RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer

ARTICLE TYPE: Original Article

TITLE: RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA mutated breast cancer

AUTHOR LIST: C. Cruz1,2,3, M. Castroviejo-Bermejo1, S. Gutiérrez-Enríquez4, A. Llop-Guevara1, Y. H. Ibrahim1, A. Gris-Oliver1, S. Bonache4, B. Morancho5, A. Bruna6, O. M. Rueda6, Z. Lai7, U. M. Polanska8, G. N. Jones8, P. Kristel9, L. de Bustos1, M. Guzman1, O. Rodriguez1, J. Grueso1, G. Montalban4, G. Caratú10, F. Mancuso10, R. Fasani11, J. Jiménez11, W. J. Howat8, B. Dougherty7, A. Vivancos10, P. Nuciforo11, X. Serres-Créixams12, I. T. Rubio13, A. Oaknin3,14, E. Cadogan8, J. C. Barrett7, C. Caldas6,15, J. Baselga16, C. Saura3,17, J. Cortés18, 19, J. Arribas5,20,21, J. Jonkers9, O. Díez4,22, M. J. O’Connor23, J. Balmaña2,3#,, V. Serra1, 21,#,* AFFILIATION LIST: 1

Experimental Therapeutics Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

2

High Risk and Familial Cancer, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

3

Department of Medical Oncology, Hospital Vall d’Hebron, Universitat Autònoma de

Barcelona, Barcelona, Spain; 4

Oncogenetics Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

5

Growth Factors Laboratory, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

6

Department of Oncology and Cancer Research UK Cambridge Institute, Li Ka Shing

Centre, Cambridge, UK;

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© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 [email protected]

7

AstraZeneca, Gatehouse Park, Waltham, Massachusetts, USA;

8

DNA Damage Response Biology Area, Oncology iMed, AstraZeneca, Cancer Research

UK Cambridge Institute, Cambridge, UK; 9

Division of Molecular Pathology and Cancer Genomics, The Netherlands

Cancer Institute, Amsterdam, The Netherlands; 10

Cancer Genomics Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

11

Molecular Oncology Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

12

Department of Radiology, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona,

Barcelona, Spain; 13

Breast Surgical Unit, Breast Cancer Center, Hospital Vall d’Hebron, Universitat

Autònoma de Barcelona, Barcelona, Spain; 14

Gynecological Malignancies Group, Vall d’Hebron Institute of Oncology, Barcelona,

Spain; 15

Breast Cancer Programme, Cancer Research UK Cambridge Cancer Centre, Cambridge,

UK; 16

Human Oncology and Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer

Center, New York, USA. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA; 17

Breast Cancer and Melanoma Group, Vall d’Hebron Institute of Oncology, Barcelona,

Spain; 18

Ramón y Cajal University Hospital, Madrid, Spain;

19

Vall d’Hebron Institute of Oncology, Barcelona, Spain;

20

Department of Biochemistry and Molecular Biology, Building M, Campus UAB,

Bellaterra (Cerdanyola del Vallès), Spain. Institució Catalana de Recerca i Estudis 2 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

Avançats (ICREA), Barcelona, Spain; 21

CIBERONC, Vall d’Hebron Institute of Oncology, Barcelona, Spain;

22

Clinical and Molecular Genetics Area, Hospital Vall d’Hebron, Universitat Autònoma de

Barcelona, Barcelona, Spain 23

DNA Damage Response Biology Area, Oncology Innovative Medicine and Early

Development Biotech Unit, AstraZeneca, Cambridge, UK #

Authors contributed equally

*To whom correspondence should be addressed

FULL ADDRESS FOR CORRESPONDENCE: Dr. Violeta Serra Experimental Therapeutics Group Vall d’Hebron Institute of Oncology Carrer Natzaret 115-117, Barcelona, 08035, Spain Phone: +34 93 2543450 Email: [email protected]

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ABSTRACT: Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and Methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, was the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ATM inhibitor.

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Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

KEYWORDS: germline BRCA, PARP inhibitor resistance, homologous recombination, RAD51, TP53BP1, ATM

KEY MESSAGE: Tumors from germline BRCA1/2-mutation carriers may restore homologous recombination repair (HRR) and acquire PARPi resistance through diverse mechanisms. The direct detection of RAD51 nuclear foci in tumor samples, as a functional biomarker of HRR, may help to refine the selection of patients eligible for PARPi-monotherapy, and to propose new combination therapies for PARPi-resistant patients.

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INTRODUCTION: BRCA1 and BRCA2 encode essential proteins for DNA homologous recombination repair (HRR)[1]. Loss of function of either gene impairs this high-fidelity DNA repair pathway and results in genetic instability and an increased risk of breast or ovarian cancer in germline BRCA1/2 (gBRCA) mutation carriers[2, 3]. Defective HRR increases sensitivity of gBRCA-mutated tumors to DNA damaging agents including anthracyclines, platinum salts, or to novel agents that block parallel DNA repair pathways, including PARPi[4-6]. PARP inhibition blocks the repair of DNA single strand breaks and results in stalling of replication fork progression by trapping PARP on the DNA break[7]. Both contribute to the accumulation of DNA double strand breaks (DSBs) that HRR-deficient cells cannot repair efficiently. PARPi are well-tolerated agents and elicit anti-cancer efficacy in metastatic gBRCA tumors. Their use has been approved for advanced ovarian cancer (olaparib (Lynparza®), rucaparib (Rubraca®) and niraparib (Zejula®)) and for gBRCA breast cancer (BC)[8-10]. Final results from other phase III clinical trials are awaited, both in the early and advanced BC setting (NCT01905592, NCT01945775, NCT02032823). Primary resistance to PARPi in a subset of gBRCA patients limits the potential of gBRCA status as the only biomarker of response to that of an enrichment strategy[11]. In addition, acquired resistance in monotherapy responders is a challenge. Work by several groups using in vitro models, transgenic mice and human tumor samples have delineated two types of resistance mechanisms to PARPi in gBRCA cells: a) independent of HRR (cellular extrusion of the PARPi, PARP1 loss, FANCD2 overexpression, SLFN11 inactivation or CHD4 loss), and b) dependent on HRR recovery, either by BRCAindependent mechanisms (loss of 53BP1, REV7, PAXIP1/PTIP, Artemis) or BRCA6 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

dependent[12-22]. The latter include secondary BRCA1/2 mutations that restore the reading frame and the expression of partially functional hypomorphic BRCA1 proteins (BRCA1∆11q alternative splice isoform, the RING-less BRCA1 generated by downstream translation initiation, or HSP90-mediated stabilization of BRCA1 C-terminal mutants. Most work in gBRCA clinical samples has focused on ovarian cancer and has established that HRR recovery through secondary BRCA1/2 mutations may act as a resistance mechanism to platinum salts and PARPi. Conversely, little is known about in vivo PARPi resistance mechanisms in gBRCA BC[22]. Patterns of DNA aberrations in the tumors (genomic scars) resulting from HRR deficiency may aid in distinguishing HRR-deficient from HRR-proficient tumors[23-25]. However, genomic scars in gBRCA tumors may persist after restoration of HRR function[26]. In order to improve patient selection for PARPi monotherapy among gBRCA mutation carriers, especially in the metastatic setting, there is a clear need for a functional biomarker of HRR status to be used in the clinic. Previous work by others showed that induction of nuclear foci of the HRR protein RAD51 after neoadjuvant chemotherapy is a measure of HRR functionality in BC biopsies and predicts treatment response[27]. Here we sought to investigate RAD51 foci as an indicator of functional HRR and its correlation with PARPi resistance in the gBRCA setting. We further explored potential treatment strategies for PARPi-resistant BC.

