo colony formation assays using hematopoietic 316 progenitors derived from human bone marrow. We evaluated the cytotoxic potency of 317 veliparib, olaparib, talazoparib, rucaparib and A-935935 towards erythroid and myeloid

318 progenitors. The IC50s for each of the clinical stage compounds fell within range of plasma 319 exposures reported in monotherapy clinical trials at the recommended phase II doses (RP2D, 320 Supplementary table S2), highlighting the relevance of this approach to model a clinically

321 relevant toxicity associated with this class of inhibitors. Comparison of IC50s for PARP catalytic 322 inhibition in bone marrow mononuclear cells (BMN) shows a 40-fold difference between the

323 most potent and least potent PARP inhibitors while the IC50 differences for BMN cytotoxicity 324 was greater than 600-fold (Supplementary tables S3 and S4). These relative differences 325 generally reflect those observed in cancer cell lines treated with alkylating agents (7-9). As has 326 been reported for PARP trapping activity, different compounds displayed significant variation in 327 the resolution between catalytic inhibition and bone marrow toxicity. For veliparib, olaparib, 328 rucaparib and A-935935 the resolution between PAR inhibition and cytotoxicity spans >100- 329 fold, but for the potent trapping agent talazoparib there was less than a 2-fold difference 330 between IC50s for PAR inhibition and BMN cytotoxicity (Figure 4, Supplementary table S3). 331 Strikingly, talazoparib was 73-fold and 26-fold more potent at killing hematopoietic progenitor 332 cells than rucaparib and A-934935, respectively. These results suggest that PARP1 trapping 333 activity is a primary driver of bone marrow toxicity associated with single-agent PARP inhibition 334 and that PARP inhibitors will differ in their ability to achieve robust catalytic inhibition in the 335 absence of such toxicity.

336 To better understand the relationship between trapping and therapeutic index under 337 monotherapy conditions, we compared the cytotoxicity of veliparib, olaparib, talazoparib, 338 rucaparib and A-934935 in a panel of cancer cell lines relative to bone marrow (Figure 5). 339 Talazoparib is significantly more potent than all other compounds in every cell line tested, 340 suggesting that PARP1 trapping contributes significantly to its single agent activity in most 341 contexts. However, all of the PARP inhibitors had similar patterns of cytotoxicity induction in 342 the different cell lines. HeyA8 and DLDwt cells are relatively insensitive to all of the PARP 343 inhibitors, while in DLD BRCA2 (-/-) cells, all the PARP inhibitors had IC50s that were below the 344 IC50s in BMN cells (Figure 5A). As reported previously (23,24), small cell lung cancer (SCLC) lines 345 are more sensitive to PARP inhibition, as demonstrated by lower cytotoxicity IC50s for all the 346 PARP inhibitors in the SCLC lines (Figure 5B) relative to HeyA8 and DLD1 cells. Synthetic lethality 347 occurred in the breast cancer cells lines containing BRCA mutations, SUM149PT and MDA-MB- 348 436, which were killed at much lower concentrations than the wild-type breast cancer lines 349 (Figure 5C). The ratio of cytotoxic potency in bone marrow to cytotoxic potency in a given 350 cancer cell line is comparable for all compounds tested, suggesting that the therapeutic index of

351 a PARP inhibitor in the monotherapy setting may be independent of its trapping potency. This 352 is reflective of preclinical data we previously reported for a combination regimen with TMZ (9).

