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Choosing wisely: Selecting PARP inhibitor combinations to promote anti-tumor immune responses beyond BRCA mutations

Open AccessPublished:October 17, 2019DOI:https://doi.org/10.1016/j.ygyno.2019.09.021

      Highlights

      • PARP inhibitors (PARPi) have transformed the management of advanced ovarian cancer.
      • With increased PARPi use, the resistance to PARPi will become a increasing clinical concern.
      • Combinatorial approaches can overcome de novo and acquired resistance to PARPi by exploiting DDR mechanisms.
      • PARPi combinations can induce “BRCAness”, inhibit DNA repair, promote replication stress, or enhance immunomodulation.
      • Selection of the appropriate combination for the clinical context could overcome, or prevent, PARPi resistance.

      Abstract

      PARP inhibitors have transformed the management of advanced high-grade serous ovarian cancer. Despite the overwhelming success of PARP inhibition, particularly in BRCA-mutated ovarian cancer, several limitations and unanswered questions remain. With PARP inhibitors now being used in earlier treatment settings, the issue of both de novo and acquired resistance mechanisms and appropriate post-PARP management are pressing concerns. In addition, the population appropriate to target with PARP inhibitors and their use in patients without BRCA mutations is controversial and evolving. In this review we will discuss exciting PARP combinations and biologic rationale for the development and selection of PARP inhibitor combinations.

      Keywords

      1. Introduction

      Treatment of ovarian cancer with PARP inhibitor monotherapy has been enormously successful, particularly in patients with germline or somatic alterations in BRCA1 and BRCA2. PARP combination therapies are under active development in an effort to deepen responses in patients with BRCA-mutated tumors, and to overcome resistance to PARP inhibitors in patients with de novo or acquired resistance. Combinations of PARP inhibitors with chemotherapy, anti-angiogenic agents, immunotherapy, and PI3Kinase inhibitors, and other components of the DNA damage repair machinery are areas of active therapeutic investigation and exciting combinations are emerging. The focus of this review is to highlight novel combinations using PARP inhibitors.
      At present, 4 PARP inhibitors are FDA-approved for the treatment of patients with breast or ovarian cancer – olaparib, rucaparib, niraparib, and talazoparib (Table 1). In October 2018, olaparib was approved for use as frontline maintenance therapy in ovarian cancer patients with germline or somatic BRCA1 or BRCA2 mutations following adjuvant chemotherapy, based on SOLO-1 which demonstrated a 3-year improvement in progression free survival (PFS) compared to placebo [
      • Moore K.
      • et al.
      Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer.
      ]. Other indications for the use of PARP inhibitors in ovarian cancer include approval as monotherapy in patients with recurrent ovarian cancer and deleterious BRCA mutations. For patients with ovarian cancer with or without BRCA mutations, olaparib, rucaparib, and niraparib are all approved for the maintenance treatment of recurrent platinum-sensitive ovarian cancer.
      Table 1FDA approvals for PARP inhibitor monotherapy.
      OlaparibRucaparibNiraparibTalazoparib
      Frontline maintenancegermline or somatic BRCA1/2 mutation------
      Recurrent maintenancePlatinum-sensitive maintenancePlatinum-sensitive maintenancePlatinum-sensitive maintenance--
      Recurrent treatmentGermline BRCA1/2 mutation, 3 priorsGermline or somatic BRCA1/2 mutation, 2 priors----
      Non-ovarian indicationsBreast cancer (germline BRCA1/2 mutation)----Breast cancer (germline BRCA1/2 mutation)
      Monotherapy dose300 mg BID (tab)

      400 mg BID (capsule)
      600 mg BID300 mg daily1 mg daily
      Ongoing and recently completed phase III studies are further expanding the use of PARP inhibitor monotherapy. The phase III PRIMA study investigating the use of frontline niraparib maintenance in patients with ovarian cancer in patients with and without BRCA mutations has completed accrual and results anticipated in late 2019. PARPs have also demonstrated success beyond ovarian cancer. Olaparib [
      • Robson M.E.
      • et al.
      OlympiAD final overall survival and tolerability results: olaparib versus chemotherapy treatment of physician’s choice in patients with a germline BRCA mutation and HER2-negative metastatic breast cancer.
      ] and talazoparib [
      • Litton J.K.
      • et al.
      Talazoparib in patients with advanced breast cancer and a germline BRCA mutation.
      ] are approved for the treatment of locally advanced or metastatic breast cancer in patients with germline BRCA mutations. In addition, PARP inhibitors have demonstrated efficacy as maintenance therapy in BRCA-mutated pancreatic cancer in the recently reported phase III POLO study [
      • Golan T.
      • et al.
      Maintenance olaparib for germline.
      ] and as monotherapy in prostate cancer in patients with alterations in DNA damage repair genes [
      • Mateo J.
      • et al.
      DNA-repair defects and olaparib in metastatic prostate cancer.
      ].

      2. Predicting response to PARP inhibitor monotherapy

      PARP1 and PARP2 repair single-strand DNA (ssDNA) breaks. PARP1 also repairs double strand DNA (dsDNA) breaks and stalled replication forks. During DNA replication, PARP1 binds to ssDNA, relaxing the chromatin and allowing recruitment of other DNA damage repair proteins. Auto-PARylation by PARP catalytic activity releases PARP from the DNA permitting access of additional repair proteins and resolution of DNA breaks and stalled replication forks [
      • Pommier Y.
      • O’Connor M.J.
      • de Bono J.
      Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action.
      ,
      • Konstantinopoulos P.A.
      • et al.
      Homologous recombination deficiency: exploiting the fundamental vulnerability of ovarian cancer.
      ]. PARP inhibitors antagonize PARP function by inhibiting PARP catalytic activity and by trapping PARP on the DNA. Despite similar potency in inhibiting PARP catalytic function, PARPs vary in PARP trapping ability, with talazoparib having the most PARP trapping ability and veliparib the least, with intermediate PARP trapping of olaparib, niraparib, and rucaparib. These differences may be helpful in combination therapy strategies to modulate toxicity and promote therapeutic synergy.
      The use of PARP monotherapy in these HR-deficient settings exploits the synthetic lethal interaction between PARP inhibition and HR-deficiency. Responses in HR-proficient cancers and platinum-resistant cancers have been far more modest. Gelmon et al. assessed the impact of mutations in BRCA1 and BRCA2 and platinum-sensitivity on response to olaparib in recurrent ovarian cancer or TNBC. Among patients with platinum-sensitive recurrent ovarian cancer, 60% of those with BRCA1 and BRCA2 mutations and 50% of those without BRCA1 or BRCA2 mutations had an objective response [
      • Gelmon K.A.
      • et al.
      Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.
      ]. Of those with platinum-resistant tumors, 33% with BRCA1 and BRCA2 mutations had an objective response, compared to only 4% (1 of 26) without a germline BRCA mutation (Table 2). This is not unique to olaparib, and similar relationships have been identified between responses and BRCA mutation status with rucaparib [
      • Oza A.M.
      • et al.
      Antitumor activity and safety of the PARP inhibitor rucaparib in patients with high-grade ovarian carcinoma and a germline or somatic BRCA1 or BRCA2 mutation: integrated analysis of data from Study 10 and ARIEL2.
      ,
      • Swisher E.M.
      • et al.
      Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): an international, multicentre, open-label, phase 2 trial.
      ] and niraparib [
      • Moore K.N.
      • et al.
      Niraparib monotherapy for late-line treatment of ovarian cancer (QUADRA): a multicentre, open-label, single-arm, phase 2 trial.
      ]. In addition to platinum-response and BRCA mutation status, the number of prior regimens influences likelihood of response, with ORR declining as the number of prior regimens increases [
      • Matulonis U.A.
      • et al.
      Olaparib monotherapy in patients with advanced relapsed ovarian cancer and a germline BRCA1/2 mutation: a multistudy analysis of response rates and safety.
      ] (Table 2).
      Table 2Determinants of response to PARP inhibitor monotherapy in ovarian cancer.
      StudyPARPiPriorsOvarian CancerBRCA1/2 mutantBRCA Wild type
      Gelmon et al. (8)Olaparib3 (1–10)Plt-sensitive60%50%
      Plt-resistant33%4%
      Matulonis et al. (12)Olaparib≥1Plt-sensitive48%
      Plt-resistant28%
      Oza et al. ARIEL2/Study 10 (9)Rucaparib≥2Plt-sensitive66%
      Plt-resistant25%
      Swisher et al. ARIEL2 (10)Rucaparib≥1Plt-sensitive80%29% (LOH high),