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METHODS: Study Design A collection of PDX models was generated by implanting tumor samples from patients with a germline BRCA1/2 mutation and breast or ovarian cancer. Their sensitivity to PARPi was evaluated, and the functionality of the HRR pathway was analyzed and compared between the PARPi-sensitive vs. the PARPi-resistant PDX samples to find a functional test correlating with response. An exploratory analysis in a set of twenty tumor samples including patients treated with PARPi at our institution was employed to confirm the findings and the potential clinical interest of the functional test. A new therapeutic PARPi combination was tested in vivo in PARPi-resistant PDX models.

See further Methods in Supplementary Material.

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RESULTS: gBRCA PDX panel Fresh tumor samples prospectively collected for implantation into nude mice yielded a total of twelve PDX models (11 gBRCA1 and 1 gBRCA2) (Supplementary Table 1). Five models were derived from patients with metastatic disease who had been treated with PARPi, three of which prior to olaparib treatment and two at progression after a sustained PR (Supplementary Table 1). Persistence of the gBRCA mutations were confirmed in all models but PDX274, and they were associated with loss of heterozygosity, i.e. loss of the wild type allele (Fig. S1). Olaparib treatment in the gBRCA PDX collection distinguished a subset of PARPiresistant tumors (Fig. 1A) (assessed by modified Response Evaluation In Solid Tumors (mRECIST), see Supplementary Methods). Treatment with olaparib exhibited antitumor activity in three gBRCA models: two CR and one SD. The remaining nine PDX models were olaparib-resistant (PD). An additional PDX model with acquired resistance (PDX230OR) was generated from its PARPi-sensitive counterpart (PDX230) after >100 days exposure to olaparib (Fig. S2), totaling 13 gBRCA1/2 models. Among the four PDX derived from gBRCA primary tumors, 50% showed CR. The sensitivity to PARPitreatment in the PDXs from metastatic patients previously treated with olaparib mirrored the patients’ clinical response to olaparib (Supplementary Table 1; Fig. S3).

BRCA1/2 sequencing, BRCA1 expression and nuclear foci formation in gBRCA PDX samples No frameshift-correction or genetic reversion of the inherited mutation –the so called secondary BRCA mutations– occurred in the gBRCA1 PARPi-resistant PDXs. 9 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

BRCA1 mRNA expression was variable across models and absent in PDX280, a model with a large deletion encompassing the complete BRCA1 gene (Fig. S4). To investigate the potential expression of hypomorphic BRCA1 isoforms and their recruitment to DNA damage sites, we set up immunostaining assays for the DNA damage response (DDR) proteins: BRCA1, RAD51 (as functional HRR marker) and γH2AX (as DNA damage marker), with geminin (as S/G2-cell cycle marker) (Fig. S5). PDX124 and PDX196 harbor a c.1961delA mutation in BRCA1 exon 11 and express the BRCA1-∆11q splice isoform (p.Ser264_Gly1366del) [20] which forms nuclear foci detected with both B1-NT and B1-CT antibodies, as expected (Fig. 1B, Fig. S6). BRCA1 nuclear foci were also detected in five additional gBRCA PDX models: PDX179, STG316, PDX274, PDX221 and PDX236 (Supplementary Table 1; Fig 1B). WB confirmed the expression of BRCA1 isoforms at the respective predicted sizes (∆11q, RING-less, and Cterminal truncated mutant proteins (Fig. S7)). In summary, hypomorphic BRCA1 isoforms were detected by IF to form nuclear foci in seven PDX models, six with primary or acquired resistance to PARPi and one model showing disease stabilization (PDX124).

Analysis of 53BP1 loss and exome sequencing in gBRCA PDX samples The assessment of 53BP1 nuclear foci by IF in olaparib-treated PDX samples identified 53BP1 loss in two PARPi-resistant models: PDX230OR and STG316 (Fig. 1B). Exome sequencing unveiled somatic mutations in TP53BP1 in both models (Supplementary Table 1). The PD PDX280 harbors a non-previously reported missense mutation in the PARPi resistance gene SLFN11 p.H661D. The SD model PDX124 displays a focal RAD51 amplification and high protein expression (Fig. S8A, B). Mutations in other

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known PARPi resistance genes (PARP1, REV7, PAXIP1/PTIP, Artemis, CHD4) were not identified.

Nuclear foci formation of the HRR protein RAD51 The observed recruitment of hypomorphic BRCA1 isoforms to DNA damage sites and/or 53BP1 loss in PARPi-resistant PDXs may help restore their ability to accomplish HRR. As a functional surrogate of HRR, we sought to detect RAD51 nuclear foci in geminin-positive cells and nuclear co-localization with BRCA1. RAD51 nuclear foci were detected in eleven PDXs in olaparib-treated samples, including all models expressing hypomorphic BRCA1 isoforms and/or lacking 53BP1 (Fig. 1B). RAD51 foci co-localized with BRCA1 foci in all PDX models expressing hypomorphic BRCA1 isoforms (Fig. S9). The three PARPi-resistant models that lacked hypomorphic BRCA1 isoform expression or 53BP1 loss (PDX127, PDX252 and PDX280) exhibited RAD51 foci suggesting that recovery of HRR occurs via BRCA1-independent mechanisms in these models (Fig. 1B). Olaparib-treated samples from PARPi-resistant PDXs showed higher percentage of RAD51-positive cells vs. those from PARPi-sensitive models (36 ± 2% in PARPi-resistant vs. 5 ± 3% in PARPi-sensitive, P=0.0017) (Fig. 1C). Analysis of γH2AX foci ruled out PARPi pharmacodynamic differences as the reason for this differential response (Fig. S10). The unexpected evidence of endogenous DNA damage and repair markers in untreated samples (Fig. S5,S10) prompted us to also score RAD51 foci in untreated tumors (Fig. 1C). Importantly, untreated samples of PARPi-resistant tumors showed a significantly higher baseline percentage of RAD51-positive cells compared to PARPi-sensitive tumors (24 ±

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2% vs. 3 ± 2%, P=0.0025). Thus, HRR functionality was frequent and associated with PARPi resistance in this gBRCA PDX panel.