353 In vivo study comparing veliparib, olaparib, rucaparib, and talazoparib, in SUM149PT

354 We next extended the analysis of efficacy of PARP inhibitors with different trapping 355 potencies to the in vivo setting. Mice bearing flank SUM149PT xenografts were treated for 42 356 days with the maximum tolerated doses of veliparib, olaparib, and talazoparib (Figure 6). 357 Modest but significant tumor growth inhibition was exhibited in groups treated with all of the 358 PARP inhibitors. Veliparib, olaparib, and talazoparib treatment resulted in 29%, 32%, and 35% 359 growth delay, respectively (Figure 6A). No weight loss or other adverse events were observed 360 for all of the dosing groups (Figure 6A). To determine whether compounds reached sufficient 361 levels to inhibit PARP in the plasma and tumors, a parallel PK/PD study in mice bearing 362 SUM149PT xenografts was performed. Mice were treated for 5 days with the same dose and 363 schedule for each PARP inhibitor that was used for the efficacy study. Plasma and tumor 364 samples were collected at 1, 4, 24, 32, and 48 hours after the last dose, and compound and PAR 365 levels were measured. Veliparib and olaparib treated tumors had >99% inhibition and 366 talazoparib treated tumors had 94% inhibition of PAR formation at 1 hour after the last dose of 367 compound (Figure 6B). PAR levels partially recovered at 4 hours, and continued to increase 368 over the rest of the time course. Recovery of PAR formation appeared faster for talazoparib 369 compared to veliparib and olaparib. Although compound concentrations were below the limit 370 of quantification in plasma for talazoparib after 4 hours, tumor compound levels decreased 371 more slowly, with only a 65% decrease at 24 hours and 82% at 48 hours. Compound 372 concentrations in the plasma were not detectible after 24 hours for veliparib and olaparib. 373 Tumor levels for these compounds were still detectible at 48 hours, but the levels at 24 and 48 374 hours were decreased by 97% and 99% for veliparib and 96 and 98% for olaparib.

375

376 DISCUSSION

377 Although PARP trapping has only been demonstrated in cells treated with combinations 378 of PARP inhibitors and high concentrations of temozolomide or MMS (7-9), it is also 379 hypothesized to be important for the single-agent activity of PARP inhibitors (6). Previously, 380 there have only been correlative data supporting the hypothesis that PARP1 trapping plays a 381 key role in cytotoxicity from PARP inhibitor monotherapy. Published methods (i.e., chromatin 382 fractionation and Western blot) are not sensitive enough to detect endogenous PARP1 trapping 383 in the monotherapy setting or trapping at clinically relevant exposures of temozolomide (9). 384 Development of a proximity ligation assay utilizing PARP1 and H2A.X has provided a 96 well 385 based assay with sufficient sensitivity to measure PARP1 trapping induced by PARP 386 inhibitor/alkylating agent combinations as well as PARP inhibitor monotherapy under 387 endogenous cellular conditions. Since the Duolink proximity ligation assay has been shown to 388 work in situ, we propose that this methodology has potential application for evaluation of PARP 389 trapping in clinical tumor samples.

390 The difference in trapping potency between talazoparib and other PARP inhibitors is up 391 to 10,000-fold, while catalytic potency only differs by up to 40-fold (9). In order to better 392 understand this difference, we characterized the trapping ability of two additional PARP 393 inhibitors with PARP1 binding and catalytic potencies equal to talazoparib. Data from these 394 studies showed that talazoparib is a more potent trapping agent than otherwise equipotent 395 PARP inhibitors, with the following rank order of PARP1 trapping: talazoparib >> A-934935 = 396 rucaparib (Figure 1D). Based on these and previous data (9), talazoparib is unique in that its 397 catalytic inhibition and trapping activities are not resolved. All other PARP inhibitors evaluated 398 trap only at concentrations much higher than those required to inhibit PAR synthesis. Further 399 studies are needed to identify the mechanism underlying the enhanced trapping potency of 400 talazoparib.

401 Trapping contributes significantly to monotherapy activity of PARP inhibitors. The rank 402 order of trapping potency for clinical PARP inhibitors in HeyA8 cells treated with 1 mM MMS is 403 talazoparib >> olaparib = rucaparib > veliparib= niraparib. The same rank order is observed 404 when comparing the cytotoxic potency for PARP inhibitor monotherapy treatment in HeyA8,

405 DLD1, and DLD1-BRCA2-/- cells as well as SCLC and breast cancer lines. Our in vitro data 406 indicate that PARP trapping is toxic to cancer cells with HRD, cancer cells with normal HR 407 capacity and normal cells (i.e. healthy human bone marrow). In vivo, we observed comparable 408 tumor growth inhibition at MTD in SUM149PT xenograft models with PARP inhibitors spanning 409 a broad range of trapping potency. Together, these data suggest that the impact of trapping 410 potency on the therapeutic index of PARP inhibitors in the monotherapy setting may be 411 negligible.

