Posts Tagged ‘DNA repair or transcription factor genes’

New studies link cell cycle proteins to immunosurveillance of premalignant cells

Curator: Stephen J. Williams, Ph.D.

The following is from a Perspectives article in the journal Science by Virinder Reen and Jesus Gil called “Clearing Stressed Cells: Cell cycle arrest produces a p21-dependent secretome that initaites immunosurveillance of premalignant cells”. This is a synopsis of the Sturmlechener et al. research article in the same issue (2).

Complex organisms repair stress-induced damage to limit the replication of faulty cells that could drive cancer. When repair is not possible, tissue homeostasis is maintained by the activation of stress response programs such as apoptosis, which eliminates the cells, or senescence, which arrests them (1). Cellular senescence causes the arrest of damaged cells through the induction of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 (2). Senescent cells also produce a bioactive secretome (the senescence-associated secretory phenotype, SASP) that places cells under immunosurveillance, which is key to avoiding the detrimental inflammatory effects caused by lingering senescent cells on surrounding tissues. On page 577 of this issue, Sturmlechner et al. (3) report that induction of p21 not only contributes to the arrest of senescent cells, but is also an early signal that primes stressed cells for immunosurveillance.Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).

Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).

Sturmlechner et al. found that activation of p21 following stress rapidly halted cell cycle progression and triggered an internal biological timer (of ∼4 days in hepatocytes), allowing time to repair and resolve damage (see the figure). In parallel, C-X-C motif chemokine 14 (CXCL14), a component of the PASP, attracted macrophages to surround and closely surveil these damaged cells. Stressed cells that recovered and normalized p21 expression suspended PASP production and circumvented immunosurveillance. However, if the p21-induced stress was unmanageable, the repair timer expired, and the immune cells transitioned from surveillance to clearance mode. Adjacent macrophages mounted a cytotoxic T lymphocyte response that destroyed damaged cells. Notably, the overexpression of p21 alone was sufficient to orchestrate immune killing of stressed cells, without the need of a senescence phenotype. Overexpression of other CDKIs, such as p16 and p27, did not trigger immunosurveillance, likely because they do not induce CXCL14 expression.In the context of cancer, senescent cell clearance was first observed following reactivation of the tumor suppressor p53 in liver cancer cells. Restoring p53 signaling induced senescence and triggered the elimination of senescent cells by the innate immune system, prompting tumor regression (5). Subsequent work has revealed that the SASP alerts the immune system to target preneoplastic senescent cells. Hepatocytes expressing the oncogenic mutant NRASG12V (Gly12→Val) become senescent and secrete chemokines and cytokines that trigger CD4+ T cell–mediated clearance (6). Despite the relevance for tumor suppression, relatively little is known about how immunosurveillance of oncogene-induced senescent cells is initiated and controlled.

Source of image: Reen, V. and Gil, J. Clearing Stressed Cells. Science Perspectives 2021;Vol 374(6567) p 534-535.


2. Sturmlechner I, Zhang C, Sine CC, van Deursen EJ, Jeganathan KB, Hamada N, Grasic J, Friedman D, Stutchman JT, Can I, Hamada M, Lim DY, Lee JH, Ordog T, Laberge RM, Shapiro V, Baker DJ, Li H, van Deursen JM. p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science. 2021 Oct 29;374(6567):eabb3420. doi: 10.1126/science.abb3420. Epub 2021 Oct 29. PMID: 34709885.

More Articles on Cancer, Senescence and the Immune System in this Open Access Online Scientific Journal Include

Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy

Natural Killer Cell Response: Treatment of Cancer

Issues Need to be Resolved With ImmunoModulatory Therapies: NK cells, mAbs, and adoptive T cells

New insights in cancer, cancer immunogenesis and circulating cancer cells

Insight on Cell Senescence

Immune System Stimulants: Articles of Note @pharmaceuticalintelligence.com

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Targeting PARP in Various Endocrine Cancers: Prostate, Cervical, Endometrial

Curator: Larry H. Bernstein, MD, FCAP


UPDATED 12/05/2020

Targeting PARP in Prostate Cancer: Novelty, Pitfalls, and Promise    
Review ArticleMay 15, 2016Oncology Journal, Prostate Cancer




© 2016 Steve Oh and Myriam Kirkman-Oh, KO Studios

Metastatic prostate cancer remains a highly lethal disease with no curative therapeutic options. A significant subset of patients with prostate cancer harbor either germline or somatic mutations in DNA repair enzyme genes such as BRCA1, BRCA2, or ATM. Emerging data suggest that drugs that target poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) enzymes may represent a novel and effective means of treating tumors with these DNA repair defects, including prostate cancers. Here we will review the molecular mechanism of action of PARP inhibitors and discuss how they target tumor cells with faulty DNA repair functions and transcriptional controls. We will review emerging data for the utility of PARP inhibition in the management of metastatic prostate cancer. Finally, we will place PARP inhibitors within the framework of precision medicine–based care of patients with prostate cancer.

In 2016, prostate cancer is expected to be diagnosed in 180,890 men, and 26,120 will die of metastatic disease.[1] While the majority of localized prostate cancers can be controlled with surgery and/or radiation, metastatic disease remains a lethal disease with no curative options. Moreover, prostate cancer is a heterogeneous disease that can be highly lethal but also slow and indolent, as reflected by a 10-year estimated survival of 17% (S9346 trial, unpublished data). The advent of affordable and efficient techniques for profiling tumors molecularly represents an unprecedented opportunity to better characterize the molecular factors that result in indolent and/or lethal disease and to tailor therapy accordingly. Many clinical trials are already underway to examine whether molecularly targeted therapies can improve outcomes.[2] In this review, we will specifically examine the molecular rationale for one of these targeted approaches, poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibition, in prostate cancer. We will review how PARP inhibitors function as a class, review the molecular features that sensitize cancer cells to this therapy, and discuss the data supporting its potential for patients with prostate cancer. We will then outline a strategy for further development of PARP inhibitors in the prostate cancer field.
Metastatic prostate cancer is typically categorized as hormone-sensitive prostate cancer (HSPC), which responds to androgen ablation, or castration-resistant prostate cancer (CRPC), which develops resistance to gonadal suppression. Although bilateral orchiectomy is the historic gold-standard treatment for metastatic HSPC, gonadal suppression is currently accomplished with gonadotropin-releasing hormone agonists or antagonists with or without androgen receptor blockade. This approach remains the cornerstone of therapy for men with metastatic HSPC.[3] Emerging data from large phase III trials (CHAARTED and Systemic Therapy in Advancing or Metastatic Prostate Cancer: Evaluation of Drug Efficacy [STAMPEDE]) have also revealed a large survival benefit for the combination of docetaxel and androgen deprivation in metastatic HSPC.[4,5]

Despite these initially effective treatments, the vast majority of men with metastatic HSPC will progress to CRPC, which is the lethal stage of the disease. For these patients, several additional therapies provide benefit by further suppression of androgen signaling (enzalutamide, abiraterone), disruption of the cell cycle in replicating cells (docetaxel, cabazitaxel), targeting of bone metastases (radium-223), or activation of antitumor immunologic response (sipuleucel-T).[6] While these therapies have undoubtedly extended the median survival of patients with metastatic CRPC, their impact on survival is modest and they clearly do not work for all men. In addition, we lack validated genomic markers that would allow better selection of patients for these therapies. Therefore, a better approach that leverages the individual and unique aspects of a patient’s cancer and utilizes therapy based on these factors may allow us to improve patient outcomes.