RAD51/geminin score and response to PARPi in patients’ samples We confirmed the feasibility of detecting RAD51 and γH2AX foci in FFPE tumor samples from patients, by firstly staining for RAD51 and geminin in 20 patients’ tumor samples including some matched with the gBRCA PDX models (N=7) (Fig. S11A,B,C). These results prompted us to further assess the potential clinical utility of the RAD51/geminin score as a functional biomarker of PARPi treatment in patients treated with various PARPi at our institution, (N=10 tumors), including two paired pre/post-PARPi samples. This cohort included eight patients with germline mutations in BRCA1/2 (BRCA1, N=5; BRCA2, N=3; diagnosis of BC, N=6; ovarian cancer, N=2). The samples had been collected prior to (N=7) or at progression to treatment with a PARPi (N=3). We stained and scored for RAD51 (Fig. 1D,E). Importantly, PARPi-resistant tumor samples showed an inverse relationship between the RAD51 score and clinical efficacy of the PARPi. Exome sequencing identified a BRCA2-secondary mutation in one tumor with acquired resistance and RAD51 foci (Fig. S12).

Platinum salts in olaparib-resistant tumors HRR recovery/retention that limits PARPi efficacy may not imply resistance to platinum-based treatments in gBRCA cancers. A previous study in gBRCA ovarian cancer showed a 40% response rate to platinum chemotherapy in the setting of resistance to olaparib[28]. We next assessed the efficacy of cisplatin in the two HRR proficient, RAD51-

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positive ovarian cancer PDX models (PDX196 and PDX280) (Fig. 2A). Response to cisplatin was confirmed in both PDX models and in the clinic for Pt280 (data not available for Pt196 due to carboplatin hypersensitivity). Next, we assessed the activity of platinumbased chemotherapy in advanced/metastatic BC in the context of PARPi resistance. We previously reported that PDX127 showed resistance to PARPi but response to platinum, in agreement with the clinical response of the patient[29]. Similarly, the PARPi-resistant, RAD51-positive model PDX252 exhibited significant tumor regression when treated with cisplatin (Fig. 2A). In the PARPi-resistant PDX236 and PDX274, cisplatin-only slowed tumor growth as compared to vehicle, while its combination with olaparib achieved PR and SD, respectively (Fig. 2B). These results highlight that platinum-based therapies can be active in PARPi-resistant metastatic BC and suggests that RAD51 foci formation does not predict resistance to platinums in this setting.

ATM blockade plus PARP inhibition in olaparib-resistant tumors We further explored the potential of DDR inhibitors to enhance PARPi antitumor activity. The Ataxia Telangiectasia Mutated (ATM) kinase is activated in response to DNA DSBs, signals to cell cycle checkpoints and DNA repair pathways, and is reciprocally synthetic lethal with PARP[30]. As previously suggested, we hypothesized that ATM inhibition is a treatment option for PARPi-resistant BRCA1-deficient tumors that restore HRR through loss of 53BP1 or REV7/MAD2L by enabling ATM-dependent end resection [17, 18]. We tested this hypothesis in three ATM-expressing PDXs (Fig. S13A): STG316, a model that lacks 53BP1, and in PDX127 and PDX280, which are devoid of hypomorphic BRCA1 isoforms, and presumably achieve PARPi resistance by a “loss of 53BP1”-like mechanism. In fact, PDX127 harbors a missense mutation in PRCC p.P55T, within the 13 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

interaction domain with Rev7/MAD2L2 (Fig. S13B). The best antitumor activity of the olaparib combination with the ATM inhibitor AZD0156 was achieved in PDX127 (SD) (Fig. 2C). We investigated whether ATM inhibition resulted in restoration of HRRdeficiency by impairing RAD51 foci formation [17, 18] (Fig. S13C). Unexpectedly, RAD51 foci formation was marginally reduced in combination-treated tumors, arguing that ATM inhibition may exert a broader effect in signaling the olaparib-induced DNA damage response beyond its effects on HRR (Fig. S13C). Quantification of DNA damage by γH2AX staining (pan-nuclear, Fig. 2D and foci formation, Fig. S13D) showed a significant increase of pan-γH2AX-positive cells upon ATM plus PARPi as compared to olaparib monotherapy in combination-responders PDX127 and STG316. These results suggest an induction of replication stress in these combination-sensitive models. These results are of interest since an international phase I clinical trial testing the tolerability of olaparib in combination with AZD0156 in solid tumors is currently ongoing (NCT 02588105).

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DISCUSSION: There is a need to refine the determinants of PARPi efficacy beyond gBRCA mutations, especially in the metastatic setting. Our analysis of RAD51 foci in a total of 20 BC patient samples, ten gBRCA1 and ten gBRCA2, provides new evidence in favor of restoration of HRR functionality as a frequent mechanism of PARPi resistance, and demonstrates the potential of functional biomarkers to discriminate tumors that will fail PARPi monotherapy. The RAD51 foci assay may capture the dynamic changes in DNA repair that occur throughout tumor evolution and may, therefore, more effectively identify the HRR-proficient BRCA1/2-mutated tumors. Unexpectedly, while previous studies reported low levels of baseline DNA damage as a potential limitation to evaluate HRR[27, 31], we were able to detect it and score for RAD51 in untreated samples, which correlated with PARPi response. This highlights that an IF assay for RAD51 staining is feasible in FFPE samples and suggests that testing for RAD51 may be directly transferrable to the clinical setting when a larger study confirms our findings. Restoration of HRR can be achieved by secondary BRCA mutations and may be captured by sequencing techniques[32, 33]. We identified a BRCA2 secondary mutation in one BC patient with acquired resistance to olaparib, whereas we did not detect in-frame secondary mutations in any gBRCA1 PDX model. Our data suggest that hypomorphic BRCA1 isoforms contribute to HRR restoration in gBRCA1 BC[20, 21]. Importantly, high RAD51 score predicted poor response to PARPi monotherapy independently of the underlying mechanism of HRR restoration. Further research is needed to establish the RAD51 score cut-off that differentiates responders from non-responders to PARPi monotherapy and to evaluate the potential impact of RAD51-independent resistance mechanisms that involve replication fork stabilization[14, 16, 34, 35]. A high RAD51 foci 15 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

score may encourage the use of combination therapies with PARPi, such as those that inhibit HRR[29, 36], or that enhance DNA damage[9, 37, 38]. Here, we propose that a subset of PARPi-resistant gBRCA tumors benefit from combined PARP plus ATM blockade[17, 30, 39]. Our study unveils coexistence of various mechanisms of PARPi resistance in each individual tumor, such as hypomorphic BRCA1 isoforms together with RAD51 amplification or 53BP1 loss. In conclusion, this emphasizes the need of comprehensive functional tests for measuring HRR activity such as the RAD51 assay to better select patients who will benefit most from PARPi monotherapy and those who may benefit from a combination therapy.