412 Consistent with this hypothesis, currently available clinical data reveal no discernible 413 relationship between trapping potency and monotherapy activity. Plasma exposures in patients 414 treated with different PARP inhibitors at recommended phase 2 doses (Supplementary Table 415 S1) appear inversely related to trapping potency. Additionally, although veliparib is among the 416 least efficient PARP trapping agents, in study NCI-2011-01472, evaluating veliparib 417 monotherapy in BRCA1/2 mutated solid tumors, the overall RECIST response rate (PR + CR) was 418 40% among the 28 subjects who received at least 400 mg BID (25). Likewise, in the phase 2 419 portion of the Veli-BRCA study, an ORR of 47% was observed in 32 heavily pre-treated (median 420 4 lines of prior therapy) patients with platinum-resistant or partially platinum-sensitive gBRCA- 421 mutated epithelial ovarian cancer (26). These response rates are similar to those reported for 422 olaparib (46% in platinum sensitive ovarian cancer;(27)), niraparib (40% in gBRCA mutated 423 ovarian cancer; (28)), and talazoparib (42% in gBRCA mutated ovarian cancer; (17)).

424 Although trapping potency may not have a significant bearing on monotherapy activity 425 of PARP inhibitors in the clinic, our discovery that PARP1 trapping is a key driver of bone 426 marrow toxicity may be important for combination regimens including DNA-damaging 427 chemotherapy that also induces bone marrow toxicity. Here we have shown that veliparib kills 428 erythroid and myeloid progenitors only at concentrations well above those required to elicit 429 robust inhibition of PAR synthesis. Consistent with this, recent data from a randomized phase 2 430 study indicate that doses of veliparib (120 mg BID) that are several fold higher than those 431 required to inhibit PAR synthesis in patient tumors (29) can be combined with carboplatin at 432 AUC6 and paclitaxel (200 mg/m2, q3wk) without exacerbation of cytopenias associated with

433 the chemotherapy backbone (30). In contrast, our data reveal that talazoparib is toxic to 434 erythroid and myeloid progenitors at concentrations similar to those required to inhibit PAR 435 synthesis. Of note, recent clinical data indicate that combination of talazoparib with 436 carboplatin at AUC1.5 results in significant enhancement of hematologic toxicity (31).

437 In conclusion, we have developed a novel methodology to demonstrate directly for the 438 first time PARP1 trapping in cells treated with PARP inhibitors alone. We demonstrate that 439 PARP inhibitors spanning a broad range of trapping potency elicit comparable monotherapy 440 activity in a xenograft model of BRCA mutant breast cancer, and that PARP1 trapping activity is 441 a primary driver of toxicity of PARP inhibitors towards healthy bone marrow progenitor cells. 442 Contrary to recent suggestions (6), trapping activity appears unable to predict monotherapy 443 clinical efficacy of PARP inhibitors; however, potent trapping agents may prove difficult to 444 combine with DNA damaging chemotherapy.

445

446 ACKNOWLEDGEMENTS

447 The authors thank Don Osterling for helpful comments and discussion during the 448 preparation of this manuscript. Biochemical PARP assays conducted by BPS Bioscience Inc. 449 6042 Cornerstone Court west, Suite B, San Diego, CA 92121. Bone marrow colony formation 450 assays conducted by StemCell Technologies Canada Inc.-Vancouver office, 1618 Station Street, 451 Vancouver, BC, V6A 1B6, Canada.