The development of high-throughput sequencing technology has made it feasible to comprehensively analyze the genetic mutations and gene expression changes in individual prostate cancers with a high degree of resolution in real time. Many institutions now routinely perform these analyses in the hope that they might uncover molecular features that predict response to certain therapies or provide guidance for clinical trial selection.[7] This approach, colloquially termed “precision” medicine, offers the potential promise of providing the right therapy for the right patient at the right time. In the context of prostate cancer, it means molecularly characterizing a tumor and then offering patients drugs that may specifically promote tumor lethality based on these molecular features. The limitation of this approach is that it requires that the target be truly biologically relevant and that there are drugs that can effectively target these molecular changes. The discovery of both somatic and germline DNA repair deficiencies in prostate cancer, together with the development of PARP inhibitors that can kill cancer cells with these defects, is a potent example of targeting therapy to molecularly defined tumor subtypes. While much early work validating this approach has occurred in breast and ovarian cancer populations, emerging data suggest that PARP inhibition is a potentially important strategy for managing a significant subset of prostate cancer patients.


PARP Inhibition: Targeting DNA Repair Deficiency

Molecular mechanism

PARP1 catalyzes the addition of poly(ADP)-ribose (PAR) groups to target proteins in a process termed PARylation.[8] PARP1 is part of a superfamily of proteins that consists of 18 members (including the related tankyrase enzymes), which have many functions within normal and cancer cells. PARP1, the founding member of this family, is responsible for the majority of PARylation of protein targets within cells. It is primarily present in the nucleus in association with chromatin, where it participates in DNA repair and regulation of gene expression by modulating protein localization and activity.[9]

DNA damage occurs continuously in all living cells as a result of oxidative damage or DNA replicative stress.[10] When DNA damage occurs on one strand of the DNA double helix, a single-strand break (SSB) results, but if two SSBs occur in close proximity and on opposite strands, the result is a double-strand break (DSB) and discontinuity of the chromosome (Figures 1 and 2). Even a single DSB is lethal to a human cell if unrepaired because of the risk of large-scale loss of genetic information.


PARP1 plays a critical role in restoration of genomic integrity by facilitating efficient repair of DNA SSBs and DSBs. PARP1 senses DNA damage by binding to the site of SSBs and DSBs and inducing auto-PARylation, which in turn promotes recruitment of DNA repair factors (such as DNA ligase III, polymerase β, and x-ray repair cross-complementing protein 1[XRCC1]).[11] Loss of PARP1 function by means of pharmacologic or genetic mechanisms results in impaired SSB repair and, following initiation of DNA replication, creation of a DNA DSB (see Figure 1). PARP may also play an important role in DSB repair and is known to recruit the MRE11-RAD50-NBS1 complex and to promote PARylation of BRCA1, factors required for the homologous recombination (HR) pathway of DNA DSB repair. Therefore, pharmacologic inhibition of PARP1/2 in DNA repair–defective (DRD) cells that lack efficient HR repair capabilities (such as those harboring BRCA1, BRCA2, or ATM mutations) results in failure to resolve SSBs, which are then converted to DSBs that promote cellular death.

The activity of PARP1 is not limited to DNA damage response. PARP1 is also known to regulate gene expression by modulation of transcription factor activity and regulation of chromatin.[12] PARP1 binds to RNA polymerase II, regulating gene expression, and may also affect tumor suppressor and oncogenic gene expression. PARP1 can also modulate hormone-dependent gene transcription from hormone-responsive nuclear receptors, such as estrogen receptors α and β, progesterone receptor, and androgen receptor.[9]

Furthermore, PARP1 can modulate the transcriptional activity of ETS transcription factors, which suggests that pharmacologic targeting of PARP1 may be useful in TMPRSS2:ERG fusion–positive prostate cancer cells (~50% of prostate cancers).[13] PARP1 physically interacts with the TMPRSS2:ERG gene fusion and the DNA–protein kinase complex, and these interactions are required for ERG-related gene transcription. Interestingly, PARP inhibition with olaparib inhibited prostate cancer xenograft growth if tumors harbored a TMPRSS2:ERG fusion, which suggests that PARP might represent a therapeutic option for prostate cancer patients withTMPRSS2:ERG fusions.[13] This concept is being evaluated in a recently completed clinical trial (National Cancer Institute [NCI] 9012).

PARP inhibitors

Given the biologic importance of PARP1 in the context of cancer, several pharmacologic agents that target this enzyme are currently under development (Table). Most PARP inhibitors mimic the NAD+ substrate of PARP1, competitively bind to the catalytic domain, and inhibit PAR synthesis.[14] PARP inhibitors require the expression of PARP1 and PARP2, and cells that lack expression of both genes are not sensitive to these agents. PARP inhibitors all appear to block catalytic activity and PAR synthesis in a roughly equivalent manner but may show differential ability to trap PARP1/2 at the site of DNA damage (niraparib > olaparib > veliparib), an event that blocks repair and promotes cellular lethality.[15,16] Whether these effects observed in vitro translate into clinically meaningful differences in efficacy is less clear. Furthermore, it is also now clear that the putative PARP inhibitor iniparib may not promote cytotoxicity via PARP inhibition. Several initial studies focused on iniparib, but when phase III trials failed to demonstrate the efficacy of this compound, additional mechanistic work demonstrated that iniparib may not truly be an effective PARP inhibitor.[17,18] These data illustrate the necessity of careful mechanistic characterization of any targeted agent prior to large-scale and expensive studies.

Germline DNA repair deficiency

Inherited defects in DNA repair pathways result in increased susceptibility to the development of malignancy.[19] Defects in mismatch repair proteins promote the development of tumors, including colon and uterine,[20] whereas inherited inactivating mutations in BRCA1 and BRCA2, which are required for efficient HR-based DNA DSB repair, significantly increase the risk of breast, ovarian, prostate, and other cancers.[21] Patients with these tumor types typically demonstrate homozygous inactivation of these genes, the first event occurring in the germline, with subsequent clonal somatic inactivation of the remaining allele.[21] These events presumably occur early in tumorigenesis and, by loss of robust DNA DSB repair, induce genomic instability, which causes loss of tumor suppressors, activation of oncogenes, and acceleration of tumorigenesis.

A germline mutation in BRCA1 or BRCA2 increases the risk of prostate cancer and thus may be found in 2% to 5% of prostate cancers.[22,23] The relative risk of development of prostate cancer for men ≤ age 65 with BRCA1 mutations is 1.8, but BRCA2 mutations in particular seem to increase the risk of prostate cancer formation by age 65 by about 8.6-fold. Mutations of BRCA1, BRCA2, and ATM (and perhaps other DNA repair genes) may also play a role in progression to the lethal castration-resistant state.[22,24-26] The frequency of BRCA2 germline mutations in prostate cancer alone may be as high as 2%.[22] Therefore, the development of therapies to target DNA repair is likely to benefit a relatively large and relatively young population.

Somatic DNA repair deficiency

In addition to germline defects, tumors can acquire defective DNA repair processes through somatic loss of DNA damage response genes, and these somatic mutations can also confer sensitivity to PARP inhibition.[27] This has led to the concept of “BRCAness,” which refers to somatically acquired defects in HR that, as a group, could predict tumor response to PARP inhibitors and cisplatin.[21] Somatic alterations can include either acquired mutations or epigenetic events that silence genes such as ATM; ATR; BRCA1 or –2; CHEK1 or -2; FANCA, -C, -D2, -E, -F; PALB2; MRE11 complex; or RAD51, which prevent efficient HR repair of DNA DSBs.

It is likely that a substantial proportion of men with prostate cancer may demonstrate aspects of BRCAness that could predict sensitivity to PARP inhibitors. Beltran et al performed targeted next-generation sequencing of tumors from men with advanced prostate cancer and found that 12% demonstrated BRCA2 loss and that 8% harbored ATM loss.[28] Furthermore, up to 19.3% of CRPCs demonstrate aberrations in BRCA1, BRCA2, or ATM; these events become more frequent as the disease progresses from hormone-sensitive to castration-resistant.[29] Together these data suggest that BRCAness is a reasonably frequent event in patients with advanced prostate cancer, which makes PARP inhibition an attractive target in this disease.