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ACKNOWLEDGEMENTS: The authors thank Dr. Geoffrey Shapiro, Dr. Peter Bouwman, Dr. Neil Johnson, the Molecular Pathology Group at VHIO and the Breast Cancer and Melanoma Group at VHIO for helpful discussions. The Breast Surgical Unit from Vall d’Hebron Hospital, as well as Cristina Bernadó, Pilar Antón, Maite Calvo, Patricia Cozar and Ambar Ahmed have provided technical support. The authors acknowledge the Cellex Foundation for providing research facilities and equipment.

FUNDING: This work was supported by Spanish Instituto de Salud Carlos III (ISCIII) funding, an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds [FIS PI17/01080 to V.S., PI12-02606 to J. Balmaña, PI15-00355 to O.D., PI13/01711 to S.G.-E.]; European Research Area-NET, Transcan-2

[AC15/00063],

Asociación

Española

contra

el

Cáncer

(AECC)

[LABAE16020PORTT] and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) [2014 SGR 1331 and 2017 SGR 540] to V.S.; Conquer Cancer Foundation of ASCO Young Investigator Award to C. C. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the American Society of Clinical Oncology or the Conquer Cancer Foundation. We acknowledge “Tumor Biomarkers Collaboration” supported by the Banco Bilbao Vizcaya Argentaria (BBVA) Foundation, GHD, the FERO Foundation and the Orozco Family for supporting this study (to V.S. and J. Baselga). C.C. is recipient of a Sociedad Española de Oncología

Médica

(SEOM) -

BUCKLER0’0

grant

and an

AECC

grant

[AIOC15152806CRUZ]. M.C.-B. [LABAE16020PORTT] and S.B. are recipient of AECC 17 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

grants. A.L.-G. is recipient of a grant from the Generalitat de Catalunya, Health Department [PERIS, SLT002/16/00477]. V.S. and S.G.E. are supported by the Miguel Servet Program [ISCIII, CP14/00228 and CP10/00617, respectively]. The xenograft program in the Caldas laboratory is supported by Cancer Research UK, and also received funding from an EU H2020 Network of Excellence (EuroCAN).

DISCLOSURE: V.S. declares a non-commercial research agreement with AstraZeneca. J. Balmaña is an advisory board member for Clovis, Tesaro and Medivation, and has received speaker bureau honoraria by AstraZeneca. B.D., Z.L., U.P., E.C., G.J., W.H., C.B. and M.J.O. are employees of AstraZeneca. No potential conflicts of interest were disclosed by the other authors.

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and Acquired PARP Inhibitor Resistance in BRCA Wild-Type and Mutated Models of Triple-Negative Breast Cancer. Cell Rep 2016; 17: 2367-2381. 30.

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SUPPLEMENTARY MATERIAL

SUPPLEMENTARY METHODS Generation of PDX models and in vivo treatment experiments Fresh tumor samples from patients with gBRCA breast or ovarian cancer were prospectively collected for implantation into nude mice under an institutional IRBapproved protocol and the associated informed consent, or by the National Research Ethics Service, Cambridgeshire 2 REC (REC reference number: 08/H0308/178)[1]. Five patients had received olaparib as single agent or in combination for their advanced disease; 2 were obtained after acquired resistance to olaparib. Experiments were conducted following the European Union’s animal care directive (2010/63/EU) and were approved by the Ethical Committee of Animal Experimentation of the Vall d’Hebron Research Institute. Surgical or biopsy specimens from primary tumors or metastatic lesions were immediately implanted in mice. Fragments of 30 to 60 mm3 were implanted into the mammary fat pad (surgery samples) or the lower flank (metastatic samples) of 6-week-old female athymic HsdCpb:NMRI-Foxn1nu mice (Harlan Laboratories). Animals were continuously supplemented with 1 µM 17β-estradiol (Sigma-Aldrich) in their drinking water. Upon growth of the engrafted tumors, the model was perpetuated by serial transplantation onto the lower flank. In each passage, flash-frozen and formalin-fixed paraffin embedded (FFPE) samples were taken for genotyping and histological studies. To evaluate the sensitivity to the drugs, tumor-bearing mice were equally distributed into treatment groups with tumors ranging 100 to 300 mm3. As single agent, olaparib 50 mg/kg p.o. was administered five times per week unless otherwise specified (in 10%v/v DMSO /

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10%w/v Kleptose [HP-β-CD]). AZD0156 was administered p.o. three times per week at 2 or 2.5 mg/kg in 10% DMSO plus 90% Captisol (30% w/v). Cisplatin (6 mg/kg) was administered weekly unless the relative tumor volume (RTV) was below 0.5 or weight loss >20%. Tumor growth was measured with caliper bi-weekly from first day of treatment to day 21 and every 7-10 days in the acquired resistance setting. To generate PDX models with acquired resistance to PARPi, olaparib treatment was maintained for up to 150 days in olaparib-sensitive tumors until individual tumors regrew. In all experiments, mouse weight was recorded twice weekly. The tumor volume was calculated as V = 4π/3/Lxl2, “L” being the largest diameter and “l” the smallest. Mice were euthanized when tumors reached 1500 mm3 or in case of severe weight loss, in accordance with institutional guidelines. The antitumor activity was determined by comparing tumor volume at 21 days to its baseline: % tumor volume change = (V21days -Vinitial)/Vinitial x100. For olaparib-sensitive PDX, the best response was defined as the minimum value of % tumor volume change sustained for at least 10 days. To classify the response of the subcutaneous implants we modified the RECIST criteria, to be based on the % tumor volume change [2, 3]: CR (complete response), best response<-95%; PR (partial response), -95%+20%.

Analysis of the BRCA1/2 genes and loss of heterozygosity in PDX tumors DNA was extracted from PDX samples using DNeasy Blood & Tissue Kit (Qiagen). All exons and flanking intronic regions of the BRCA1 and BRCA2 genes were sequenced by the Multiplicom MASTR T Dx methodology in an Illumina MiSeq Sequencer. Coverage range was 100-2240X. Results were analyzed with Sophia DDM® platform, which integrates 2 Downloaded from https://academic.oup.com/annonc/advance-article-abstract/doi/10.1093/annonc/mdy099/4959904 by guest on 07 April 2018

PEPPER™, MUSKAT™ and MOKA™ technologies (minimum coverage of 100 reads per base). Pathogenic variants were confirmed by Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit in a 3130xl sequencer (Applied Biosystems, Foster City, CA, USA). Screening for large genomic rearrangements was done using MLPA commercial kit #P002 for BRCA1 (MRC-Holland, Amsterdam, The Netherlands), in accordance with the manufacturer’s instructions. PCR products were analyzed on an ABI 3130xl capillary sequencer using GeneMapper (Applied Biosystems) and Coffalyser.Net (MRC-Holland, Amsterdam, The Netherlands) software. The deletion of exons 3-7 of BRCA1 in PDX274 was confirmed by sequencing the PCR product (736bp) after amplification of cDNA with exon 1 oligonucleotide 5’-ACAGGCTGTGGGGTTTCTC-3’ and exon 10 reverse oligonucleotide 5’- TTCATCCCTGGTTCCTTGAG-3’. Nomenclature of variants was according to the Human Genome Variation Society.