452 REFERENCES

453 1. Lord CJ, Ashworth A. PARP inhibitors: Synthetic lethality in the clinic. Science 454 2017;355(6330):1152-8 doi 10.1126/science.aam7344. 455 2. Scott LJ. Niraparib: First Global Approval. Drugs 2017;77(9):1029-34 doi 10.1007/s40265-017- 456 0752-y. 457 3. Karlberg T, Langelier MF, Pascal JM, Schuler H. Structural biology of the writers, readers, and 458 erasers in mono- and poly(ADP-ribose) mediated signaling. Molecular aspects of medicine 459 2013;34(6):1088-108 doi 10.1016/j.mam.2013.02.002. 460 4. Ogata N, Ueda K, Kawaichi M, Hayaishi O. Poly(ADP-ribose) synthetase, a main acceptor of 461 poly(ADP-ribose) in isolated nuclei. The Journal of biological chemistry 1981;256(9):4135-7. 462 5. Satoh MS, Lindahl T. Role of poly(ADP-ribose) formation in DNA repair. Nature 463 1992;356(6367):356-8 doi 10.1038/356356a0. 464 6. Pommier Y, O'Connor MJ, de Bono J. Laying a trap to kill cancer cells: PARP inhibitors and their 465 mechanisms of action. Science translational medicine 2016;8(362):362ps17 doi 466 10.1126/scitranslmed.aaf9246. 467 7. Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Trapping of PARP1 and 468 PARP2 by Clinical PARP Inhibitors. Cancer research 2012;72(21):5588-99 doi 10.1158/0008- 469 5472.CAN-12-2753. 470 8. Murai J, Huang SY, Renaud A, Zhang Y, Ji J, Takeda S, et al. Stereospecific PARP trapping by BMN 471 673 and comparison with olaparib and rucaparib. Molecular cancer therapeutics 472 2014;13(2):433-43 doi 10.1158/1535-7163.MCT-13-0803. 473 9. Hopkins TA, Shi Y, Rodriguez LE, Solomon LR, Donawho CK, DiGiammarino EL, et al. Mechanistic 474 Dissection of PARP1 Trapping and the Impact on In Vivo Tolerability and Efficacy of PARP 475 Inhibitors. Molecular cancer research : MCR 2015;13(11):1465-77 doi 10.1158/1541-7786.MCR- 476 15-0191-T. 477 10. Murai J, Zhang Y, Morris J, Ji J, Takeda S, Doroshow JH, et al. Rationale for Poly(ADP-ribose) 478 Polymerase (PARP) Inhibitors in Combination Therapy with Camptothecins or Temozolomide 479 Based on PARP Trapping versus Catalytic Inhibition. The Journal of pharmacology and 480 experimental therapeutics 2014;349(3):408-16 doi 10.1124/jpet.113.210146. 481 11. Webber SE, Canan-Koch, S.S., Tikhe, J., Thoresen, L.H. Tricyclic Inhibitors of Poly(ADP- 482 ribose)Polymerases. Agouron Pharmaceuticals, Inc/Cancer Research Campaign Technology 483 Limited 2000;WO2000042040. 484 12. Penning TD, Zhu, G., Gandhi, V.B., Gong, J., Giranda, V.L. Pyrazoloquinolones are Potent PARP 485 Inhibitors. AbbVie, Inc 2013;U.S. Patent No 8,546,368. 486 13. Ji J, Kinders RJ, Zhang Y, Rubinstein L, Kummar S, Parchment RE, et al. Modeling 487 pharmacodynamic response to the poly(ADP-Ribose) polymerase inhibitor ABT-888 in human 488 peripheral blood mononuclear cells. PloS one 2011;6(10):e26152 doi 489 10.1371/journal.pone.0026152. 490 14. Kummar S, Chen A, Ji J, Zhang Y, Reid JM, Ames M, et al. Phase I study of PARP inhibitor ABT-888 491 in combination with topotecan in adults with refractory solid tumors and lymphomas. Cancer 492 research 2011;71(17):5626-34 doi 10.1158/0008-5472.CAN-11-1227. 493 15. Kummar S, Kinders R, Gutierrez ME, Rubinstein L, Parchment RE, Phillips LR, et al. Phase 0 494 clinical trial of the poly (ADP-ribose) polymerase inhibitor ABT-888 in patients with advanced 495 malignancies. Journal of clinical oncology : official journal of the American Society of Clinical 496 Oncology 2009;27(16):2705-11 doi 10.1200/JCO.2008.19.7681.