      10% (LOH low)
      Moore et al. QUADRA (11)Niraparib≥3Plt-sensitive39%26% (HRD-pos)

      4% (HRD-neg)
      Plt-resistant27%10% (HRD-pos)

      3% (HRD-neg)
      PARPi: PARP inhibitor. Plt: platinum. LOH: loss of heterozygosity. HRD-pos: homologous recombination deficiency positive. HRD-neg: HRD negative.

      3. Chemotherapy combinations

      Chemotherapeutic agents may synergize with PARP inhibitors by enhancing PARP catalytic activity, enhancing PARP trapping, or by enhancing chemotherapy cytotoxicity through increased formation of double-strand DNA breaks. The DNA damaging properties of platinums, topoisomerase inhibitors, and DNA alkylating agents have all been exploited in PARP combinations. Table 3 summarizes PARP inhibitor-chemotherapy combination studies.
      Table 3Selected trials of PARP inhibitor plus chemotherapy combinations.
      StudyPhasePARPiChemoDoseCancerNotes
      Ramalingam et al. (14)Ph 2Veliparib 120 mg BID D1-D7Carboplatin

      Paclitaxel
      AUC 6, q21D

      200 mg/m2, q21D
      NSCLCNo difference PFS, OS v. carbo/tax
      Han et al. (15)Ph 2Veliparib 120 mg BID D1-D7Carboplatin

      Paclitaxel
      AUC 6, q21D

      175 mg/m2, q21D
      Breast (BRCA1/2)No difference PFS, OS v. carbo/tax, ORR increased
      Loibl et al. BrighTNess (16)Ph 3Veliparib 50 mg BIDCarboplatin

      Paclitaxel
      AUC 6, q21D

      80 mg/m2, q7D
      BreastNo difference pCR rate v. carbo/tax
      Thaker et al. (19)Ph 1Veliparib 400 mg BID D1-7Cisplatin

      Paclitaxel
      50 mg/m2, q21D

      175 mg/m2, q21D
      CervixMTD not reached, ORR 7%
      NCT02470585 (VELIA)#Ph 3Veliparib 150 mg BIDCarboplatin

      Paclitaxel
      AUC 6, q21D

      175 mg/mg2, q21D
      Ovary
      Plummer et al. (20)Ph 2Rucaparib 12 mg/m2 (IV)Temozolamide200 mg/m2 D1-5, q28Dmelanomamyelosuppression 54%
      LoRusso et al. (25)Ph 1Veliparib 40 mg BID D1-14Irinotecan100 mg/m2 D1, D8, q21DOvaryORR 19%
      NCT01012817#Ph 2Veliparib 300 mg BID D1-3, 8–10, 15-17Topotecan3 mg/m2 D2, 9, 16, q28DOvary
      Kunos et al. (28)Ph1/2Veliparib 10 mg BIDTopotecan0.6 mg/m2 D1-5, q21DCervixORR 7%, myelosuppression 59%
      Ph: phase. AUC: Area under the curve. MTD: maximum tolerated dose. pCR: pathological complete response. BID: twice daily. D: days. #: ongoing clinical trial.

      3.1 Platinums

      Tumors with HR-deficiency are highly sensitive to platinum analogues. Platinum agents enter the cell and bind to neutrophilic groups containing nitrogen, oxygen, and sulfur thus readily bind nucleic acids and form platinum-DNA adducts and inter- and intra-strand DNA crosslinks. These complexes interfere with transcription and DNA replication, and lead to activation of DNA damage repair pathways, including base excision repair (BER) and HR [
      • Martin L.P.
      • Hamilton T.C.
      • Schilder R.J.
      Platinum resistance: the role of DNA repair pathways.
      ]. By combining PARP inhibition with targeting of BER by platinum considerable synergy could be generated. Several combinations in clinical trials have been performed, however dosing has been limited by overlapping toxicities, particularly myelosuppression. Thus, attenuated doses of either PARP inhibitor or the platinum have often been needed to improve tolerability, potentially at the expense of efficacy.
      Veliparib has less trapping ability than the other clinically available PARP inhibitors, and therefore may be better tolerated in chemotherapy combination studies. In a small randomized phase II study, 160 patients with non-small cell lung cancer (NSCLC) were treated with the combination of carboplatin (AUC 6) and paclitaxel (200 mg/m2) on day 3 with veliparib 120 mg or placebo on days 1–7 of a 3-week cycle [
      • Ramalingam S.S.
      • et al.
      Randomized, placebo-controlled, phase II study of veliparib in combination with carboplatin and paclitaxel for advanced/metastatic non-small cell lung cancer.
      ]. Although the regimen was generally tolerated, no difference in PFS or OS was observed with veliparib at this attenuated schedule. In patients with recurrent BRCA1/2-mutated breast cancer, the addition of veliparib to carboplatin and paclitaxel increased overall response rate, but did not improve PFS or OS [
      • Han H.S.
      • et al.
      Veliparib with temozolomide or carboplatin/paclitaxel versus placebo with carboplatin/paclitaxel in patients with BRCA1/2 locally recurrent/metastatic breast cancer: randomized phase II study.
      ]. Similarly, in patients with high-risk triple negative breast cancer, addition of veliparib (given continuously at 50 mg twice daily) to carboplatin and paclitaxel did not improve outcomes [
      • Loibl S.
      • et al.
      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ].
      So far these platinum-PARP combinations have not demonstrated improved responses over chemotherapy alone, but all studies administer the PARP inhibitor well below the recommended monotherapy dose, which is veliparib 400 mg PO twice daily continuously [
      • Coleman R.L.
      • et al.
      A phase II evaluation of the potent, highly selective PARP inhibitor veliparib in the treatment of persistent or recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer in patients who carry a germline BRCA1 or BRCA2 mutation - an NRG Oncology/Gynecologic Oncology Group study.
      ]. Based on a successful dose-finding study [
      • Armstrong D.
      • et al.
      A phase I study of Veliparib incorporated into front-line platinum based chemotherapy and Bevacizumab in epithelial ovarian cancer (NCT00989651):a GOG/NRG trial.
      ], the ongoing randomized phase III VELIA study in ovarian cancer is incorporating veliparib at 150 mg twice daily continuously into standard frontline carboplatin and paclitaxel therapy (GOG-3005/VELIA/M13-694 trial (NCT02470585)). The study has accrued and results are pending. Veliparib has also been successfully incorporated into cisplatin and paclitaxel regimen in a phase I dose finding study in cervical cancer which achieved full doses of veliparib 400 mg twice daily for days 1–7 and MTD was not reached [
      • Thaker P.H.
      • et al.
      A phase I trial of paclitaxel, cisplatin, and veliparib in the treatment of persistent or recurrent carcinoma of the cervix: an NRG Oncology Study (NCT#01281852).
      ]. Despite the rational mechanism, success of PARP-platinum combinations are impaired by overlapping toxicities and attenuated dosing and are unlikely to be extensively developed.