Analysis of BRCA1 mRNA expression RNA was extracted from PDX samples (15-30 mg) by using the PerfectPure RNA Tissue kit (5 Prime). The purity and integrity was assessed by the Agilent 2100 Bioanalyzer system and cDNA was obtained using the PrimeScript RT Reagent kit (Takara). Quantitative RT-PCR was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Universal Master Mix II (Applied Biosystems) and predesigned human

specific

primers

and

TaqMan

probes

(Hs99999908_m1

for

GUSB,

Hs99999903_m1 for ACTB and Hs01556193_m1 for BRCA1). The comparative CT method was used for data analysis, in which geNorm algorithms were applied to select the most stably expressed housekeeping genes (GUSB and ACTB) and geometric means were calculated to obtain normalized CT values [4].

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Antibodies and immunofluorescence/immunohistochemistry experiments The following primary antibodies were used for immunofluorescence: mouse anti-γH2AX (Millipore JBW301, 1:200); rabbit anti-RAD51 (Santa Cruz Biotechnology H-92, 1:250); mouse anti-Geminin (NovoCastra NCL-L, 1:100 for PDX samples and 1:60 for human specimens); rabbit anti-Geminin (ProteinTech Group, 10802-1-AP, 1:400); mouse antiBRCA1 N-terminus (Abcam MS110, 1:200); mouse anti-BRCA1 C-terminus (Santa Cruz Biotechnology D-9, 1:50); rabbit anti-53BP1 (Cell Signaling 4937, 1:100); Rabbit antiATM (Abcam ab32420, 1.2 µg/ml). Goat anti-rabbit Alexa fluor 568 or 488, goat antimouse Alexa fluor 488 or donkey anti-rabbit Alexa fluor 568 (Invitrogen; 1:500) were used as secondary antibodies for immunofluorescence studies. 3 µm tissue sections were deparaffinized with xylene and hydrated with decreasing concentrations of ethanol. For target antigen retrieval, sections were microwaved for 4 min at 110ºC in DAKO Antigen Retrieval Buffer pH 9.0 (115ºC in DAKO Antigen Retrieval Buffer pH 6.0 for 53BP1 staining) in a T/T MEGA multifunctional Microwave Histoprocessor (Milestone). Sections were cooled down in distilled water for 5 min, then permeabilized with DAKO Wash Buffer (contains Tween 20) for 5 min, followed by incubation in blocking buffer (DAKO Wash Buffer with 1% bovine serum albumin) for 5 min (DAKO serum free block for 15 min for ATM). Primary antibodies were diluted in DAKO Antibody Diluent and incubated at room temperature for 1 hour (TBS/Tween for ATM). Sections were washed for 5 min in DAKO Wash Buffer followed by 5 min in blocking buffer. Secondary antibodies were diluted in blocking buffer and incubated for 30 min at room temperature. The 2-step washing was repeated followed by 5 min incubation in

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distilled water. Dehydration was performed with increasing concentrations of ethanol. Sections were mounted with DAPI ProLong Gold antifading reagent and stored at -20ºC. To be evaluable, samples must display endogenous DNA damage with a percentage of γH2AX/geminin positive cells ≥ 20% (with at least 5 foci/cell). RAD51 was quantified by scoring the percentage of geminin-positive cells with ≥5 foci/cell. Scoring was performed blindly by two independent investigators. One-hundred geminin-positive cells from at least 3 representative areas of each sample were analyzed. Three biological replicates of each PDX model (both vehicle- and olaparib-treated) were studied. Immunofluorescence images were acquired by using Olympus DP72 microscope and CellSens Entry software. For ATM staining, tissues were additionally blocked in endogenous peroxidase block (3% H2O2 in dH2O) for 10 min and antibody detection was performed with DAKO Envision HRP for 30 min with visualization using DAKO DAB solution for 10 min and counterstaining in Carazzi’s haematoxylin. ATM was scored by image analysis using Indica Labs HALO, by automated tissue classification of tumor regions and analysis of percentage positive nuclei in tumor only. For the pharacodynamic studies, the extent of DNA damage in the tumor samples was scored by the percentage of geminin-positive cells with γH2AX foci, as described. PanγH2AX was quantified by scoring the percentage of pan-nuclear γH2AX-positive cells.

Independent cohort for RAD51 assay validation The cohort consisted of gBRCA metastatic patients treated at the Vall d’Hebron University Hospital within clinical trials testing PARP inhibitors in monotherapy or combination, with

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FFPE material representative of the disease and signed IRB-approved informed consent form.

PDX protein isolation and immunoblotting Flash-frozen pieces of tumor were lysed in ice-cold buffer containing Tris–HCl pH 7.8 20 mmol/L, NaCl 137 mmol/L, EDTA pH 8.0 2 mmol/L, NP40 1%, glycerol 10%, supplemented with NaF 10 mmol/L, Leupeptin 10 mg/mL, Na2VO4 200 mmol/L, PMSF 5 mmol/L, and Aprotinin (Sigma-Aldrich). Homogenization was performed on ice with a POLYTRON® system PT 1200 E (Kinematica). Lysates were centrifuged at 13.000 rpm 4ºC during 30 min and the supernatants were collected. Protein concentration was calculated using DCTM Protein Assay (Bio-Rad). A total of 30 µg of protein were separated on 12% SDS-PAGE at 100V and transferred to nitrocellulose membrane during 1.5h at 100V. Membranes were blocked for 1 hour in 5% milk in Tris-buffered saline (TBS)-Tween and then hybridized using the primary antibodies in 5% BSA TBS-Tween: rabbit anti-RAD51 (Santa Cruz Biotechnology H-92, 1:500), rabbit anti-hGAPDH (Abcam ab128915, 1:5000), mouse anti-tubulin (Sigma-Aldrich T-9026, 1:5000). Mouse and rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; GE Helthcare) were diluted in 5% milk in TBS-Tween and proteins were detected with Immobilon Western Chemiluminiscent HRP substrate (Millipore). Images were captured with a FUJIFILM

LASS-4000

camera

system

and

quantified

with

Image

J

(http://rsb.info.nih.gov/ij/). For BRCA1, tumor lysates were made in RIPA lysis buffer complemented with 2X Complete protease inhibitor cocktail (Roche) and Pefabloc (Roche; 1mg/ml). Western blots were performed as described [5].