497 16. Kikuchi R, Lao Y, Bow DA, Chiou WJ, Andracki ME, Carr RA, et al. Prediction of clinical drug-drug 498 interactions of veliparib (ABT-888) with human renal transporters (OAT1, OAT3, OCT2, MATE1, 499 and MATE2K). Journal of pharmaceutical sciences 2013;102(12):4426-32 doi 10.1002/jps.23737. 500 17. de Bono J, Ramanathan RK, Mina L, Chugh R, Glaspy J, Rafii S, et al. Phase I, Dose-Escalation, 501 Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 502 Mutations and Selected Sporadic Cancers. Cancer Discov 2017;7(6):620-9 doi 10.1158/2159- 503 8290.CD-16-1250. 504 18. Konecny GE, Kristeleit RS. PARP inhibitors for BRCA1/2-mutated and sporadic ovarian cancer: 505 current practice and future directions. Br J Cancer 2016;115(10):1157-73 doi 506 10.1038/bjc.2016.311. 507 19. Kristeleit R, Shapiro GI, Burris HA, Oza AM, LoRusso P, Patel MR, et al. A Phase I-II Study of the 508 Oral PARP Inhibitor Rucaparib in Patients with Germline BRCA1/2-Mutated Ovarian Carcinoma 509 or Other Solid Tumors. Clinical cancer research : an official journal of the American Association 510 for Cancer Research 2017 doi 10.1158/1078-0432.CCR-16-2796. 511 20. Domchek SM, Aghajanian C, Shapira-Frommer R, Schmutzler RK, Audeh MW, Friedlander M, et 512 al. Efficacy and safety of olaparib monotherapy in germline BRCA1/2 mutation carriers with 513 advanced ovarian cancer and three or more lines of prior therapy. Gynecol Oncol 514 2016;140(2):199-203 doi 10.1016/j.ygyno.2015.12.020. 515 21. Coleman RL, Sill MW, Bell-McGuinn K, Aghajanian C, Gray HJ, Tewari KS, et al. A phase II 516 evaluation of the potent, highly selective PARP inhibitor veliparib in the treatment of persistent 517 or recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer in patients who carry 518 a germline BRCA1 or BRCA2 mutation - An NRG Oncology/Gynecologic Oncology Group study. 519 Gynecol Oncol 2015;137(3):386-91 doi 10.1016/j.ygyno.2015.03.042. 520 22. Mirza MR, Monk BJ, Herrstedt J, Oza AM, Mahner S, Redondo A, et al. Niraparib Maintenance 521 Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer. The New England journal of medicine 522 2016;375(22):2154-64 doi 10.1056/NEJMoa1611310. 523 23. Byers LA, Wang J, Nilsson MB, Fujimoto J, Saintigny P, Yordy J, et al. Proteomic profiling 524 identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets 525 including PARP1. Cancer Discov 2012;2(9):798-811 doi 10.1158/2159-8290.CD-12-0112. 526 24. Cardnell RJ, Feng Y, Diao L, Fan YH, Masrorpour F, Wang J, et al. Proteomic markers of DNA 527 repair and PI3K pathway activation predict response to the PARP inhibitor BMN 673 in small cell 528 lung cancer. Clinical cancer research : an official journal of the American Association for Cancer 529 Research 2013;19(22):6322-8 doi 10.1158/1078-0432.CCR-13-1975. 530 25. Puhalla S, Beumer JH, Pahuja S, Appleman LJ, Tawbi HA-H, Stoller RG, et al. Final results of a 531 phase 1 study of single-agent veliparib (V) in patients (pts) with either BRCA1/2-mutated cancer 532 (BRCA+), platinum-refractory ovarian, or basal-like breast cancer (BRCA-wt). Journal of Clinical 533 Oncology 2014;32(15_suppl):2570- doi 10.1200/jco.2014.32.15_suppl.2570. 534 26. Steffensen KD, Adimi P, Jakobsen AKM. Veliparib monotherapy to patients with BRCA germline 535 mutation and platinum-resistant or partially platinum-sensitive selapse of epithelial ovarian 536 cancer: A phase I/II study. Journal of Clinical Oncology 2016;34(15_suppl):5532- doi 537 10.1200/JCO.2016.34.15_suppl.5532. 538 27. Fong PC, Yap TA, Boss DS, Carden CP, Mergui-Roelvink M, Gourley C, et al. Poly(ADP)-ribose 539 polymerase inhibition: frequent durable responses in BRCA carrier ovarian cancer correlating 540 with platinum-free interval. Journal of clinical oncology : official journal of the American Society 541 of Clinical Oncology 2010;28(15):2512-9 doi 10.1200/JCO.2009.26.9589. 542 28. Sandhu SK, Schelman WR, Wilding G, Moreno V, Baird RD, Miranda S, et al. The poly(ADP-ribose) 543 polymerase inhibitor niraparib (MK4827) in BRCA mutation carriers and patients with sporadic