Synthetic lethality

The concept of promoting the killing of cancer cells by simultaneously blocking SSB repair using PARP inhibition in cells that lack efficient DSB repair is called “synthetic lethality.” In this scenario, tumor cells may harbor either germline or somatically acquired homozygous inactivation of HR. Germline defects (when present) typically affect only one allele in normal cells, and therefore normal tissues retain HR function. This difference between the DNA repair capacity of normal and cancer cells can be leveraged to produce selective cell killing of tumor cells by PARP inhibitors. Treatment of patients with PARP inhibitors will then block normal SSB repair in all cells, and these SSBs are subsequently converted to DSBs by DNA replication. In normal cells, HR restores the genome and allows survival, but in DRD cancer cells, DSBs persist, inducing cellular death selectively in the tumor cell population (see Figure 2).


Early-phase studies

Ample data indicate that PARP inhibitors possess antitumor activity within diverse patient populations, particularly those with BRCA1 or BRCA2 mutations.[14] One of the first studies to validate the concept of clinical benefit in patients with BRCA mutations was a phase I trial that looked at pharmacokinetic and pharmacodynamic aspects of olaparib treatment.[24] In this study, 60 patients with solid tumors were treated with various doses of olaparib (10 mg daily to 600 mg twice daily) to determine maximum tolerated dose (MTD). The study population was intentionally enriched for BRCA mutation carriers, and 22 patients of the cohort harbored BRCA1 or BRCA2 mutations. Objective tumor activity was observed in the mutation carrier population in patients with breast, ovarian, and prostate cancers. Three patients with advanced prostate cancer were included in this study cohort; the one with a BRCA2 mutation had a greater than 50% response in prostate-specific antigen (PSA) level, resolution of bone metastases, and an extended treatment course. This study suggested that there was a benefit of olaparib therapy in BRCA mutation carriers and the potential for benefit in prostate cancer patients. Further validation of olaparib efficacy in patients with BRCA mutations came from parallel proof-of-concept studies demonstrating the activity of this agent in women with breast and ovarian cancers and BRCA1 or BRCA2 mutations.[30,31] These data ultimately led to US Food and Drug Administration (FDA) approval of olaparib for women with a BRCA mutation and metastatic ovarian cancer after chemotherapy.
Additional data that demonstrate a similar spectrum of activity are available for other PARP inhibitors. Phase I data on the safety and pharmacodynamics of single-agent veliparib have been reported as an abstract,[32] and additional studies of veliparib in combination with mitomycin,[33] irinotecan,[34] and other agents have been reported.[35] VanderWeele et al published a case report of a patient with metastatic CRPC and BRCA2 mutation who had a sustained complete response to veliparib and carboplatin/gemcitabine.[36] It seems likely that many of the available PARP inhibitors may have overlapping activities, and further data will be needed to clarify which agent to use in which tumor type and the relative toxicities of each agent.

emozolomide and veliparib in metastatic CRPC

Compelling data implicate PARP1 in the mediation of DNA repair responses to alkylating agents,[37] cellular survival in BRCA-deficient cells,[24,38] and androgen receptor–mediated prostate cancer cellular proliferation.[9,39] Furthermore, data suggest that prostate cancers that harbor the TMPRSS2:ERG fusion (present in up to 50% of prostate cancers) may be more sensitive to PARP inhibition.[13] Therefore, Hussain et al carried out a single-arm pilot study to assess the safety and efficacy of veliparib with the alkylator temozolomide (TMZ) in patients with metastatic CRPC following docetaxel therapy.[40] In this study, patients with a PSA level of ≥ 2 ng/mL were treated with veliparib, 40 mg twice daily, on days 1 to 7 and TMZ, 150 to 200 mg/m2, on days 1 to 5 on a 28-day cycle, based on tolerance data from a phase I study (ClinicalTrials.gov identifier: NCT00526617). The primary endpoint was PSA response rate (30% decline). Of the 25 patients who were evaluable for response, 2 had a confirmed response, 13 had stable PSA, and 10 had progression. The most frequent toxicities were thrombocytopenia, anemia, fatigue, neutropenia, nausea, and constipation. The investigators did assess frequency of TMPRSS2:ERG fusion but found it in only one of eight evaluable patients. Although this patient had stable disease, no conclusions could be drawn regarding the contribution of the fusion product to veliparib sensitivity. Overall, while the combination was considered tolerable, it had only modest activity. No preselection was done in the study, and because BRCAness exists in 20% of patients, it is perhaps not surprising that activity was modest. The lower dose of PARP inhibitor and the lack of established benefit for TMZ may also have contributed to less than robust clinical activity for this combination. Given the emerging molecular data, it seems that future studies will be more likely to identify activity if done in preselected patient populations.


The Trial of PARP Inhibition in Prostate Cancer (TOPARP-A) sought to determine whether patients with prostate cancers with molecularly identified defects in DNA repair benefited from full-dose olaparib therapy.[25] In this phase II study, 50 men with CRPC underwent biopsy of metastatic disease and targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase chain reaction. The primary endpoint was response rate (either objective response or reduction of 50% in PSA level or reduction in circulating tumor cells). All had previously received docetaxel, and most had been treated with abiraterone or enzalutamide (98%) and cabazitaxel (58%). Patients were grouped according to the presence or absence of a homozygous deletion of or deleterious mutation in DNA damage response genes, which predict sensitivity to PARP inhibition. Overall, 16 of 49 evaluable patients (33%) were biomarker positive (indicative of homozygous deleterious changes in BRCA1/2, ATM, Fanconi anemia genes, or CHEK2). Of these, five patients had germline and somatic events (three patients with germline BRCA2 and three patients with germline ATM deletions or mutations). Of the 16 patients with deleterious changes in DNA repair genes, 14 (88%) responded to olaparib. The median overall survival for patients with biomarker-positive DRD tumors who received olaparib was 13.8 months, compared with 7.5 months for those with biomarker-negative tumors (P = .05). Interestingly, two biomarker-negative patients also met criteria for response to olaparib. Although one was a longer-term responder still on therapy at the time of publication, this particular patient did harbor monoallelic deletions of both BRCA2 and PALB2 that did not meet criteria for the prespecified biomarker-positive category but that may have contributed to tumor sensitivity. Toxicity was as expected, with patients displaying grade 3 or 4 anemia (10/50), fatigue (6/50), leukopenia (3/50), thrombocytopenia (2/50), and neutropenia (2/50). These results illustrate the feasibility of using molecular profiling to identify prostate cancers that display molecular features suggestive of sensitivity to PARP inhibition (BRCAness).

NCI 9012

ETS gene fusions—which result from gene rearrangement and juxtaposition of an androgen-responsive gene, such as TMPRSS2, to an ETS transcription factor gene, such as ERG or ETV1—occur in 50% to 60% of prostate cancers.[41,42] ETS transcription factors may also physically interact with PARP1, and PARP1 activity may be required for ETS-mediated invasion, transcription, and metastasis.[13] Androgen receptor–mediated transcription may also promote DNA DSBs and requires PARP activity for efficient repair.[43-45] Therefore, therapeutic targeting of androgen receptor signaling and PARP1 activity using abiraterone and veliparib is an attractive strategy in the management of metastatic prostate cancer.