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Exome sequencing All laboratory methods were performed using the manufacturer’s protocols. Genomic DNA was isolated from fresh frozen PDX tissue using the Promega Maxwell 16® Tissue SEV DNA Purification Kit (catalog #AS1030) and the Maxwell® 16 MDx Instrument (Promega Corp., Madison, WI, USA). Specifically, samples were loaded into well #1 of the Maxwell cartridge, run using the “DNA/tissue” protocol, and genomic DNA was eluted with 300 µl Elution Buffer. All samples were quantified using the Qubit® dsDNA HS Assay Kit (catalog #Q32851) and Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Exome libraries were constructed using the KAPA Hyper Prep Library Preparation Kit (Kapa Biosystems Inc., Wilmington, MA, USA) and genes were captured using the xGen® Exome Research Panel v1.0 (Integrated DNA Technologies, Coralville, IA, USA). Paired end 150 bp sequencing was performed on an Illumina HiSeq 4000 using TruSeq SBS reagents (Illumina) with approximately 10 Gbp per sample for ~200-fold average sequence depth. Data analysis followed standard methodologies. Briefly, sequencing reads were aligned to both human hg19 and mouse mm10 genomes using BWA, then mouse-derived sequences in the human .bam file were removed using Disambiguate[6]. Variants were called in the human .bam files using VarDict [7]. Copy number analysis was performed using Seq2C [8].

Statistical analysis All statistical tests were performed with GraphPad Prism version 7.0. Paired, unpaired t test (two-tail) or one-way ANOVA were used and P values given. For treatment efficacy

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comparisons of the in vivo experimental data, two-way ANOVA was used. Error bars represent SEM of at least three biological replicates, unless otherwise stated.

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SUPPLEMENTARY FIGURE AND TABLE LEGENDS: Supplementary Table 1. Patient’s and PDX’s characteristics. 13 models were derived from 12 patients harboring germline BRCA1/2 mutations (samples at early disease or metastatic setting, with or without prior exposure to PARPi). PDX230OR derives from PDX230 at PARPi progression. The patient characteristics are summarized, including the germline BRCA1/2 mutation, prior therapies and response to PARPi when available. The PDX information include timepoint of implantation, response to PARPi, loss of heterozygosity in BRCA1 (LOH), genetic reversions in BRCA1 and relevant mutations found in the PDXs. For missense mutations, the variant allele frequency and functional effect is indicated. HGVS, Human Genome Variation Society. TNBC, triple negative BC. Met, metastasis. Pap, papillary. OvCa, ovarian cancer. ER+, estrogen receptor positive BC. Adj, adjuvant; NeoAdj, neoadjuvant. SD, stable disease. PD, progressive disease. CR, complete response.

Supplementary figure 1. Confirmation of the germline BRCA1/2 mutations in the PDX models. Orthogonal validation of the germline BRCA1/2 mutations identified by MiSeq using Sanger sequencing and/or MLPA is shown. PDX127 is shown as an example for the BRCA1 mutation c.68_69delAG, also identified in PDX179 and PDX230. Of note, PDX274 lacks the germline BRCA1 c.211A>G mutation in exon 5 due to an out-of-frame deletion of exons 3-7 of BRCA1 in the pathogenic allele, unveiled by massively parallel sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). This deletion was confirmed at the somatic level in the original patient´s tumor and thus was not acquired in the PDX. The out-of-frame deletion of BRCA1 exons 3-7 in PDX274 was predicted to cause a premature stop codon and a non-functional protein, thus it is not a secondary

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mutation restoring the open reading frame (Supplementary Table 1). Electropherograms are shown. Arrows indicate the specific mutations. Vertical lines indicate exon-intron boundaries, when applicable.

Supplementary figure 2. The acquired-resistance PDX model PDX230OR derives from PDX230 after prolonged exposure to olaparib. The tumor volume of PDX230 treated with olaparib (50mg/kg, 6 days/week) is plotted. For vehicle-treated tumors, the mean +/- SEM is represented (squares). The individual tumors of the olaparib arm are shown (different triangle patterns). The PDX230OR model was established by implanting the tumor overgrowing with olaparib treatment (black triangles).

Supplementary figure 3. Sensitivity of the PDX vs. the patient’s clinical response to olaparib. Time to PARPi progression of patients and PDXs is shown. Blue bars, time point at which the % of tumor volume change was ≥20% following olaparib treatment in the PDX models. Patients’ time on PARPi therapy until progression is plotted in light grey bars (Pt127, Pt179 and Pt124). Blue bars show mean +/- SEM of at least 3 individual tumors.

Supplementary figure 4. Analysis of BRCA1 mRNA expression by quantitative RTPCR. BRCA1 mRNA of the gBRCA PDX models measured by real-time qRT-PCR is represented and compared to the mean of two non-gBRCA TNBC PDX models. Mean +/- SEM of biological triplicates is represented. The domain of the BRCA1 mutation is indicated. PDX280 harbors a complete deletion in BRCA1.

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Supplementary figure 5. Setup of HRR markers by IF in FFPE samples from breast cancer PDX tumors selectively detects DNA damage proteins. A) IF of γH2AX and RAD51, markers of DNA DSBs and HRR functionality, respectively, following cisplatin or olaparib treatment. PDX131 is a luminal B BRCA1/2-wild type breast cancer model. PDX94 is a triple negative BRCA1/2-wild type breast cancer model. B) Assessment of the HRR markers BRCA1 (with antibodies towards the N-terminus of BRCA1 (B1-NT) or C-terminus (B1-CT)) and RAD51 in G2/S-phase cells (gemininpositive cells) by IF in FFPE samples as in A). DAPI staining is shown in blue.

Supplementary figure 6. Assessment of HRR markers by IF in FFPE samples from the gBRCA1 PDX196 model, harboring an exon 11 mutation. A) Detection of hypomorphic BRCA1-∆11q isoform in G2/S-phase cells (gemininpositive) by IF in control or olaparib-treated FFPE samples using an antibody towards the N-terminus of BRCA1 (B1-NT) or C-terminus (B1-CT). B) Detection of γH2AX, a marker of DNA DSBs induced by olaparib and the HRR protein RAD51. Co-localization is shown (merge). DAPI staining is shown in blue.

Supplementary figure 7. Western blot of hypomorphic BRCA1 isoforms. WB detected with an antibody towards the exon 11 of BRCA1 (B1-exon 11); loading control is GAPDH. PDX179, STG316 and PDX274 express a low molecular weight BRCA1 isoform. BRCA1 nuclear foci were observed exclusively with the B1-CT antibody in these models, consistent with the BRCA1 RING-less protein isoform being expressed

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(Fig. 1B). Remarkably, PDX274 is able to express a RING-less BRCA1 isoform due to a preserved translation initiation downstream of the BRCA1 3-7 exon deletion. BRCA1 isoforms are detected in two out of the three C-terminal BRCA1 mutant models, PDX221 and PDX236. This is consistent with the detection of BRCA1 nuclear foci with the B1-NT antibody but not with the B1-CT BRCA1 antibody (Fig. 1B), and the hypothetical stabilization of the gBRCA1 mutant protein via HSP90.