544 cancer: a phase 1 dose-escalation trial. The Lancet Oncology 2013;14(9):882-92 doi 545 10.1016/S1470-2045(13)70240-7. 546 29. LoRusso PM, Li J, Burger A, Heilbrun LK, Sausville EA, Boerner SA, et al. Phase I Safety, 547 Pharmacokinetic, and Pharmacodynamic Study of the Poly(ADP-ribose) Polymerase (PARP) 548 Inhibitor Veliparib (ABT-888) in Combination with Irinotecan in Patients with Advanced Solid 549 Tumors. Clinical cancer research : an official journal of the American Association for Cancer 550 Research 2016;22(13):3227-37 doi 10.1158/1078-0432.CCR-15-0652. 551 30. Ramalingam SS, Blais N, Mazieres J, Reck M, Jones CM, Juhasz E, et al. Randomized, Placebo- 552 Controlled, Phase II Study of Veliparib in Combination with Carboplatin and Paclitaxel for 553 Advanced/Metastatic Non-Small Cell Lung Cancer. Clinical cancer research : an official journal of 554 the American Association for Cancer Research 2017;23(8):1937-44 doi 10.1158/1078-0432.CCR- 555 15-3069. 556 31. Dhawan MS, Bartelink IH, Aggarwal RR, Leng J, Kelley RK, Melisko ME, et al. A phase I study of 557 carboplatin and talazoparib in patients with and without DNA repair mutations. Journal of 558 Clinical Oncology 2017;35(15_suppl):2527- doi 10.1200/JCO.2017.35.15_suppl.2527.

559

560 FIGURE LEGENDS

561 Figure 1. Biochemical and cellular characterization of talazoparib, A-934935 and rucaparib. 562 (A), Talazoparib, A-934935 and rucaparib have similar PARP1 binding properties as determined 563 by surface plasmon resonance. (B), Inhibition of PAR synthesis in HeyA8 cells by the 564 compounds. (C), Analysis of talazoparib, rucaparib and 934935 trapping of PARP1 onto single- 565 strand breaks in a biochemical system. (D), Trapping of PARP1 onto chromatin after treatment 566 of HeyA8 cells with combinations of 1 mM MMS plus talazoparib, rucaparib or A-934935. (E),

567 Potentiation of MMS-induced DNA damage measured by H2A.X staining after treatment of 568 HeyA8 cells with talazoparib, rucaparib or A-934935. Data in panels B-E are presented as means 569 ± SEM from at least 3 independent experiments.

570 Figure 2. Cytotoxicity of talazoparib is driven by PARP1. Cell viability dose response curves for 571 talazoparib, A-934935 and rucaparib following treatment of HAP1 wt and HAP1 PARP1 572 knockout cells for 5 days. Data points are means ± SEM from 3 independent experiments.

573 Figure 3. PARP1 trapping induced by veliparib can be detected by PLA in BRCA1 and BRCA2 574 synthetic lethal cell line models. Dose dependent inhibition of auto-PARylation (black line) and 575 dose dependent increased activation of apoptosis marker caspase-3/7 (blue), DNA damage

576 marker H2A.X (orange) and PLA (purple) in DLDwt (A), DLD BRCA2 (-/-) (B) and SUM149PT (C).

577 Time and dose dependent cytotoxicity induced by veliparib in DLDwt (D), DLD BRCA2 (-/-) (E) 578 and SUM149PT (F). Data are presented as means ± SEM from at least 3 independent 579 experiments.

580 Figure 4. PARP inhibitors differ in their resolution between catalytic inhibition and bone 581 marrow toxicity. Bone marrow CFC data were normalized to DMSO controls and are presented 582 as means with standard errors of total colony forming cells (erythroid and myeloid lineages) 583 from three independent donors. Cellular PAR levels were determined by ELISA in bone marrow 584 mononuclear cells treated with PARP inhibitors. Data are means ± SEM from at least 4 585 independent experiments.

586 Figure 5. PARP1 trapping activity is associated with greater in vitro cytotoxic potency 587 towards multiple cancer cell lines. In vitro cytotoxic IC50s of PARP inhibitors across cancer cell 588 lines compared to inhibition of PAR formation and inhibition of colony formation of bone 589 marrow cells. IC50s for HeyA8, DLD1, and DLD1 BRCA2 (-/-) cells (A), SCLC lines (B), and breast 590 cancer cell lines (C). For cell lines more sensitive than bone marrow, a 2-sided student’s t-test 591 was performed and *p < 0.05, **p < 0.01, ***p < 0.001. Dashed lines are clinical Cmax/min at 592 monotherapy RP2D. Data points are from a minimum of 3 independent experiments and 593 means ± SEM are indicated.