      3.2 Alkylating agents (temozolomide)

      Some of the first uses of PARP inhibitor combinations were as chemosensitizing agents for temozolomide. Temozolomide is an alkylating agent that adds methyl-groups to guanines at O6 and N7 positions and adenines at N3 position. Single strand DNA breaks generated by N7 guanine methylation lead to PARP1 activation and recruitment of DNA repair factors. PARP inhibition prevents repair of temozolomide-induced ssDNA breaks, resulting in accumulated DNA damage. Here too, administration of the combination has been impaired by poor tolerance and overlapping toxicities. In a study of rucaparib and temozolomide in melanoma at the RP2D, 54% of patients required a dose reduction due to cytopenias [
      • Plummer R.
      • et al.
      A phase II study of the potent PARP inhibitor, Rucaparib (PF-01367338, AG014699), with temozolomide in patients with metastatic melanoma demonstrating evidence of chemopotentiation.
      ].
      These combinations may have new life in targeting primary CNS tumors. Methylguanine DNA methyltransferase (MGMT) resolves temozolomide-generated O6-methylguanine lesions. Approximately 60% of glioblastoma multiform (GBM) exhibit lack of methylation of the MGMT promoter and have high tumor levels of MGMT. Lack of MGMT methylation predicts a poor response to temozolomide and carries a poor prognosis. In the presence of PARP inhibitors, however, repair of N7-methylguanine and N3-methyladenine is impaired, restoring temozolomide-sensitivity in GBM without MGMT promoter hypermethylation. The OPARTIC study tested the hypothesis that olaparib could penetrate the disrupted GBM blood brain barrier to potentiate temozolomide-mediated cytotoxicity against GBM tumors, and detected olaparib in 95% of tumor core specimens (NCT01390571) [
      • Chalmers A.
      • et al.
      Phase I clinical trials evaluating Olaparib in combination with radiotherapy (RT) adn/or temozolomide (TMZ) in glioblastoma patients: results of OPARATIC and PARADIGM phase I and early results of PARADIGM-2.
      ]. A phase I/II study in combination with radiation, is now underway (NCT03212742).

      3.3 Topoisomerase inhibitors

      PARP inhibitors synergize with topoisomerase I inhibitors by potentiating topoisomerase-I-mediated DNA damage and cytotoxicity [
      • Pommier Y.
      • O’Connor M.J.
      • de Bono J.
      Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action.
      ]. Topoisomerase I proteins bind and cleave DNA, generating ssDNA breaks to relax DNA supercoils formed during transcription. Topoisomerase I poisons induce covalent binding of topoisomerase I to DNA 3’ phosphate group, thereby trapping topoisomerase I on the DNA, leading to stalled replication forks and formation of dsDNA breaks. High fidelity repair of the dsDNA breaks relies on intact HR pathways and PARP1 catalytic activity.
      This mechanism has been tested in cell culture and mouse model systems. Patel et al. demonstrated that PARP inhibitor veliparib could sensitize ovarian cancer cells to topoisomerase I poison at PARP concentrations achieved clinically [
      • Patel A.G.
      • et al.
      Enhanced killing of cancer cells by poly(ADP-ribose) polymerase inhibitors and topoisomerase I inhibitors reflects poisoning of both enzymes.
      ]. Similar synergy has been demonstrated with the combination of olaparib and irinotecan [
      • Tahara M.
      • et al.
      The use of Olaparib (AZD2281) potentiates SN-38 cytotoxicity in colon cancer cells by indirect inhibition of Rad51-mediated repair of DNA double-strand breaks.
      ] in colon cancer cells and in mouse xenograft models, supporting the dominant negative model. Further evidence has demonstrated that inhibition of PARP1 catalytic activity impairs stabilization and recruitment of tyrosyl-DNA-phosphodiesterase 1 (TDP-1), an enzyme required to cleave topoisomerase I covalent complexes from the DNA [
      • Das B.B.
      • et al.
      PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage.
      ]. This may explain why poor PARP trappers such as veliparib can result in synergy with topoisomerase I inhibitors even at low PARP concentrations.
      Several clinical studies have combined topoisomerase inhibitors with PARP inhibitors [
      • LoRusso P.M.
      • et al.
      Phase I safety, pharmacokinetic, and pharmacodynamic study of the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888) in combination with irinotecan in patients with advanced solid tumors.
      ,
      • Wahner Hendrickson A.E.
      • et al.
      A phase I clinical trial of the poly(ADP-ribose) polymerase inhibitor veliparib and weekly topotecan in patients with solid tumors.
      ,
      • Hjortkjaer M.
      • et al.
      Veliparib and topotecan for patients with platinum-resistant or partially platinum-sensitive relapse of epithelial ovarian cancer with BRCA negative or unknown BRCA status.
      ,
      • Kunos C.
      • et al.
      A phase I-II evaluation of veliparib (NSC #737664), topotecan, and filgrastim or pegfilgrastim in the treatment of persistent or recurrent carcinoma of the uterine cervix: an NRG Oncology/Gynecologic Oncology Group study.
      ] with limited success largely due to overlapping toxicity requiring attenuated dosing and schedule. In the phase I study of veliparib and topotecan in ovarian cancer, 45 patients were enrolled, of whom 35% harbored mutations in HR genes [
      • Wahner Hendrickson A.E.
      • et al.
      A phase I clinical trial of the poly(ADP-ribose) polymerase inhibitor veliparib and weekly topotecan in patients with solid tumors.
      ]. There was a trend toward increased responses in patients with tumor HR defects, however this was not statistically significant. Doses of up to ¾ of the individual MTDs for olaparib and topotecan were achieved with intermittent dosing schedule. A phase II study in platinum resistant ovarian cancer is underway (NCT01012817). Like other PARP-chemotherapy combinations, development of PARP inhibitor and topoisomerase inhibitor combinations is limited by overlapping toxicities.