Supplementary figure 8. RAD51 amplification and overexpression in PDX124. A) Copy number plots for chromosome 15 (RAD51 is marked) in PDX124. B) Western blot of RAD51 and quantification relative to human GAPDH in PDX124 and other gBRCA models. One-way ANOVA: *, P < 0.05.

Supplementary figure 9. Hypomorphic BRCA1 isoforms co-localize with RAD51. FFPE tumor samples from the indicated PDXs were co-stained for BRCA1 (red) and RAD51 (green). Right panels show the merge images. The domain of the BRCA1 mutation is indicated.

Supplementary figure 10. Quantification of γH2AX nuclear foci. DNA damage following treatment with olaparib was quantified in sensitive (SD+CR) and resistant (PD) PDXs, as scored as the percentage of geminin-positive cells with γH2AX nuclear foci staining. Paired t test: ****, P<0.0001; *, P <0.05. Veh, vehicle; Olap, olaparib.

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Supplementary figure 11. RAD51 and γH2AX foci in patient’s tumor samples. A) Quantification of RAD51 and γH2AX in treatment-naïve patient tumor samples, and in tumor samples with prior treatment. Ten treatment-naïve primary breast tumors from gBRCA1/2 patients, either from the diagnostic biopsy (N=6) or the surgery specimen (N=4) were scored for RAD51 and B) γH2AX. Ten primary or metastatic breast tumors with prior treatment from gBRCA1/2 patients, either from their surgery specimen after having received conventional neoadjuvant chemotherapy (N=3) or from the metastatic setting (N=7), were scored for RAD51 and B) γH2AX. In total, we analyzed 10 BRCA1- plus 10 BRCA2-mutated tumors. Paired t test: not significant. C) IF of RAD51 and geminin in FFPE patient’s tumor samples and corresponding PDXs. Empty arrowheads show gemininpositive cells devoid of RAD51 staining (RAD51-). Solid arrowheads indicate double RAD51/geminin-positive cells (RAD51+). DAPI staining is shown in blue.

Supplementary figure 12. Genomic alterations acquired in two BRCA2 mutation carriers after PARPi treatment. IGV (Integrative Genomics Viewer) views of tumor exome sequencing results showing the BRCA2 gene from patients Pt310 and Pt201, two BRCA2 mutation carriers. Pt310 achieved a sustained PR for 17 months to the PARPi talazoparib and Pt201 achieved a sustained PR for >2 years after cisplatin plus olaparib followed by olaparib monotherapy maintenance. Of note, tumor cellularity in Pt310’s post-treatment (post-) tumor sample was 30%, and its pre-treatment (pre-) counterpart was 95%.

Supplementary figure 13. Targeting ATM in PARPi-resistant PDXs.

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A) ATM expression in PDXs by immunohistochemistry. Percentage of ATM-expressing tumor cells in the PDXs quantified by immunohistochemistry staining. B) Sanger sequencing of PRCC (DNA and retrotranscribed RNA) from PDX127. C) RAD51- and D) γH2AX-foci formation in PDX127 treated with olaparib, the ATM inhibitor AZD0156 and the combination of olaparib and AZD0156. Mean +/- SEM are shown. Two-way ANOVA: *, P<0.05; **, P<0.01.

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Figure 1. HRR markers and PARPi response. A) Antitumor activity of olaparib in gBRCA PDXs. Best response to olaparib is plotted as the percentage of tumor volume change after at least 21 days of treatment. Striped bars, model derived from a PARPi-treated patient. +20%, -30% and -95% are marked by dashed lines to indicate the range of CR (complete response), PR (partial response), SD (stable disease) and PD (progressive disease). Mut, mutation; B1, mutation in BRCA1; B2, mutation in BRCA2; Metastatic, PDX derived from a metastatic lesion (otherwise, derived from a primary tumor); TNBC, triple negative BC; ER+: estrogen receptor positive BC; OvCa, ovarian cancer. B) Immunofluorescence staining of BRCA1, 53BP1 and RAD51 across the PARPi-sensitive and PARPi-resistant gBRCA PDX models. Detection of BRCA1 (with an antibody towards the N-terminus of BRCA1 (B1-NT) or C-terminus (B1-CT)), 53BP1 and RAD51 nuclear foci in olaparib-treated PDX models. CR, PD, SD are indicated. For BRCA1, the location of the mutation within the gene is indicated. DAPI staining is shown in blue. Green nuclei indicate geminin-positive cells (S/G2 phase of the cell cycle). C) RAD51 nuclear foci formation discriminates PARPi-resistant tumors. Quantification of geminin positive cells with RAD51 nuclear foci detected by IF in FFPE samples from tumors

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treated with vehicle (black bars) or olaparib (green bars). The graph displays mean +/- SEM from three independent tumors. The association with PARPi-response is shown in the table below. Dark grey, PD; light grey, SD; white, CR. For RAD51, dark grey means high RAD51 score; light grey, intermediate RAD51; white, low RAD51. Expression of hypomorphic BRCA1 isoforms and loss of 53BP1 is depicted in dark grey. D) RAD51 in patient’s tumors is associated with PARPi clinical response. IF of RAD51 and geminin in the pretreatment setting using a pre-treatment tumor sample (or the most recent metastatic sample). Samples from three PARPi-resistant patients (Pt179, skin metastasis of TNBC; Pt183, dermal lymphatic carcinomatosis of ovarian cancer; Pt034, lymph node metastasis of ER+ BC) and four PARPi-sensitive patients (Pt310pre, liver metastasis of ER+ BC; Pt124pre, primary TNBC; Pt280, peritoneal implant of ovarian cancer; Pt04, lymph node metastasis TNBC) are shown. For acquired resistance, samples obtained from three patients at PARPi progression (Pt310post, liver metastasis; Pt124post, skin metastasis; Pt201, skin metastasis of ER+ BC) are shown. Empty arrowheads show geminin-positive cells devoid of RAD51 staining. Solid arrowheads indicate RAD51/geminin-positive cells. DAPI staining is shown in blue. E) Quantification of RAD51/geminin-positive cells from tumor samples shown in panel D. Pt183 was not scored as the tumor did not contain 100 geminin-positive cells. Unpaired t test: *, P<0.05; **, P<0.01. 190x274mm (284 x 284 DPI)

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Figure 2. Cisplatin or ATM inhibition overcomes PARPi resistance. A) Relative tumor volume (RTV) of vehicle, cisplatin or olaparib in PDX196, PDX280 and PDX252. Cisplatin was administrated 6mg/kg weekly unless RTV<0.5. Olaparib was administrated daily at 50 mg/kg (5 doses/week). N of tumors per arm is indicated. B) Relative tumor volume of vehicle, cisplatin, olaparib or its thereof combination in PDX236 and PDX274. Cisplatin and olaparib were administrated as in panel A). C) Relative tumor volume of vehicle, olaparib, AZD0156 or the combination of treatments in PDX127, STG316 and PDX280. Olaparib was administrated daily at 50 mg/kg (5 doses/week) and AZD0156 was administered three times per week at 2 or 2.5 mg/kg. D) Quantification of pan-nuclear γH2AX-positive cells in PDX127, STG316 and PDX280 treated with vehicle, olaparib, AZD0156 or the combination of drugs at the endpoint of experiments shown in panel C). All figures show mean and SEM. Statistical P-values are shown when relevant: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (two-way ANOVA). 190x254mm (300 x 300 DPI)