A randomized phase II clinical trial in patients with metastatic CRPC was recently completed; it examined whether ETS fusion is a biomarker of response to abiraterone or abiraterone plus veliparib. In this study, 148 patients with metastatic CRPC underwent biopsy followed by assessment of ETS fusion status and then random assignment to either abiraterone alone or abiraterone plus veliparib. The primary endpoint was confirmed PSA response in patients receiving either abiraterone alone or combination therapy, stratified by ETS status. Secondary endpoints included safety, objective response rate, progression-free survival, and whether DNA repair gene deficiency (homozygous deletions of or deleterious mutations in: BRCA1, BRCA2, ATM, FANCA, PALB2, RAD51B, RAD51C) predicts response. This trial has now completed enrollment, and preliminary results will be presented at the American Society of Clinical Oncology 2016 Annual Meeting. Although final results are pending, the study does illustrate the feasibility of a large-scale metastatic tissue–based, biomarker-driven trial involving PARP inhibition in patients with metastatic CRPC. This study will also begin to ascertain the role of ETS fusions in determining response to PARP inhibitor therapy and will further explore the contribution of DRD to patient outcomes in those treated with standard therapy (abiraterone arm) and those treated with PARP inhibition (abiraterone plus veliparib arm).

Future studies

Given the data from the studies discussed previously and the enthusiasm for molecularly targeted trials in oncology, there is interest in further testing of PARP inhibition in prostate cancer patients. Multiple trials have recently been completed, are actively enrolling, or are nearing activation within this space (see Table, ClinicalTrials.gov).

Olaparib. Olaparib is the agent that is farthest along in clinical development and has an FDA indication in ovarian cancer. Olaparib also has the most active or pending studies in prostate cancer patients. TOPARP continues to enroll patients with metastatic CRPC, with a target accrual of 98 patients (ClinicalTrials.gov identifier: NCT01682772). There is a randomized double-blind, placebo-controlled phase II study of abiraterone plus olaparib or placebo for patients with metastatic CRPC who received prior docetaxel therapy (ClinicalTrials.gov identifier: NCT01972217). This trial, which is similar to the NCI 9012 study, has completed enrollment, but results are pending. Another trial is examining the biologic effect of olaparib on prostate cancer specimens when given alone or in combination with degarelix prior to prostatectomy (ClinicalTrials.gov identifier: NCT02324998). Furthermore, there is an open-label phase II study to assess the efficacy and safety of olaparib in patients with BRCA1 or BRCA2 mutations (regardless of tumor type), which is ongoing but no longer enrolling patients (ClinicalTrials.gov identifier: NCT01078662).

Veliparib. NCI 9012 (discussed previously) will help determine whether veliparib has potential therapeutic activity in metastatic CRPC and may identify molecularly determined subsets of disease (ie, ETS fusion–positive, DRD-positive) that might be expected to show the most benefit. The results of this study may help determine whether additional studies of this agent within the prostate cancer space are warranted.

Niraparib. The Hoosier Cancer Research Network has a planned phase I study of the combination of enzalutamide and niraparib for patients with metastatic CRPC (ClinicalTrials.gov identifier: NCT02500901), which has not yet begun enrollment. The primary endpoint of this study will be determination of MTD and dose-limiting toxicity.

Talazoparib. Although no prostate cancer–specific trials using other PARP inhibitors are currently active, several trials for molecularly targeted patient populations or phase I trials for toxicity assessment in combination with chemotherapy are ongoing; these provide some information on prostate cancer populations, depending on the types of solid tumors enrolled. There is a phase I trial of talazoparib in combination with carboplatin and paclitaxel (ClinicalTrials.gov identifier: NCT02317874) and another for patients with solid tumors and hepatic and renal dysfunction (ClinicalTrials.gov identifier: NCT02567396).

Precision Targeting of the PARP Pathway in Prostate Cancer

PARP inhibitors are a promising therapeutic option for men with prostate cancer. There is good evidence that men with either germline or somatic mutations in DNA repair pathways can derive therapeutic benefit from inhibition of PARP1/2, which blocks repair of SSB, driving persistent DSBs that lead to cancer cell lethality. Preclinical data also suggest that PARP inhibition may produce benefits by targeting chromatin and gene transcription, which implies that clinical benefits may extend beyond patients with DRD tumors.[12] To continue to develop PARP inhibitors within the prostate cancer field, we will need to develop and refine a set of biomarkers for use in selecting the right patient populations for these agents and then incorporate these biomarkers into prospective studies. As part of a precision therapy strategy, PARP inhibitors will likely play an important role in the management of prostate cancer in the near future.

It is now feasible to comprehensively profile the mutational, epigenetic, and gene expression changes in men with prostate cancer, and we are beginning to use this information to guide treatment choices.[7] Unfortunately, the functional relevance of many of the molecular features uncovered in these profiles is not completely understood. DNA repair processes are complex and require many genes for efficient repair of various types of DNA damage. Most past and ongoing studies focused on patients with specific molecular features, such as BRCA1, BRCA2, ATM, FANCA, PALB2, RAD51B, and RAD51C mutations. While mutations of these genes are likely to affect sensitivity to PARP inhibitors, mutations in other DNA repair or transcription factor genes may as well, and identification of those genes could expand the patient population that could benefit from therapy. Determination of whether other genes are susceptible to PARP inhibitor therapy will require robust preclinical models with a wide selection of genetic changes that reflect human disease; such models can be used to determine whether additional mutations and epigenetic or gene expression changes also result in PARP inhibitor sensitivity. Given the potential infrequency of many of the individual mutations that might sensitize to PARP inhibitors, large-scale registries that catalog mutations and their responsiveness to therapies may be needed.

As we define the molecular features that suggest sensitivity to PARP inhibition, the challenge will then become understanding the best strategy for incorporating these targeted agents into our standard treatment algorithms. In the context of prostate cancer, PARP inhibitors could be considered in high-risk patient populations in an adjuvant manner, before or with androgen deprivation therapy (ADT) in patients with newly metastatic disease, or in the setting of castration-resistant disease before or after the many other therapeutic options. To date, most trials in the prostate cancer space have been in the castration-resistant setting, perhaps because mutations in DNA damage genes may become more common as the disease progresses.[25] Nonetheless, there is no reason to assume that patients who harbor mutations may not benefit earlier in the disease course. Adjuvant use of PARP inhibitors in those with high-risk or micrometastatic disease could conceivably render patients disease free. Similarly, the combination of ADT and PARP inhibitors in early metastatic disease may provoke prolonged progression-free intervals similar to the situation with early docetaxel therapy but with less toxicity.[4,5] In the context of castration-resistant disease, it is reasonable to hypothesize that the combination of PARP inhibitors with hormonal agents such as abiraterone or enzalutamide or with chemotherapies might act synergistically to promote disease control.

The trials to examine these questions may be more challenging to design and execute because patients with sensitizing molecular changes represent a limited subset of total patients with prostate cancer. This means that in order to identify the subset that will benefit, many will need to be screened.[25] Because most molecular analyses are done using biopsy tissue, screening and cost may be challenging factors. In addition, the natural history of patients with DNA damage pathway mutations may also be distinct from those without such mutations. It is conceivable that mutations in DNA damage response genes may modulate patient response to standard hormonal agents, chemotherapy, or radium because all three of these therapeutic modalities have the potential to induce DNA damage in prostate cancer cells. Given these caveats, it will be essential to design an efficient precision medicine clinical trial pipeline that can rapidly molecularly profile patient tumors, assign to a therapeutic intervention, and then assess the complex resulting data and analyze results according to molecular categories.

PARP inhibitors have the potential to be a promising addition to the therapeutic arsenal used to treat prostate cancer and other solid tumors that harbor the appropriate molecular features. The transition from a standard, one-size-fits-all approach to a targeted, precision medicine strategy in which an individual prostate cancer patient’s tumor biology will guide choice of therapy will require careful planning and thought. The inclusion of PARP-targeted therapies before, after, with, or in place of standard hormonal therapies and chemotherapies will need to be defined so as to maximize antitumor effect and patient survival. Hopefully, application of these novel combinations in those most likely to benefit will ultimately lead to longer and better lives for patients with prostate cancer.