      4. Combination with anti -angiogenesis agents

      Combinations with biologic agents are attractive on a mechanistic level and they also may avoid the issue of myelosuppression. An exciting pairing is the combination of PARP inhibitors with antiangiogenic agents. Clinical experience with these pairings is summarized in Table 4. The biological rationale stems from preclinical findings that demonstrate crosstalk between the hypoxic tumor microenvironment and HR repair .
      Table 4Selected trials of PARP inhibitor plus anti-angiogenesis agents.
      StudyPhaseControlCombinationCancerNotes
      Liu et al. (34)Ph 2Olaparib 400 mg BID (c)Olaparib 200 mg BID (c)

      Cediranib 30 mg daily
      Plt-sensitiveORR 47.8 v 79.6

      BRCA wt 32 v 76

      BRCA mut 63 v 84 (p = 0.19)
      Liu et al. (36)Ph 2Olaparib 200 mg BID (t)

      Cediranib 30 mg daily
      Plt-sensitive and resistantPlt-sensitive: 77%

      Plt-resistant: 20%

      Plt-resistant/BRCA wt: 18%
      NCT02446600,

      NRG-GY004#
      Ph 3SOC (platinum-doublet) v. Olaparib 300 mg BID (t)Olaparib 200 mg BID (t)

      Cediranib 30 mg daily
      Plt-sensitive
      NCT02502266,

      NRG-GY005#
      Ph 2/3SOC (non-platinum)Olaparib 200 mg BID (t)

      Cediranib 30 mg daily
      Plt-resistant
      NCT02889900,

      CONCERTO#
      Ph 2Olaparib 200 mg BID (t)

      Cediranib 30 mg daily
      Plt-resistant
      Lheureux et al. EVOLVE (37)Ph 2Olaparib 150 mg BID (t)

      Cediranib 20 mg daily
      Post-PARPORR 12% post-PARP
      Mirza et al. AVANOVA2 (40)Ph 3Niraparib 300 mg dailyNiraparib 300 mg daily

      Bevacizumab 15 mg/kg IV q21D
      Plt-sensitivePFS 5.5–11.2 mo
      NCT02477644,

      PAOLA-1#
      Ph 3PlaceboOlaparib 300 mg BID (t)

      Bevacizumab 15 mg/kg IV q21D
      Frontline maintenance
      NCT03462212,

      MITO25#
      Ph 1/2Rucaparib v. BevacizumabRucaparib 400–600 mg

      Bevacizumab 15 mg/kg IV q21D
      Frontline maintenance
      c: capsule. t: tablets.
      Antiangiogenic agents and PARP inhibitors have shown at least additive effects in preclinical model systems. Interruption of the tumor blood supply can lead to hypoxia in the tumor microenvironment. Hypoxic conditions alter gene transcription, and induce changes in gene expression. Expression of BRCA1 and RAD51C are reduced under hypoxic conditions generating impairing HR repair [
      • Bindra R.S.
      • et al.
      Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells.
      ,
      • Bindra R.S.
      • et al.
      Hypoxia-induced down-regulation of BRCA1 expression by E2Fs.
      ,
      • Lim J.J.
      • et al.
      VEGFR3 inhibition chemosensitizes ovarian cancer stemlike cells through down-regulation of BRCA1 and BRCA2.
      ]. Inhibition of VEGFR3 is also associated with decreased expression of BRCA1 and BRCA2 [
      • Lim J.J.
      • et al.
      VEGFR3 inhibition chemosensitizes ovarian cancer stemlike cells through down-regulation of BRCA1 and BRCA2.
      ], and the antiangiogenic agent cediranib was recently shown to directly represses BRCA1/2 and RAD51C via inhibition of PDGFR [
      • Kaplan A.R.
      • et al.
      Cediranib suppresses homology-directed DNA repair through down-regulation of BRCA1/2 and RAD51.
      ]. Therefore, agents that interfere with tumor blood supply and block VEGF signaling, may induce an HR-deficient state and promote responses to PARP inhibitors.

      4.1 Cediranib

      Cediranib is an orally available tyrosine kinase inhibitor with activity against VEGFR1, VEGFR2 and VEGFR3 as well as PDGFR. As a single agent, it has demonstrated responses of 17% or so in recurrent ovarian cancers [
      • Matulonis U.A.
      • et al.
      Cediranib, an oral inhibitor of vascular endothelial growth factor receptor kinases, is an active drug in recurrent epithelial ovarian, fallopian tube, and peritoneal cancer.
      ]. In a dose finding study of cediranib in combination with the PARP inhibitor olaparib, response rates of 44% were achieved in patients with recurrent ovarian cancer [
      • Liu J.F.
      • et al.
      A Phase 1 trial of the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) in combination with the anti-angiogenic cediranib (AZD2171) in recurrent epithelial ovarian or triple-negative breast cancer.
      ]. In the phase II study, patients with recurrent platinum-sensitive ovarian cancer were randomized to receive olaparib capsule at the RP2D of 400 mg twice daily versus the combination of olaparib capsules 200 mg twice daily with cediranib 30 mg daily [
      • Liu J.F.
      • et al.
      Combination cediranib and olaparib versus olaparib alone for women with recurrent platinum-sensitive ovarian cancer: a randomised phase 2 study.
      ]. An improvement in PFS was noted from 9 months (95% CI 5.7–16.5) for olaparib alone to 17.7 months (95% CI 14.7-NR) for the combination. Investigators also observed an increase in ORR from 47.8% in the olaparib group to 79.6% in the combination group, including 5 complete responses.
      Post hoc analysis of this study demonstrated substantial treatment heterogeneity between patients with and without BRCA mutations. In the 43 patients with wild type or unknown BRCA mutation status, patients randomized to the combination arm had prolonged PFS compared to those receiving olaparib alone (16.5 versus 5.7 months, respectively, HR 0.32, 95% CI 0.14–0.74, p = 0.008). The ORR was 32% for olaparib alone versus 76% in the combination. Women with BRCA mutations, however, did relatively well regardless of treatment assignment, with no difference in PFS in this small sample size (16.5 versus 19.4 months, HR 0.55 95% CI 0.24–1.27, p = 0.16) and objective responses observed in most patients (63% versus 84%, p = 0.19). One interpretation of these results is that addition of cediranib promotes responses to PARP inhibitors in HR-proficient tumors but does not add benefit for those with HR-deficient tumors.
      Investigations of the combination are ongoing in both platinum-sensitive and platinum-resistant recurrent ovarian cancer. In an NCI-sponsored phase II study, patients with platinum-sensitive and platinum-resistant recurrent ovarian cancer were enrolled to treatment with the combination; confirmed response rates of 74% in platinum-sensitive and 20% in platinum-resistant cancers were observed, including a response rate of 18% (4 out of 22) in patients with wild-type germline BRCA and platinum-resistant recurrence [
      • JF L.
      • et al.
      A phase 2 biomarker trial of combination cediranib and Olaparib in relapsed platinum sensitive and platinum resistant ovarian cancer.
      ]. A randomized phase III study of this combination in platinum-sensitive recurrent ovarian cancer of fully accrued in November 2017, but has not yet reported (NRG-004, NCT02446600). The CONCERTO trial (NCT02889900) and the NRG-sponsored randomized phase III study evaluate the combination in platinum-resistant BRCA wild type ovarian cancer (NRG-005, NCT02502266).
      An additional role for the olaparib and cediranib combination may be in the post-PARP setting, where patients have developed acquired resistance to PARP inhibitors and often have restored HR proficiency. In the EVOLVE study, 34 patients with progression after PARP inhibitors were enrolled to receive olaparib and cedirinib. There were 4 partial responses: 2 in the platinum-resistant ovarian cancer group, and 2 in the group who received an intervening chemotherapy regimen between PARP and study enrollment [
      • S L.
      • et al.
      Evolve: a post PARP inhibitor clinical translational Phase II trial of cediranib-olaparib in ovarian cancer - a Princess Margaret Consortium-GCIG Phase II trial.
      ], suggesting olaparib-cedarinib combination may be promising for patients with progression on prior PARP inhibitor.
      Cediranib has had a complex regulatory path in ovarian cancer and shares toxicities with other receptor-tyrosine kinase inhibitors (TKI)s. Other agents in this class (such as lenvatinib) are moving forward in gyn cancers and potentially paving a way for other oral TKIs like cediranib. Due to the potential of cedirinib to restore HR and the significant synergy observed with the combination in HR-proficient tumors, the olaparib-cedirinib combination is likely to play a future role in ovarian cancer and offer an all-oral option. The greatest utility of this combination may be in the platinum-resistant setting, enriched for patients with HR-proficient tumors with de novo PARP resistance, and in the post-PARP setting, where patients often have acquired resistance to PARP monotherapy. (Fig. 1).
      Fig. 1
      Fig. 1Choosing the right combination – PARP combination strategies. Multiple PARP inhibitor combinations are under development. Combinations exploit one of four key mechanisms to induce synergy with PARP inhibitors – 1) Induction of “BRCA-ness”, 2) Inhibition of DNA repair, 3) Immunomodulation, or 4) Induction of “replication stress-ness”. Relevant combinations and potential optimal demographic are listed.