594 Figure 6. Anti-tumor activity and PK/PD for veliparib, olaparib, and talazoparib in SUM149PT 595 xenografts. (A) Efficacy plots for SUM149PT xenografts in mice dosed orally with PARP 596 inhibitors. PARP inhibitors were administered PO, QD or BID for 42 days (d30-71). Data are 597 presented as means with standard errors. Maximum percent weight loss and percent tumor 598 growth inhibition (TGI) on day 54 vs. vehicle are indicated. P values are from 2-sided t-tests for 599 mean difference in log10 (tumor volume) on specified day. (B) Pharmacokinetics and 600 pharmacodynamics in SUM149PT xenograft tumor-bearing mice. Mice were dosed orally BID 601 or QD as indicated with compounds for 5 days. At 1, 4, 24, 32, and 48 hours after the last dose, 602 plasma and tumors samples were harvested. Plasma and tumor drug concentrations (PK) and 603 tumor PAR levels (PD) were determined. Data are presented as means with standard errors.

Figure 1

B 120 C 6 Talazoparib 100 Rucaparib 80 4 A-934935 60 Talazoparib 40 Rucaparib 2 20 A-934935 TR-FRET Ratio TR-FRET

% Inhibition (PAR) Inhibition % 0 0 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 10 [PARPi] (µM) [PARPi] (µM)

D 10 E 2.5 Talazoparib 8 Rucaparib 2.0 A-934935 6 1.5 4 Talazoparib H2Ax Signal H2Ax Normalized γ Normalized 1.0 Rucaparib

PARP1Trapping 2 A-934935 0.5 0.00010.001 0.01 0.1 1 10 Downloaded from mcr.aacrjournals.org on September0.00010.001 26, 2021. 0.01 © 2018 0.1 American 1 10Association for Cancer Research. [PARPi] (µM) [PARPi] (µM) Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2

120 Talazoparib HAP WT Talazoparib HAP PARP KO 100 Rucaparib HAP WT Rucaparib HAP PARP KO 80 A-934935 HAP WT A-934935 HAP PARP KO

60 Viability (%) Viability 40

0 0.001 0.01 0.1 1 10 100 1000 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. [PARPi] (µM) Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3

A D 80 PAR yH2AX 160 120 120hr Caspase 3/7 PLA Induction PAR % 100 96hr 60 120 80 72hr 48hr H2A.X γ Caspase 3/7 40 80 60 24hr o r

% Control 40 20 40

PLA 20 0 0 0 % Cells with 0.0001 0.01 1 100 0.01 0.1 1 10 100 1000 [Veliparib] (µM) [Veliparib] (µM) B E 80 160 120 120hr % PAR Induction PAR % 100 96hr 60 120 80 72hr 48hr H2A.X γ Caspase 3/7 40 80 60 24hr o r

% Control 40 20 40

PLA 20 0 0 0 % Cells with 0.0001 0.01 1 100 0.01 0.1 1 10 100 1000 [Veliparib] (µM) [Veliparib] (µM) C F

80 160 120 120hr % PAR Induction PAR % 100 96hr 60 120 80 72hr 48hr H2A.X γ Caspase 3/7 40 80 60 24hr o r

% Control 40 20 40

PLA 20 0 0 0 % Cells with 0.01 0.1 1 10 100 1000 1 0.01 0.0001Downloaded 0.01 from 1 mcr.aacrjournals.org 100 on September 26, 2021. © 2018 American Association for Cancer Research. [Veliparib] (µM) [Veliparib] (µM) Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4

Veliparib Olaparib 140 140 140 140

BM-CFU (%Control) CFC Total (%Control) CFC Total BM-CFU 120 PAR 120 120 120 PAR 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40

PAR (% Inhibition) (% PAR 20 20 Inhibition) (% PAR 20 20 0 0 0 0 -5 0 -5 0 Log[PARPi] (µM) Log[PARPi] (µM)

Talazoparib Rucaparib 140 140 140 140

To BM-CFU (%Control) CFC Total BM-CFU

120 120 120 120 t PAR PAR C al 100 100 100 100

80 80 80 80 (%C

Inhibition)