Financial Disclosure: Dr. Hussain is the principal investigator for a clinical trial of veliparib through the Cancer Therapy Evaluation Program (for AbbVie), and is collaborating on a clinical trial of olaparib for AstraZeneca.

David B. Solit, MD
Philip W. Kantoff, MD
Memorial Sloan Kettering Cancer Center, New York, New York

How an Ovarian Cancer Drug Came to Have ‘Breakthrough Therapy Designation’ for Prostate Cancer

With the emergence of precision medicine, clinicians can now take advantage of high-throughput tumor sequencing to identify driver mutations in individuals with cancer, with the goal of matching these with effective therapies. Since driver mutations can be shared across cancer types, precision medicine has also challenged the notion that cancer types, as defined by site of origin, are completely separate entities. One such example is the use of vemurafenib in multiple BRAF V600–mutant cancers. Another example is that of poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitors and prostate cancer. It is now recognized that DNA repair abnormalities, including and most notably BRCA2 mutations, are found frequently in the germline and as somatic mutations in the tumors in men with metastatic prostate cancer. Moreover, recent studies have demonstrated promising activity for olaparib—a drug approved for use in BRCA-mutated ovarian cancer—in men with castration-resistant disease and germline or somatic DNA repair abnormalities. This has led the US Food and Drug Administration to confer “breakthrough therapy designation” on olaparib, based on the strong belief that the drug will ultimately be approved for this indication.

What Questions Should Future Research on PARP Inhibitors for Prostate Cancer Focus on?

Many questions still remain unanswered. These include:

1) Given the pleiotropic effects of PARP inhibitors, which activities are the most critical and which PARP inhibitors are best for each disease/mutation scenario?

2) Have we identified the full gamut of DNA repair abnormalities that might respond to PARP inhibition?

3) Can we extend the spectrum of patients eligible for PARP inhibitors to those who are homologous recombination–proficient, by combining PARP inhibitors with therapies such as alkylating agents or antiangiogenic agents like cediranib?

4) Can we identify patients early on in their disease course in whom PARP inhibition may contribute to a curative strategy?

UPDATED 12/05/2020

Targeting PARP in Cervical Cancer


Associated Data

Supplementary Materials



Cervical cancer (CC) remains a major health problem worldwide. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutics in ovarian cancer. We explored the preclinical in vitro and in vivo activity of olaparib against multiple primary whole exome sequenced (WES) CC cells lines and xenografts.


Olaparib cell-cycle, apoptosis, homologous-recombination-deficiency (HRD), PARP trapping and cytotoxicity activity was evaluated against 9 primary CC cell lines in vitro. PARP and PAR expression were analyzed by western blot assays. Finally, olaparib in vivo antitumor activity was tested against CC xenografts.


While none of the cell lines demonstrated HRD, three out of 9 (33.3%) primary CC cell lines showed strong PARylation activity and demonstrated high sensitivity to olaparib in vitro treatment (cutoff IC50 values < 2μM, p=0.0012). Olaparib suppressed CC cell growth through cell cycle arrest in the G2/M phase and caused apoptosis (p<0.0001). Olaparib activity in CC involved both PARP enzyme inhibition and trapping. In vivo, olaparib significantly impaired CC xenografts tumor growth (p=0.0017) and increased overall animal survival (p=0.008).


A subset of CC primary cell lines is highly responsive to olaparib treatment in vitro and in vivo. High level of PARylation correlated with olaparib preclinical activity and may represent a useful biomarker for the identification of CC patients benefitting the most from PARPi.


Despite the implementation of prophylactic vaccination strategies against Human Papillomavirus (HPV) infection and advances in chemoradiation and immunotherapy, cervical cancer (CC) remains a major health problem in the United States with 13,240 new cases and 4,170 related deaths in 2018 []. Chemoradiation represents the standard of care for patients with locally advanced disease not suitable for curative surgery [] while the usual treatment for recurrent/metastatic CC is a combination of paclitaxel and cisplatin or paclitaxel, cisplatin and bevacizumab. These chemotherapy treatments, although not curative, result in median survival times of approximately one to 1.5 years []. Once patients progress after this initial therapy for recurrent or metastatic disease, options are limited (there are no FDA approved or NCCN level 1 or 2A therapies available). Identification of novel, effective therapies for CC patients with disease resistant to standard treatment modalities remains an unmet medical need.

In recent years, Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutic agents for ovarian cancer associated with defects in homologous recombination DNA repair (HRR) system []. PARP1 is one of the most abundant proteins among several members of the PARP family and multiple studies implicated PARP1 as having pleiotropic cellular functions, such as maintenance of genomic integrity, DNA repair and regulation of apoptotic and survival balance in cells []. Furthermore, the enzyme is involved in the PARylation of nuclear proteins (i.e., the post-translational modification process by which polymers of ADP-ribose (poly(adenosine diphosphate-ribose)) are covalently attached to proteins by PAR polymerase enzymes), recruitment of DNA repair factors and stabilization of chromatin for transcriptional regulation []. Importantly, since PARPi prevents repair of single strand breaks, causing DNA destabilization and eventual double strand breaks, cancer cells with deficient double strand repair (HRR) are particularly sensitive to PARPi []. Accordingly, based on preclinical and clinical results, in 2014 the US Food and Drug Administration (FDA) approved the first PARPi (i.e., olaparib) for treatment of patients with germline BRCA-mutated advanced ovarian cancer, who have been treated with three or more prior lines of chemotherapy. Since 2017, three PARP inhibitors (i.e., olaparib, rucaparib and niraparib), have received FDA approval in the ovarian cancer recurrent setting as maintenance therapy following platinum-based therapy [].

Although several clinical trials are currently underway investigating the clinical efficacy and safety of PARPi for various human malignancies, limited preclinical and clinical information is currently available on the potential activity of olaparib in CC patients []. Accordingly, in this study, we evaluated the preclinical activity of olaparib against multiple homologous recombination competent (HRD) primary CC cell lines (i.e., both squamous and adenocarcinoma) and xenografts. Furthermore, we also investigated possible mechanisms behind CC sensitivity to PARPi and elucidated the correlation between sensitivity to olaparib and PARylation activity.


Establishment of CC cell lines

Study approval was obtained from the Institutional Review Board (IRB), and all patients signed consent prior to tissue collection according to the institutional guidelines. Nine primary CC cell lines (Table 1) were established from fresh tumor biopsy samples and maintained at 37 °C, 5% CO2 in Keratinocytes-SFM (Gibco®, Life Technologies™), supplemented with prequalified human recombinant Epidermal Growth Factor 1–53 (EGF 1–53), Bovine Pituitary Extract (BPE), 10%, 1% penicillin/streptomycin (Mediatech, Manassas, VA), and 1% Fungizone (Life Technologies, Carlsbad, CA). Briefly, cervical tumor biopsies were obtained from all patients and viable tumor tissue was mechanically minced under sterile conditions in enzyme solution [0.14% Collagenase Type I (Sigma St. Louis, MO) and 0.01% DNAse (Sigma, 2000 KU/mg)] in RPMI 1640, and incubated on a magnetic stirring apparatus 40’ at room temperature. The resultant cell suspension was washed in RPMI 1640 plus 10% FBS and then washed in PBS. Tumors were staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Patient characteristics are noted in Table 1

Table 1

Characteristics and demographic data of cervical cancer cell lines.