      4.2 Bevacizumab

      PARP inhibition is also being combined with the anti-VEGFR antibody bevacizumab. Both olaparib and niraparib have been combined with bevacizumab in phase I studies, while maintaining their single agent recommended doses and schedules [
      • MR M.
      A phase 1 study of bevacizumab in combination with niraparib in patients with platinum-sesnstive epithelial ovarian cancer: the ENGOT-OV24/AVANOVA1 trial.
      ,
      • Dean E.
      • et al.
      Phase I study to assess the safety and tolerability of olaparib in combination with bevacizumab in patients with advanced solid tumours.
      ]. Results of the AVANOVA2 study were recently reported [
      • MR M.
      Combination of niraparib and bevacizumab versus niraparib alone as treatment of recurrent platinum-sensitive ovarian cancer. A randomized controlled chemotherapy-free study - NSGO-AVANOVA2/ENGOT-OV24.
      ]. Women with measurable platinum-sensitive recurrent ovarian cancer were randomized to receive niraparib 300 mg once daily or the combination of niraparib 300 mg once daily and bevacizumab 15 mg/kg every three weeks, with improvement in PFS from 5.5 to 11.2 months in unselected patients (p < 0.001). Pre-planned exploratory analyses evaluated responses in HR-proficient and HR-deficient subgroups. While all subgroups benefited from the combination compared to monotherapy, there was a trend toward increased benefit observed for the combination in HR-proficient (hazard ratio 0.36), compared to HR-deficient tumors (hazard ratio 0.47). Patients without BRCA mutation had the greatest benefit from the combination with hazard ratio of 0.33, compared to 0.53 for patients with BRCA mutations. The magnitude of benefit was also somewhat greater in patients with platinum-free interval less than 12 months (2.2 versus 11.3 months) compared to those with a platinum-free interval greater than 12 months (6.1 versus 13.1 months).
      The recent approvals of both PARP inhibitors and bevacizumab for maintenance in the frontline and in platinum-sensitive recurrence have generated interest in bevacizumab-PARP inhibitor combinations in the maintenance setting. The combination of olaparib and bevacizumab after first line platinum chemotherapy is being studied in the randomized phase III PAOLA-1 trial (ENGOT OV25, NCT02477644) in patients with and without BRCA mutations. The combination of rucaparib and bevacizumab in frontline maintenance following platinum-taxane chemotherapy will be compared to maintenance with either agent alone in the phase I/II MITO25 study (NCT03462212). These studies may address the critical question regarding the optimal maintenance strategy for ovarian cancer, particularly in patients with HR-proficient tumors, where the best choice among observation, bevacizumab, and PARP inhibition is unclear in the absence of overall survival benefit for monotherapy in the maintenance setting.

      5. Combinations with immunotherapy

      Immune checkpoint inhibitors including anti-PD-1/PD-L1 and anti-CTLA4 antibodies have demonstrated potent and durable responses across a variety of tumor types. Response to PD-1/PD-L1 inhibitor monotherapy has been modest in recurrent ovarian cancer, despite evidence of immune cell infiltration, with single agent anti-PD-1 (avelumab, atezolizumab, durvalumab) and anti-PD-L1 strategies (nivolumab, pembrolizumab) demonstrating response rates of 8–15% [
      • Hamanishi J.
      • et al.
      Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer.
      ,
      • Matulonis U.A.
      • et al.
      Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phase 2 KEYNOTE-100 study.
      ,
      • Varga A.
      • et al.
      Pembrolizumab in patients with programmed death ligand 1-positive advanced ovarian cancer: analysis of KEYNOTE-028.
      ,
      • Disis M.L.
      • et al.
      Efficacy and safety of avelumab for patients with recurrent or refractory ovarian cancer: phase 1b results from the JAVELIN solid tumor trial.
      ,
      • Liu J.F.
      • et al.
      Safety, clinical activity and biomarker assessments of atezolizumab from a Phase I study in advanced/recurrent ovarian and uterine cancers.
      ]. Strategies to harness the immune system in ovarian cancer are high priority.
      Several lines of evidence point to interaction between HR repair and immunogenicity. On the one hand, in cells with HR-deficiency, double strand DNA breaks are repaired by error prone DNA repair mechanisms such as non-homologous end joining (NHEJ), leading to somatic mutations. These mutations can result in neoantigen formation and activation of the immune system. BRCA1/2-mutated ovarian cancers harbor a higher number of mutations compared to BRCA wild type ovarian cancers [
      • Birkbak N.J.
      • et al.
      Tumor mutation burden forecasts outcome in ovarian cancer with BRCA1 or BRCA2 mutations.
      ]. BRCA-deficient and HR-deficient ovarian cancers demonstrate increased neoantigen formation and increased CD3+ and CD8+ immune cell infiltration compared to HR-proficient ovarian cancers [
      • Strickland K.C.
      • et al.
      Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.
      ]. Tumors with BRCA1/2 mutations also exhibit increased expression of PD-L1 and PD-1 in the intraepithelial and peritumoral immune cell compartment compared to expression in HR proficient ovarian cancers.
      Another mechanism by which defective DNA repair can lead to activation of the immune system is by release of damaged DNA resulting in activation of the innate immune system. The cyclic GMP-AMP synthase (cGAS)-STING pathway is a major mechanism of the innate immune system, which protects cells from cytosolic DNA derived from any source including viruses as well tumor cells. Recent data have demonstrated that antitumor efficacy of PARP inhibition is dependent on activation of the STING pathway. In mouse models, PARP inhibition by olaparib triggers robust anti-tumor immune activity that is diminished by loss of cytotoxic T-cells or loss of STING pathway signaling in BRCA-deficient [
      • Ding L.
      • et al.
      PARP inhibition elicits STING-dependent antitumor immunity in brca1-deficient ovarian cancer.
      ] and BRCA wild type models [
      • Shen J.
      • et al.
      PARPi triggers the STING-dependent immune response and enhances the therapeutic efficacy of immune checkpoint blockade independent of BRCAness.
      ].
      These data suggest that PARP inhibitors, by interfering with HR repair, may increase neoantigen production and stimulate the tumor immune microenvironment to generate synergy with immune checkpoint inhibitors. This concept is being tested in several clinical trials (Table 5). The TOPACIO study is a phase I/II study to evaluate the safety and efficacy of the combination of niraparib and pembrolizumab in patients with advanced or metastatic triple-negative breast cancer or recurrent ovarian cancer (KEYNOTE-162, NCT02657889). Results of the ovarian cohort were recently reported [
      • Konstantinopoulos P.A.
      • et al.
      Single-arm phases 1 and 2 trial of niraparib in combination with pembrolizumab in patients with recurrent platinum-resistant ovarian carcinoma.
      ]. Patients with platinum-resistant or platinum-intolerant ovarian cancer with up to 5 prior lines of treatment were eligible with an overall response rate of 25%. For patients without BRCA mutations, 20% had an objective response. For comparison, responses to individual agents in platinum-resistant recurrence is 8% to pembrolizumab monotherapy [
      • Matulonis U.A.
      • et al.
      Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: results from the phase 2 KEYNOTE-100 study.
      ] and 4% for BRCA wild type PARP inhibition [
      • Gelmon K.A.
      • et al.
      Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.
      ]. In the TOPACIO study, patients with BRCA mutations had a response rate of 45% to the combination of niraparib and pembrolizumab, supporting synergy in both HR-deficient and HR-proficient settings.
      Table 5Selected trials of PARP inhibitor plus immunotherapy (IO) and triplets with anti-angiogenesis agents (AA).
      StudyPhasePARPiIOAACancerNotes
      Konstantinopoulos et al., (TOPACIO) (50)Ph 2Niraparib