60 60 60 60 Co (% 40 n 40 40 40 t

PAR (% Inhibition) (% PAR PAR PAR 20 20 20 20 l) 0 0 0 0 -5 0 -5 0 Log[PARPi] (µM) Log[PARPi] (µM)

A-934935 140 140 BM-CFU (%Control) CFC Total 120 120 PAR 100 100 80 80 60 60 40 40

PAR (% Inhibition) (% PAR 20 20 0 0 -5 Downloaded0 from mcr.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Log[PARPi] (µM) B A Figure 5 C 0.0001 0.0001 0.0001 0.001 0.001 0.001 IC50 (µM) IC50 (µM) IC50 (µM) 0.01 0.01 0.01 100 100 100 0.1 0.1 0.1 10 10 10 1 1 1

PAR PAR PAR Veliparib Veliparib BM-CFU Veliparib BM-CFU BM-CFU T47D H69 HeyA8 UACC812 H1048

* Downloaded from SUM149PT H1930 DLD1 ** MDA-MB-436 H2081 *** DLD1-BRCA2-/- Author ManuscriptPublishedOnlineFirstonNovember14,2018;DOI:10.1158/1541-7786.MCR-18-0138 PAR PAR PAR Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Olaparib Olaparib BM-CFU BM-CFU BM-CFU Olaparib T47D H69 HeyA8 UACC812 H1048 SUM149PT H1930 DLD1 * MDA-MB-436 H2081 * DLD1-BRCA2-/- mcr.aacrjournals.org

PAR PAR PAR Talazoparib Talazoparib Talazoparib BM-CFU BM-CFU BM-CFU T47D H69 HeyA8 UACC812 H1048 SUM149PT H1930 DLD1 * MDA-MB-436 H2081 * DLD1-BRCA2-/-

PAR PAR PAR Rucaparib Rucaparib Rucaparib BM-CFU BM-CFU BM-CFU on September 26, 2021. © 2018American Association for Cancer Research. T47D H69 HeyA8 UACC812 H1048 SUM149PT H1930 DLD1 * MDA-MB-436 H2081 * DLD1-BRCA2-/-

PAR PAR PAR A-934935 A-934935 BM-CFU BM-CFU A-934935 BM-CFU T47D H69 HeyA8 UACC812 H1048 * SUM149PT H1930 DLD1 * MDA-MB-436 H2081 ** DLD1-BRCA2-/- Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6

A Max % Wt Loss% change

1000 Vehicle (0) -8 ABT-888 (300) -6 Olaparib (300) -5 Talazoparib (0.5) -4

p < 0.05 vs. vehicle ) * 3 *

500 * * * *

Tumor Volume (mm %TGI d:54 vs Vehicle Veliparib (300) = 29* Olaparib (300) = 32* Talazoparib (0.5) = 35* * p < 0.05 0 25 35 45 55 65 PARPi, PO Day

B Tumor PD Tumor PK Plasma PK Veliparib (300mkd, BIDx5) Olaparib (300mkd, BIDx5) ) ) 100 100 Tumor PAR (pg/mL) Tumor PAR (pg/mL) g/g g/g 1000 1000 µ µ 10 10 or or 750 750 1 1 500 500 µg/ml µg/ml 0.1 0.1 250 250 0.01 0.01

0 0 0.001 0.001 Exposure ( Exposure ( 0 4 8 8 2 0 4 8 6 8 2 6 12 16 20 24 2 3 36 40 44 48 12 1 20 24 2 3 3 40 44 48 Time (hrs) Time (hrs)

Talazoparib (0.5mkd, qdx5) ) 100 Tumor PAR (pg/mL)

g/g 1000 µ 10

or 750 1 500 µg/ml 0.1 250 0.01

0 0.001 Exposure ( 0 4 8 4 8 2 4 12 16Downloaded20 2 2 3 from36 mcr.aacrjournals.org40 4 48 on September 26, 2021. © 2018 American Association for Cancer Research. Time (hrs) Author Manuscript Published OnlineFirst on November 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0138 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

PARP1 trapping by PARP inhibitors drives cytotoxicity both in cancer cells and healthy bone marrow

Todd A Hopkins, William B Ainsworth, Paul A Ellis, et al.

Mol Cancer Res Published OnlineFirst November 14, 2018.

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