Cell line Age RACE FIGO stage Histology HPV
CVX3 35 B IB2 SCC 16
CVX4 40 W IIA SCC 16
CVX5 42 W IB2 SCC 18
CVX7 22 H IB2 SCC 16
CVX8 29 W IB1 SCC 16
ADX1 33 W IB ADSQ 18
ADX2 33 B IB ACA 18
ADX3 25 W IB ACA 18
ADX4 47 B IB SCC 45

Homologous recombination deficiency (HRD) evaluation in CC cell lines

Log2-ratios of read counts in exonic intervals in whole exome sequenced (WES) tumor and normal samples [], were tabulated (Figure 1S). Intervals were determined from high coverage regions in the normal samples, and intervals that did not overlap with RefSeq annotations were removed, to ensure remaining intervals corresponded to known genic loci. SNP allele frequencies were calculated in these exonic intervals, using SNPs defined in the phase 3 1000 Genomes dataset (Figure 2S). The log2-ratios and allele frequencies were used to assess HRD status for each sample using an ad hoc scoring algorithm, similar to the one used in the ARIEL2 trial [].

Immunoblotting and antibodies

Cells were washed twice in ice-cold PBS and harvested with radioimmunoprecipitation assay buffer (RIPA) (50 mmol/L Tris–HCl, pH 8, 150 mmol/L NaCl, Triton X-100 1%, Na deoxycholate 0.5%, SDS 0.1%, MgCl 5 mmol/L in H2O) supplemented with Protease and Phosphatase Inhibitor (cat#78430, Thermo Fisher Scientific). Protein concentrations were measured by BCA Protein Assay Kit (Pierce™ #23225) to ensure equal loading. Proteins were denatured at 95°C for 5 minutes in Laemmli sample buffer (S3401; Sigma-Aldrich) and then resolved in SDS-PAGE electrophoresis, transferred on nitrocellulose, and blotted with corresponding antibodies. The antibodies used for western blotting were as follows: PAR (#4336, Trevigen), PARP (#9532, Cell Signaling Technology, Inc.), and GAPDH (#2118, Cell Signaling Technology, Inc.).

Cell viability assay

CC cell lines were plated at log phase of growth in 6-well tissue culture plates at a density of 80,000–100,000 cells/well. After 24 hours of incubation, cells were treated with Olaparib (AZD2281, LYNPARZA™, AstraZeneca) at a concentration of 0, 0.15, 1.5, 3, 12 μM. 72 hours after drug treatment, cells were harvested in their entirety, centrifuged and stained with propidium iodide (2 μl of 500 μg/ml stock solution in PBS). Count was performed using a flow-cytometry based assay to quantify percent viable cells as a mean ± SEM relative to untreated cells as 100% viable control. A minimum of three independent experiments per cell line was performed.

Cell-cycle analysis

After 48h incubation at the conditions described in Figure 4, cells were harvested and washed with ice-cold PBS, fixed in ice-cold 70% ethanol at −20°C for a minimum of 30 minutes to overnight. Subsequently, cells were washed in PBS, incubated with ribonuclease A (100 μg/ml) for 5 minutes at room temperature and stained with propidium iodide (20 μg/ml) in PBS. Cell-cycle phase distributions were analyzed with Flow-Jo software program (v. 8.7).


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A) Cell cycle assay on CVX5 after 48h Olaparib treatment at the following concentrations: 0.15, 1.5, 3 μM (p=0.00005) B) Cell cycle assay on CVX8 (representative resistant cell line) after 48h Olaparib treatment at the following concentrations: 0.15, 1.5, 3 μM (p>0.05).

Annexin V-FITC/PI double staining

Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) double staining was used to quantify apoptosis. Adherent cells were incubated with 0, 0.15, 1.5, 3 μM of olaparib for 72 hours, then harvested and collected. Cells were washed twice with ice-cold PBS and resuspended in 1× Binding Buffer at a concentration of 1×106 cells/ml. 5 μl of Annexin V-FITC and 5 μl of propidium iodide were added to 100 μl of the cell suspension. After 15 minutes of incubation, 400 μl of Binding Buffer were added to each cell suspension. Cells were analyzed by flow cytometry within 1 hour.

siRNA transfection

Cells were plated in 6 well plate in Keratinocytes-SFM (Gibco®, Life Technologies™), supplemented with prequalified human recombinant Epidermal Growth Factor 1–53 (EGF 1–53), Bovine Pituitary Extract (BPE), 1% penicillin/streptomycin (Mediatech, Manassas, VA), and 1% Fungizone (Life Technologies, Carlsbad, CA). 70–80% confluent cells were subjected to transfection. PARP1 siRNA and negative control siRNA were purchased from Ambion®, Life Technologies™. Briefly, the siRNA was incubated with Lipofectamine RNAiMAX reagent (Invitrogen, CA, USA) in OptiMEM medium for 20 minutes, then added to a monolayer of cells in Keratinocytes-SFM without antibiotics. Twenty-four hours after the transfection, cells were treated with scalar amounts of olaparib ranging from 1.5 μM to 400 μM. Cells were then counted by flow cytometry.

Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

RNA was obtained from cells after 48 hours of incubation with olaparib (Table 1S) using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (5 μg) was reverse-transcribed using Superscript III (Invitrogen). Quantitative PCR was carried out to evaluate the expression level of PARP-1 (PARP-1, Assay ID: Hs00242302_m1, Applied Biosystems) in all samples with a 7500 Real-Time PCR System (Applied Biosystems) following the manufacturer’s protocol. Each reaction was run in duplicate. The internal control GAPDH (Assay ID: Hs99999905_ml, Applied Biosystems) was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle (Ct) method was used for the calculation of amplification fold as specified by the manufacturer. Analyses were performed using SDS software 2.2.2 (Applied Biosystems/Life Technologies).

In vivo treatment

The in vivo antitumor activity of olaparib was tested in xenograft models. Briefly, four to six-week-old CB17/SCID mice were given a single subcutaneous injection in the abdominal region of 7 × 106 CVX5 cells in approximately 300 μl of a 1:1 suspension of sterile PBS containing cells and Matrigel® (BD Biosciences). Xenografted mice were randomized into treatment groups (6 mice each group) when mean tumor burden was 0.15–0.25 cm3, and dosing (vehicle PO or olaparib 10 mg/kg BID, PO) was delivered to the CVX5 xenografts for 4 weeks (7 days/week). Drug dosage was chosen according to previous studies []. Tumor and weight measurements of each mouse were recorded twice weekly. Mice were humanely euthanized when tumor volume reached 1.5 cm3 using the formula (width2 × height)/2. Animal care and euthanasia were carried out according to the rules and regulations as set forth by the Institutional Animal Care and Use Committee (IACUC).

Statistical analysis

Statistical analysis was performed using Graph Pad Prism version 8 (Graph Pad Software, Inc. San Diego, CA). The inhibition of proliferation in the CC cell lines after exposure to olaparib was evaluated by the two-tailed unpaired student t-test. Unpaired t-test was used to evaluate significant differences in the tumor volumes at specific time points in the in vivo experiments. Overall survival data was analyzed and plotted using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Differences in all comparisons were considered statistically significant at p-values < 0.05.

Olaparib suppresses CC cell lines growth

To evaluate the potential of PARP inhibitors on CC, we investigated the in vitro effects of olaparib on the growth of 9 primary CC cell lines using flow cytometric-based assay as described in the methods. As shown in Figure 1A,1B,1B, after 72 hours of incubation with increasing concentrations of olaparib, we found a progressive, dose-response decrease in cell proliferation in 33% of CC lines tested, with a significant difference in IC50 values between the sensitive and resistant group (p= 0.0012).


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A) In vitro proliferation assay overview of the established primary CC cell lines (n=9) B) Violin scatter dot plot representing grouped sensitive cell lines and resistant cell lines (p=0.0012) C) Western blot analysis displaying basal expression of PARP, PAR, and GAPDH in all nine CC cell lines.