      300 mg daily
      Pembrolizumab

      200 mg IV q21D
      Recurrent ovarian (plt-resistant), breastBRCAmut ORR 45%

      BRCAwt ORR 20%
      Drew et al., (MEDIOLA) (51)Ph 2Olaparib

      300 mg BID
      Durvalumab

      1.5 g IV q28D
      ASTPlt-sens, BRCAmut ORR 72%
      Lee et al. (52)Ph 2Olaparib

      300 mg BID
      Durvalumab

      1.5 g IV q28D
      Recurrent ovarianORR 15%
      NCT03330405 (JAVELIN PARP medley)#Ph 1b/2Talazoparib (dose esc)Avelumab

      800 mg IV q14D
      Recurrent ovarian
      NCT03602859, (FIRST)#Ph 3Niraparib

      300 mg daily
      TSR042

      500 mg IV q21D
      Upfront maintenance
      NCT03522246, (ATHENA)#Ph 3Rucaparib

      600 mg BID
      Nivolumab

      480 mg IV q28D
      Upfront maintenance
      NCT03740165#Ph 3Olaparib

      300 mg BID
      Pembrolizumab

      200 mg IV q21D
      Upfront maintenance (BRCA wild type)
      NCT03574779, (OPAL)#Ph 2Niraparib

      300 mg daily
      TSR042

      500 mg IV q21D
      BevacizumabRecurrent ovarian (Plt-resistant)
      NCT03737643, (DUO-O)#Ph 3Olaparib

      300 mg BID
      Durvalumab

      1.5 g IV q28D
      BevacizumabUpfront maintenance
      Other PARP inhibitor and checkpoint inhibitor combinations are also being tested. The MEDIOLA study is a phase II study of the PARP inhibitor olaparib in combination with the PD-L1 inhibitor durvalumab in patients with solid tumors (NCT02734004). Preliminary results in the cohort of patients with germline BRCA mutations and platinum-sensitive recurrent ovarian cancer demonstrated a 72% overall response rate and 81% disease control rate in the 34 patients in the initial safety analysis including 6 complete, and 17 partial responses as well as 3 additional patients with stable disease [
      • Y D.
      An open-label, phase II basket study of olaparib and durvalumab (MEDIOLA): results in germline-mutated (gBRCAm) platinum-sensitive relapsed ovarian cancer.
      ]. A phase II trial of durvalumab and olaparib reported a response rate of 15% (all partial responses) in 35 patients with recurrent ovarian cancer [
      • J-M, L.
      A phase 2 study of durvalumab, a PD-L1 inhibitor and olaparib in recurrent ovarian cancer (OvCa).
      ] The combination of avelumab and talazoparib is being investigated in the JAVELIN PARP Medley study in advanced recurrent solid tumors (NCT03330405). Combination of PARP inhibition and immune checkpoint inhibition is also being explored in the upfront maintenance setting in two large randomized phase III studies with target enrollment of approximately 1000 patients. ATHENA is a large randomized phase III trial with a target enrollment of 1012 patients, of the combination of rucaparib with nivolumab in patients with newly diagnosed ovarian cancer after completion of frontline platinum/taxane combination (NCT03522246). The FIRST trial evaluates the addition of anti-PD1 agent TSR-042 and niraparib combination in frontline maintenance (NCT03602859), and KEYLYNK-001/ENGOT-OV43 evaluates pembrolizumab and olaparib in this space in BRCA wild type ovarian cancer (NCT03740165).

      6. Triplet combinations

      Triplet regimens are also being explored which combine immunotherapy, PARP inhibitors, and antiangiogenic agents. The first cohort of the phase II OPAL study is underway which combines TSR-042, niraparib, and bevacizumab in patients with platinum-resistant recurrent ovarian cancer (NCT03574779), while the phase III DUO-O study includes an arm of durvalumab, olaparib, and bevacizumab after frontline chemotherapy with carboplatin, paclitaxel, and bevacizumab (AGO-OVAR23/ENGOT-OV46, NCT3737643). An additional phase II study combines nivolumab, rucaparib, and bevacizumab in the recurrent setting (NCT02873962). The addition of immunotherapy and bevacizumab to PARP inhibitor therapies has demonstrated tolerability and has familiar and manageable side effect profiles. If these combination studies are positive, these combinations are likely to have powerful impact on ovarian cancer treatment strategies, and expand options for patients with both newly diagnosed and recurrent ovarian cancer.

      7. Combinations with DNA damage repair targets

      Additional synergy may be generated by simultaneously targeting more than one DNA repair pathway. This is the proposed mechanism behind the efficacy of PARP inhibitors in BRCA-mutated cancers, where the BRCA mutation inhibits HR repair, and PARP inhibitors impair base-excision repair (BER), generating synthetic lethality. In cells with proficient HR, PARP inhibitors may be combined with agents targeting other HR components downstream of BRCA1/2 or by targeting other DNA repair pathways such as non-homologous end-joining (NHEJ). Agents against DNA repair machinery proteins including ATR, ATM, Chk1, Wee1 and DNA-PK are all rational targets to combine with PARP inhibitors to promote synthetic lethality with the combination.