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PDX

Patient Diagnosis

implanted at progression to PARPi

PARPi treated

implanted prior to exposure to PARPi

PARPi treatment

ID

Sample origin

Germline BRCA1/2 mutation (HGVS)

BRCA1

TNBC Skin Met

c.1961delA

Pt127

TNBC Skin Met

BRCA1

BRCA1

c.68_69delAG

p.Glu23Valfs*17

Pt179

TNBC sc. Met

Pt196

Pap. Serous OvCa Pericardial Met

Pt280

Pap. Serous OvCa Lymph node Met

Pt221

TNBC Primary

Pt230

TNBC Primary

BRCA1

c.68_69delAG

p.Glu23Valfs*17

BRCA1 Complete deletion

BRCA1

BRCA1

c.5027delT

p.Leu1676*

BRCA1

BRCA1

c.68_69delAG

p.Glu23Valfs*17

1. NeoAdj doxorubicin + cyclophosphamide ‘ docetaxel

PDX127

Prior to cisplatin and Primary PARPi combination resistance

PD

Bilateral metachronic BC. 1. 1st Adj: epirubicin + cyclophosphamide + 5FU; 2. 2nd. Adj: capecitabine; 3. cisplatin; 4. cisplatin

PDX179

Prior to cisplatin and Primary PARPi combination resistance

PD

PDX196

At PARPi progression

Acquired resistance

PD

1. Adj carboplatin + paclitaxel; 2. carboplatin + paclitaxel; 3. liposomal doxorubicin; 4. paclitaxel + bevacizumab; 5. cisplatin + olaparib ‘ olaparib monotherapy; 6. carboplatin + pegylated liposomal doxorubicin; 7. gemcitabine; 8. paclitaxel

PDX280

At PARPi progression

Acquired resistance

PD

1. NeoAdj doxorubicin + cyclophosphamide + 5FU → paclitaxel

PDX221

Post-neoadjuvant chemotherapy

Untreated

PD

PDX230

Post-neoadjuvant chemotherapy

Untreated

CR

1. NeoAdj non-pegylated liposomal doxorubicin + cyclophosphamide ‘ paclitaxel

PARPi untreated

1. NeoAdj doxorubicin + cyclophosphamide + docetaxel; BRCA1 BRCA1 2. paclitaxel + gemcitabine; c.5194-12G>A p.His1732Phefs*5 3. capecitabine; 4. pegylated irinotecan (NKTR102)

TNBC Skin Met

Pt252

TNBC Lymph node Met

Pt274

TNBC Skin Met

STG316

TNBC Primary

BRCA1

BRCA1

c.134+3A>C

p.Cys27*

Pt71

ER+BC Primary

BRCA1

BRCA1

c.5123C>A

p.Ala1708Glu

BRCA1

BRCA1

c.211A>G

p.Cys64*

BRCA2

BRCA2

c.3264dupT

p.Gln1089Serfs*10

PDX

SD

mutation (HGVS)

BRCA1 c.1961delA

BRCA1 c.68_69delAG

BRCA1 c.68_69delAG

BRCA1 c.1961delA

Genetic Reversion

RAD51 PARP1

TP53BP1

MAD2L2 (REV7)

PAXIP1 (PTIP)

Artemis

Complete deletion

BRCA1 c.5027delT

SLFN11

CHD4

ATM gene

protein (IHC, HScore)

None

62

NA

p.I1547T, 0.52, unknown

136

3 copies, not focal

High

None

116

None

None

Low

p.V245I, 0.22, unknown; p.C532Y, 0.71, unknown

178

p.H661D, 0.97, unknown

None

None

NA

None

66

None

None

None

None

NA

None

13

None

None

None

None

None

Low

None

22

None

None

None

None

None

None

NA

None

48

None

None

None

None

None

None

None

NA

None

57

None

None

None

None

None

None

None

None

NA

p.R919T, 0.29, unknown

45

No

None

None

None

None

None

None

None

None

NA

None

140

LOH

No

None

deletion exons 12-16

None

None

None

None

None

None

NA

None

52

LOH

No

None

None

None

None

None

None

None

None

NA

None

65

LOH

BRCA1/2

LOH

No

None

None

None

(4 copies)

None

None

None

LOH

No

None

None

None

None

None

None

None

None

LOH

No

None

None

None

None

None

None

None

LOH

No

None

None

None

None

None

None

LOH

No

None

None

None

None

None

LOH

No

None

None

None

None

LOH

No

None

None

None

LOH

No

None

splice; c.5294_5305 +33del45

LOH

No

None

LOH

No

LOH

gene

BRCA1

c.68_69delAG NA

PD

PDX236

Post-pegylated irinotecan

Untreated

PD

1. NeoAdj epirubicin + docetaxel; 2. carboplatin + gemcitabine; 3. lurbinectedin (PM01183)

PDX252

Post-lurbinectedin

Untreated

PD

1. NeoAdj doxorubicin + cyclophosphamide ‘ docetaxel 2. Adj carboplatin + gemcitabine; 3. eribulin; 4. pegylated liposomal doxorubicin

PDX274

Post-pegylated liposomal doxorubicin

Untreated

PD

1. NeoAdj docetaxel x3, epirubicin + cyclophosphamide + 5FU x1, cisplatin x1, carboplatin x2

STG316

Post-carboplatin

Untreated

PD

None

BRCA1/2

protein (WB)

6 copies, Very high focal

BRCA1 PDX230OR derived from PDX230

Pt236

Patient

Sensitive (PR)

1. NeoAdj/Adj carboplatin + paclitaxel; 2. carboplatin + paclitaxel; 3. carboplatin + paclitaxel; p.Lys654Serfs*47 4. pegylated liposomal doxorubicin; 5. olaparib

p.0

Timepoint of implantation

Prior to cisplatin and PARPi combination

BRCA1

BRCA1

#

PDX124

BRCA1

BRCA1

c.1961delA

Prior therapies

1. NeoAdj doxorubicin + docetaxel; 2. Adj cisplatin + vinorelbin; 3. paclitaxel + bevacizumab; p.Lys654Serfs*47 4. vinorelbine + capecitabine + bevacizumab

Pt124

BRCA1

PDX Response to Olaparib

Predicted BRCA1/2 protein

PDX071

Treatment naïve

BRCA1 c.5194-12G>A

BRCA1 c.5123C>A

BRCA1

Untreated

CR

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deletion exons 3-7

BRCA1 c.134+3A>C

BRCA2 c.3264dupT