Sensitivity to olaparib is strongly correlated to PARP activity

To better understand the mechanisms behind the sensitivity to olaparib in a subset of primary CC, we analyzed PARP and PAR basal expression in all nine CC cell lines as well as their mutation spectrum (i.e., HRD), as defined in the methods section. None of the tested CC cell lines demonstrated HRD. Indeed, within the nine CC cell lines, genomic loss of heterozygosity (LOH) results ranged from 0–12.3% (Table 2S), which falls short of the initial ARIEL2 cutoff of 14% (and the current revised cutoff of 16%) used to classify a tumor as HRD []. In contrast, as demonstrated in Figure 1C, using immunoblot (i.e., cells lysates were loaded in order from the most sensitive to the most resistant CC based on IC50 values previously obtained by flow cytometric-based assay) we found a direct correlation between basal expression level of PARP activity (PAR) and sensitivity to olaparib treatment. Indeed, CVX5, CVX1 and CVX3 (i.e., the 3 CC primary cell lines with the higher PARP expression of both PARP isoforms 116 and 89 kDa), consistently demonstrated the higher sensitivity to olaparib exposure in the in vitro experiments.

Silencing of PARP-1 elicits resistance to olaparib

To evaluate further the correlation between PARP-1 activity and sensitivity of CC to olaparib we transiently transfected CVX5 cells with PARP-1 siRNA and negative siRNA control as described in materials and methods section. After 72 hours of olaparib treatment, IC50 values of either PARP-1 siRNA and negative control siRNA transfected CVX5 cells were evaluated through flow cytometric-based assay as described in Methods. Validation of PARP-1 mRNA silencing in tumor cells was confirmed with q-real time PCR (Table S1). As shown in Figure 2, CVX5 cells transfected with PARP-1 siRNA from sensitive become highly resistant (i.e., IC50 from 8.69 μM to 513.2 μM) to olaparib treatment (p=0.0063).


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In vitro proliferation assay in PARP-1 silenced CVX5 cell line versus non-silenced control (p=0.0063).

Olaparib triggers apoptosis of CC in a dose-dependent manner

To gain better insight into the mechanism of PARPi activity, CVX5 was exposed to increasing concentration of olaparib (0.15, 1.5, 3 μM) for 48 hours before being harvested for Annexin V/PI staining. As shown in Figure 3, we demonstrated that olaparib at the dose of 1.5 μM and 3 μM induced apoptosis in 18% to 20% of cells, respectively, and tardive apoptosis in an additional 27.5% of cells (p<0.0001).


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Up Left (UL) and Up Right (UR) quadrants show single positive events for FL1-H (ANNEXIN V-FITC) and double positive events for FL1-H and FL2-H, respectively. Double positive events stand for tardive apoptosis, corroborated by the absence of events in Down Right (DR) quadrant (single positive for FL2-H representing cell necrosis) (p<0.0001).

Olaparib sensitivity is associated with G2/M cell cycle arrest

We next examined the cell cycle profiles of CVX5 (i.e., a representative olaparib-sensitive CC cell lines) and CVX8 (a representative olaparib-resistant CC cell line) after 24 hours of olaparib treatment. As shown in Figure 4A, starting at 1.5 μM of olaparib, 67.7% of CVX5 cells demonstrated a G2/M cell cycle arrest (in comparison to non-treated cells (i.e., 22.3%) (p=0.000061). This percentage increased at the dose of 3 μM olaparib (78.3%) (p=0.00005). In contrast, as demonstrated in Figure 4B, CVX8 cell cycle was not affected by olaparib treatment at any dose tested (16.2% cells in G2-M non-treated cells vs 13.5% cells in G2-M after 3 μM olaparib treatment) (p>0.05).

Olaparib PARP inhibition and PARP trapping on sensitive CC

Next, we analyzed PARP-1 and PAR expression in CVX5 cells by immunoblotting assay after exposure to different doses of olaparib (0.15 μM – 1.5 μM) at two different time points (24–48 hours). As shown in Figure 5, PARP expression increased after exposure to 1.5 μM olaparib at both time points while no significant variation was detected in PARP-1 mRNA expression level at 24 or 48 hours (Table S1). A dramatic reduction in PAR levels was detected at both doses of olaparib (0.15 and 1.5 μM) (Figure 5).


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Western blot analysis displaying expression of PARP, PAR, and GAPDH in CVX5 cells after 24–48 hours of treatment with 0.15 and 1.5 μM Olaparib.

Olaparib impairs CVX5 xenograft tumor growth in vivo

The in vivo effects of olaparib was determined by establishing xenografts from the primary CVX5 CC cell line. Briefly, after the tumors had reached the goal size, animals were randomized into treatment groups and treated as described in Materials and Methods. Tumor size was assessed weekly and mice were sacrificed if tumors became necrotic, reached a volume of 1.5 cm3, or mice appeared to be in poor health. Twice daily oral dose of olaparib 50 mg/kg was well tolerated with no clear impact on body weight compared with vehicle control (data not shown). As shown in Figure 6, mice undergoing olaparib treatment exhibited a significantly slower rate of tumor growth, compared to vehicle control starting at day 12 (p=0.0017). Furthermore, the overall survival was significantly prolonged in the treated group (Log Rank Mantel-Cox test p=0.008).


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A) In vivo tumor growth inhibition following 19 days dosing olaparib or vehicle of CVX5 injected xenografts (p=0.0017) B) overall survival (p=0.008).


The inhibition of PARP was initially demonstrated to determine ‘synthetic lethality’ in cancer patients harboring specific DNA repair defects, (i.e., BRCA1 or BRCA2 (BRCA1/2) mutations) causing deficiency in the cell homologous recombination (HR) repair system []. Accordingly, initial FDA approval was restricted to the treatment of patients harboring deleterious or suspected deleterious germline BRCA-mutated (gBRCAm) advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy. More recently, however, PARPi approval was expanded to maintenance therapy for patients with platinum-sensitive relapsed ovarian cancer, who responded to their second line regimen, regardless of BRCA1 or BRCA2 (BRCA1/2) mutation status [] []. This broader use of PARPi stems from the evidence that tumors that share molecular features with BRCA-mutant tumors (i.e., BRCAness) also exhibit different levels of defective homologous recombination DNA repair, and therefore will respond to PARP inhibition []. Importantly however, recent results from large prospective randomized clinical trials have demonstrated significant PARPi clinical activity also against patients harboring HR-competent/BRCA wild-type tumors [].

Unfortunately, while olaparib, rucaparib and niraparib are currently FDA-approved in ovarian cancer and multiple clinical trials are currently evaluating PARPi as single agents or in combination against multiple human tumors, limited information is currently available on the role of olaparib in CC patients. Accordingly, in this study, we thoroughly investigated the preclinically activity of olaparib against multiple primary CC cell lines in vitro and in vivo.

We found three of the nine primary CC cell lines to be highly sensitive to olaparib exposure with a cutoff IC50 value < 2μM []. To gain further insight into the molecular characteristics making these CC cell lines sensitive to olaparib treatment we evaluated their mutation spectrum (i.e., HRD), as well as their level of PARP1 expression [], and the potential role played by PARylation. Using the ARIEL2 study cutoff of 14% (and the current revised cutoff of 16%) used to classify a tumor as HRD [], we found none of the tested CC cell lines to demonstrate HRD. Importantly, we found the level of PARylation but not PARP1 expression in the tumors to consistently correlate with CC cell line sensitivity to olaparib. To prove the correlation between PARylation overexpression and olaparib sensitivity was causative, we downregulated PARP1 mRNA through PARP1 siRNA transfection in a representative cell line (i.e., CVX5 cell line) and analyzed the IC50 values in comparison to transfected CVX5 with a universal negative control siRNA. We found CVX5 transfected with PARP1 siRNA to gain high resistance to olaparib treatment (p=0.0063), confirming that PARP activity (PAR) is of utmost importance in determining olaparib sensitivity in CC cell lines. These results are similar to the results obtained by Michels et al., who also found a positive correlation between cellular PARylation levels and sensitivity to PARP inhibitors in non-small cell lung carcinoma cell lines []. Moreover, in agreement with our results, other groups demonstrated that in the absence of functional HR, PARP1 or PARP2 knockout cells are resistant to PARP inhibitors []. Taken together, these data combined with our findings in CC strongly suggest that determination of the level of PARP1 protein activity (i.e., PAR expression), may represent a biomarker potentially able to identify the most sensitive CC patients for treatment with PARPi. Accordingly, testing the possible link between PARP expression/activity and sensitivity to PARP inhibitors in the clinical setting may be warranted in future CC studies.