      7.1 ATR

      Ataxia telangiectasia and Rad3-related protein (ATR) and its downstream kinase checkpoint kinase 1 (Chk1) are activated in response to DNA replication stress and DNA damage. ATR/Chk1 inhibition prevents DNA damage-induced cell cycle arrest, resulting in inappropriate entry into mitosis, chromosomal aberrations and segregation errors, and ultimately apoptosis. Cells with defects in certain DNA repair proteins (such as XRCC1, ERCC1, BRCA1/2, PALB2) or cell cycle proteins (TP53, ATM) are particularly sensitive to ATR inhibition.
      Preclinical studies have demonstrated that ATR is critical for survival of PARP-resistant BRCA-deficient cells [
      • Yazinski S.A.
      • et al.
      ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells.
      ]. Inhibition of ATR reverses resistance to PARP inhibitors in BRCA1-deficient models with acquired resistance to PARP inhibitors. ATR inhibition resolved stalled replication forks in BRCA1/2-deficient patient-derived xeongrafts (PDX) and circulating tumor cells. Inhibition of ATR also significantly sensitizes BRCA2-deficient cells to PARP inhibitors, resulting in decreased cell viability and reduced colony formation in cell line models and tumor regression in PDX model systems [
      • Kim H.
      • et al.
      Targeting the ATR/CHK1 Axis with PARP inhibition results in tumor regression in.
      ]. These studies suggest that ATR inhibitors may synergize with PARP inhibitors in both HR-proficient and HR-deficient cells, and suggest a key role for the combination clinically may be in the post-PARP setting.
      Several ATR inhibitors are in clinical development including AZD-6738 and VX-970 (also known as M6620 or berzosertib). The combination of AZD-6738 and olaparib was investigated in a phase I dose-finding study in solid tumors [
      • TA Y.
      Phase I modular study of AZD6738, a novel oral, potent and selective ataxia telangiectasia Rad3-related (ATR) inhibitor in combination with carboplatin, Olaparib, or durvalumab in patients with advanced cancers.
      ]. The investigators achieved a dose of olaparib 300 mg twice daily continuously with abbreviated dosing of the ATR inhibitor with AZD-6738 given at a dose of 160 mg on days 1–7 of a 28-day cycle as the RP2D. This dose and schedule are being studied in two phase II studies in ovarian cancer patients. In the CAPRI study, patients with platinum-sensitive or platinum-resistant recurrences, or patients with platinum-sensitive HR-deficient ovarian cancer who have progressed on prior PARP inhibitors are eligible to enroll (NCT03462342). In the OLAPCO study, the combination of AZD-6738 and olaparib is administered to patients with HR-deficient solid tumors (NCT0257644).

      7.2 Chk1

      Downstream of ATM and ATR in response to DNA damage, checkpoint kinase 1 (Chk1) and 2 (Chk2) are also therapeutic DNA damage repair targets. Chk1 is activated by ATM or ATR in response to DNA damage. Chk1 phosphorylates and inhibits Cdc25A and Cdc25C and activates Wee1, induces cell cycle arrest at the G2-M checkpoint, and promotes repair of stalled replication forks. Inhibition of Chk1 kinase thus prevents activation of the G2-M checkpoint and impairs reversal of stalled replication forks. Unresolved replication forks result in replication fork collapse into double strand DNA breaks, promoting cell death. Cells with loss of G1-S checkpoint, such by TP53 mutation, may be particularly dependent on the G2-M checkpoint for genomic integrity. Chk1 is also essential for HR repair. Therefore, targeting G2-M checkpoint may be highly relevant to high-grade serous ovarian cancers which have a high prevalence of TP53 mutations and deficient HR repair.
      Pharmacologic inhibitors of Chk1 and Chk2 kinases have been developed. Prexasertib (LY2606368) is a small molecule Chk1/2 kinase inhibitor. A phase II study of prexasertib in high grade serous and endometrioid ovarian cancer has demonstrated efficacy in a platinum-resistant population without germline BRCA mutations (NCT02203513) [
      • Lee J.M.
      • et al.
      Prexasertib, a cell cycle checkpoint kinase 1 and 2 inhibitor, in BRCA wild-type recurrent high-grade serous ovarian cancer: a first-in-class proof-of-concept phase 2 study.
      ]. Out of 28 evaluable patients, there were 8 partial responses (29%), suggesting promising activity in patients with HR-proficient tumors. Post-hoc analysis suggests activity in patients with replication stress: 4 out of 8 responders had CCNE1 upregulation, amplification, or copy number gain.
      Combinations of Chk1 inhibition and PARP inhibition are underway. In preclinical studies, the combination has demonstrated synergy in both BRCA wild type [
      • Brill E.
      • et al.
      Prexasertib, a cell cycle checkpoint kinases 1 and 2 inhibitor, increases.
      ] and BRCA-mutant ovarian cancer models [
      • Kim H.
      • et al.
      Targeting the ATR/CHK1 Axis with PARP inhibition results in tumor regression in.
      ]. Chk1 inhibition in vitro prevents PARP-induced Rad51 focus formation in both BRCA mutant and BRCA wild type ovarian cancer cells, and the combination generates increased DNA damage and apoptosis than either agent alone. A phase I dose finding trial of the combination of prexasertib and olaparib is underway; preliminary results reported 3 partial responses in 9 patients with BRCA-mutated tumors, including 2 patients who had received prior PARP inhibitor [
      • KT D.
      Phase I combination study of the CHK1 inhibitor Prexasertib (LY2606368) and Olaparib in patients with high-grade serous ovarian cancer and other advanced solids tumors.
      ].

      7.3 Wee1

      Wee1 is also involved in regulation of the G2-M checkpoint and HR repair [
      • Do K.
      • Doroshow J.H.
      • Kummar S.
      Wee1 kinase as a target for cancer therapy.
      ]. Wee1 is a tyrosine kinase activated in response to DNA damage by Chk1 kinase. Activated Wee1 phosphorylates cyclin dependent kinase 1 (Cdk1) at tyrosine 15, and results in G2-M cell cycle arrest. Cells with disrupted G1-S cell cycle checkpoint may be particularly susceptible to Wee1 inhibitors as well as to Chk1 inhibitors. Wee1 is also directly implicated in HR repair. Inactivation or hyperactivation of Cdk1 by inhibition of Wee1, both impair HR repair.
      Preclinical data supports synergy of PARP inhibition and Wee1 inhibition. The combination of Wee1 and PARP inhibition promotes DNA damage and apoptosis in vitro in numerous cell types including NSCLC and gastric cancer. Two studies combining PARP and Wee1 inhibition with AZD1775 (adavosertib) are ongoing in ovarian cancer. The multi-arm phase II OLAPCO study will enroll patients with TP53 or KRAS mutations to adavosertib with olaparib, among other olaparib combination arms (NCT02576444). A randomized phase II study in recurrent ovarian cancer will randomize patients to adavosertib in combination with olaparib versus adavosertib alone following progression on PARP inhibitors (NCT 03579316).

      7.4 PI3K

      Multiple lines of evidence implicate phosphotidyl-inositol signaling in HR repair. In BRCA1-deficient cells, treatment with the pan-PI3K inhibitor BKM120 (buparlisib) promoted dsDNA breaks, increased γ-H2AX foci and PARP activity in a dose-dependent fashion, and impaired formation of Rad51 foci [
      • Juvekar A.
      • et al.
      Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1-related breast cancer.
      ]. Inhibition of PI3K signaling by knockdown of PI3K-α (PIK3CA) decreased BRCA1/2 expression, generating HR deficiency, and sensitizing BRCA wild type cells, to PARP inhibition [
      • Ibrahim Y.H.
      • et al.
      PI3K inhibition impairs BRCA1/2 expression and sensitizes BRCA-proficient triple-negative breast cancer to PARP inhibition.
      ]. PTEN deficiency and PI3K-β inhibition can also disrupt the HR repair complex and interfere with dsDNA break repair [
      • Shen W.H.
      • et al.
      Essential role for nuclear PTEN in maintaining chromosomal integrity.
      ]. Preclinical studies have demonstrated synergy of PI3K inhibition and PARP inhibition in breast, prostate, ovarian, and endometrial cancers.
      PI3K inhibitors have been successfully combined with PARP inhibitors in early phase clinical trials. In a phase I dose escalation study of buparlisib with olaparib in high-grade serous ovarian cancers and breast cancers demonstrated that the combination can be safely administered with attenuated dosing of buparlisib [
      • Matulonis U.A.
      • et al.
      Phase I dose escalation study of the PI3kinase pathway inhibitor BKM120 and the oral poly (ADP ribose) polymerase (PARP) inhibitor olaparib for the treatment of high-grade serous ovarian and breast cancer.
      ]. The RP2D for the combination was buparlisib 50 mg daily and olaparib 300 mg twice daily. The combination was administered to 70 patients, with 17 partial responses in 59 evaluable patients. The activity of the combination at all doses was 29% in the ovarian cancer patients with 28% in breast cancer patients with responses observed in both germline BRCA-mutation carriers as well as germline BRCA-wildtype patients. Olaparib has also been safely combined with the α-specific PI3K inhibitor alpelisib in patients with ovarian cancer [
      • Konstantinopoulos P.A.
      • et al.
      Olaparib and α-specific PI3K inhibitor alpelisib for patients with epithelial ovarian cancer: a dose-escalation and dose-expansion phase 1b trial.
      ]. The MTD and RP2D for the combination was identified as alpelisib 200 mg daily with attenuated olaparib 200 mg twice daily. Ten out of 28 patients with ovarian cancer achieved a response (36%). Of note, 33% of patients with germline and somatic BRCA wild type, platinum-resistant ovarian cancer had a response, compared to the reported 4% response rate for olaparib monotherapy in this population [
      • Gelmon K.A.
      • et al.
      Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.
      ]. Other promising PI3K pathway combination studies in ovarian cancer include combinations with the AKT inhibitor AZD5363 (ComPAKT NCT02338622 [
      • S W.
      Phase I expansion of Olaparib (PARP inhibitor) and AZD5363 (AKT inhibitor) in recurrent ovarian, endometrial, and triple negative breast cancer.
      ]) and mTORC1/2 inhibitor (NCT02208375 [
      • Westin S.
      Phase I trial of Olaparib (PARP inhibitor) and vistusertib (mTORC1/2 inhibitor) in recurrent endometrial, ovarian, and triple negative breast cancer.
      ]).

      8. Other emerging combinations

      Chromatin and epigenetic modifications also have the potential to enhance responses to PARP inhibitors. The enzyme enhancer to zeste homolog 2 (EZH2) is an oncogene which catalyzes histone 3 (H3) lysine 27 (K27-me3) trimethylation, leading to gene silencing. Overexpression of EZH2 has been described in several cancer cell types, and promotes cell proliferation, tumor growth, stem cell expansion, and metastasis. In the presence of DNA damage, activated PARP1 catalyzes PARylation and inhibition of EZH2, with subsequent reduction in K27-me3. Since PARP inhibitors relieve the inhibition of EZH2, increased EZH2 oncogene activity may promote PARP inhibitor resistance. Therefore EZH2 inhibitors may sensitize to PARP inhibition, as demonstrated in preclinical BRCA-deficient model systems [
      • Yamaguchi H.
      • et al.
      EZH2 contributes to the response to PARP inhibitors through its PARP-mediated poly-ADP ribosylation in breast cancer.
      ]. Inhibitors of EZH2 including tazemostat are in early phase clinical development. Bromodomain and extraterminal (BET) proteins are epigenetic readers involved in regulation of gene transcription including cell cycle checkpoint and DNA damage repair genes. Bromodomain inhibitors may synergize with PARP inhibitors by reducing expression of BRCA1 and RAD51, among other mechanisms [
      • Yang L.
      • et al.
      Repression of BET activity sensitizes homologous recombination-proficient cancers to PARP inhibition.
      ]. Several bromodomain inhibitors are in development and combinations with PARP are emerging (NCT03901469). Hypomethylating agents have also demonstrated efficacy in preclinical model systems of breast and ovarian cancer in combination with PARP inhibitors. Coadministration of DNA methyltransferase inhibitor guadecitabine and talazoparib can overcome inherent resistance to PARP inhibition in mouse xenograft models independent of BRCA mutation status [
      • Pulliam N.
      • et al.
      An effective epigenetic-PARP inhibitor combination therapy for breast and ovarian cancers independent of BRCA mutations.
      ]. Trials with histone deactacetylases, such as entinostat, in combination with PARP inhibition are also underway (NCT03924245).
      Other areas of active development include combinations with MEK inhibitors, which sensitize cells with activating RAS pathway mutations to PARP inhibitors [
      • Sun C.
      • et al.
      Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in.
      ]. Additional strategies to promote HR deficiency include the combination of PARP inhibitors with HSP90 inhibitors, which promote degradation of BRCA1 protein, and the combination of PARP inhibitors and cdk4/6 inhibitors, which can regulate expression of HR genes including BRCA1 and BRCA2 through myc-mediated transcriptional activity.

      8.1 Future

      As development of PARP inhibitor combinations continues, selecting the optimal combination for the appropriate patient and setting becomes increasingly relevant. A potential schema utilizing the proposed biological rationale to categorize the combinatorial strategies is illustrated in Fig. 1. Rigorous translational science to understand predictors of response and resistance are required to optimize responses to these important combinations. While monotherapy may promote responses in patients with HR-deficient tumors, deeper responses may be achieved through the addition of chemotherapy or immunotherapy. Patients with HR-proficient tumors may benefit from the combination with immunotherapy, but also from combinations which generate HR deficiencies (ie. “BRCA-ness”) such as antiangiogenic combinations, PI3K combinations, and epigenetic strategies, or from combinations that exploit sensitivity to replication stress. As PARP inhibitors become standard of care earlier in the treatment history for many ovarian cancer patients, overcoming mechanisms of resistance becomes increasingly important. PARP inhibitor combinations are likely to play an important role in preventing and reversing treatment resistance.
      While overlapping toxicities likely limit the development of chemotherapy combinations and ATR and Chk1 inhibitors, antiangiogenics and immunotherapy combinations are safely combined at full doses and have encouraging efficacy and are moving into advanced stages of clinical development. Understanding the molecular mechanisms of potential synergy will help apply the right combination to the right patient, to improve outcomes for patients with recurrent ovarian cancer.

      Author contributions

      Conception and design: Jennifer Taylor Veneris, Panagiotis A. Konstantinopoulos, Joyce Liu, Ursula Matulonis. Data analysis and interpretation: Jennifer Taylor Veneris. Manuscript writing: Jennifer Taylor Veneris, Panagiotis A. Konstantinopoulos, Joyce Liu, Ursula Matulonis. All authors have approved the final article.

      Declaration of competing interest

      Dr. Veneris reports spouse employment at Takeda. Dr. Matulonis reports personal fees from AstraZeneca, Myriad Genetics, Clovis, Merck, Eli Lilly, Mersana, Geneos, Fuji Film, Cerulean, Immunogen, and other from 2X Oncology, outside the submitted work. Dr. Konstantinopoulos reports personal fees from AstraZeneca, Pfizer, Merck, and Tesaro, outside the submitted work. Dr. Liu reports personal fees (advisory board participation) for AstraZeneca, Tesaro, Clovis, Merck, Genentech and Mersana outside the submitted work.

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