To better understand the functional mechanisms of olaparib in inhibiting CC cell growth, we performed cell cycle analysis experiments. We found olaparib, in a dose-dependent manner, to consistently arrest cell cycle in G2/M phase in all sensitive cell lines, ultimately preventing cells to going through the G1 phase. In contrast, no detectable alteration was found in the cell cycle of olaparib-resistant CC cell lines (i.e., CVX8). This effect of olaparib, as previously demonstrated in ovarian cancer, is explained by the PARP trapping mechanism, by which PARP inhibitors induce the formation of cytotoxic PARP–DNA complexes, preventing DNA replication []. When we investigated the mechanism of cell death in the CC cell lines exposed to olaparib, we found that only less than 1% of total cells demonstrated necrosis, corroborating the result that olaparib triggers and induces apoptosis in olaparib-sensitive CC cell lines.

To further elucidate the mechanism of action of olaparib against PARP, we analyzed PARP-1 and PAR protein expression in a representative cell line (i.e., CVX5) during olaparib treatment. Our immunoblot experiments clearly demonstrated a dose dependent increase of PARP1, as main consequence to olaparib exposure, further supporting an olaparib-induced PARP trapping phenomenon. In agreement with this interpretation, PARP-1 mRNA levels were not increased in any of the condition tested in any CC cell line. Taken together, our results support the notion of PARP-1 accumulation in cells treated with increasing concentrations of olaparib as main mechanism of action in CC. Importantly, when we evaluated the activity of olaparib in vivo in xenografted animals injected with CVX5, our result were confirmatory of the in vitro results with significant impairment of CVX5 tumor growth, and a significant increase in animal overall survival (p=0.008).

In conclusion, we demonstrated in vitro and in vivo activity of olaparib in a significant subset of CC primary cell lines and suggest that PAR expression may represent a novel biomarker for the potential prediction of PARPi response in patients with CC. Future studies with PARPi used alone or in combination with other targeted agents in patients with CC resistant to standard treatment modalities are warranted.

  • A subset of primary CC cell lines is highly sensitive to olaparib in vitro and in vivo

  • High PARylation activity correlates with sensitivity to olaparib in CC cell lines

  • Silencing of PARP-1 reverses CC cell line sensitivity to olaparib and induce resistance


Supplementary Material



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)

P. H. Thaker,1R. Salani,2W. E. Brady,3H. A. Lankes,3D. E. Cohn,2D. G. Mutch,1R. S. Mannel,4K. M. Bell-McGuinn,5P. A. Di Silvestro,6D. Jelovac,7J. S. Carter,8W. Duan,9K. E. Resnick,10D. S. Dizon,11C. Aghajanian,5 and P. M. Fracasso12Author informationCopyright and License informationDisclaimerThis article has been cited by other articles in PMC.Go to:


Preclinical studies demonstrate poly(ADP-ribose) polymerase (PARP) inhibition augments apoptotic response and sensitizes cervical cancer cells to the effects of cisplatin. Given the use of cisplatin and paclitaxel as first-line treatment for persistent or recurrent cervical cancer, we aimed to estimate the maximum tolerated dose (MTD) of the PARP inhibitor veliparib when added to chemotherapy.

Patients and methods

Women with persistent or recurrent cervical carcinoma not amenable to curative therapy were enrolled. Patients had to have received concurrent chemotherapy and radiation as well as possible consolidation chemotherapy; have adequate organ function. The trial utilized a standard 3 + 3 phase I dose escalation with patients receiving paclitaxel 175 mg/m2 on day 1, cisplatin 50 mg/m2 on day 2, and escalating doses of veliparib ranging from 50 to 400 mg orally two times daily on days 1–7. Cycles occurred every 21 days until progression. Dose-limiting toxicities (DLTs) were assessed at first cycle. Fanconi anemia complementation group D2 (FANCD2) foci was evaluated in tissue specimens as a biomarker of response.


Thirty-four patients received treatment. DLTs (n =1) were a grade 4 dyspnea, a grade 3 neutropenia lasting ≥3 weeks, and febrile neutropenia. At 400 mg dose level (DL), one of the six patients had a DLT, so the MTD was not reached. Across DLs, the objective response rate (RR) for 29 patients with measurable disease was 34% [95% confidence interval (CI), 20%–53%]; at 400 mg DL, the RR was 60% (n =3/5; 95% CI, 23%–88%). Median progression-free survival was 6.2 months (95% CI, 2.9–10.1), and overall survival was 14.5 months (95% CI, 8.2–19.4). FANCD2 foci was negative or heterogeneous in 31% of patients and present in 69%. Objective RR were not associated with FANCD2 foci (P =0.53).


Combining veliparib with paclitaxel and cisplatin as first-line treatment for persistent or recurrent cervical cancer patients is safe and feasible.

Clinical trial information


Targeting PARP in Endometrial Cancer

PARP Inhibitors for BRCA1/2 mutation-associated and BRCA-like malignancies

J-m. Lee,1,*J. A. Ledermann,2 and E. C. Kohn1Author informationArticle notesCopyright and License informationDisclaimerThis article has been cited by other articles in PMC.Go to:


Poly(ADP-ribose)polymerase inhibitors (PARPis) have shown promising activity in patients with BRCA1/2 mutation-associated (BRCA1/2MUT+) ovarian and breast cancers. Accumulating evidence suggests that PARPi may have a wider application in the treatment of sporadic high-grade serous ovarian cancer, and cancers defective in DNA repair pathways, such as prostate, endometrial, and pancreatic cancers. Several PARPis are currently in phase 1/2 clinical investigation, with registration trials now being designed. Olaparib, one of the most studied PARPis, has demonstrated activity in BRCA1/2MUT+ and BRCA-like sporadic ovarian and breast cancers, and looks promising in prostate and pancreatic cancers. Understanding more about the molecular abnormalities involved in BRCA-like tumors, exploring novel therapeutic trial strategies and drug combinations, and defining potential predictive biomarkers, is critical to rapidly advancing the field of PARPi therapy and improve clinical outcomes.Keywords: parp inhibitor, brca-like cancers, brca1/2 mutation, brca1/2 mutation-associated cancersGo to:


Progress has been made over the past two decades in the diagnosis, treatment, and prevention of cancer. A key component of progress in women’s cancers was the cloning of the BRCA1 and BRCA2 genes [12] and reporting of The Cancer Genome Atlas’ (TCGA) comprehensive molecular analyses of high-grade serous ovarian cancer (HGSOC) and breast cancers [34]. This knowledge is being translated into clinical opportunities through application of these new molecular definitions to tailor therapeutics uniquely to the individual patient.

Knowledge of BRCA1/2 mutation status in a patient has gone from a research question to demonstrated clinical utility directly affecting patient care. Dissection of their normal roles, both critical in normal DNA damage and repair, has led to better understanding of how their loss may cause or alter the course of cancer. Interestingly, neither knock-out nor knock-in models have demonstrated BRCA-1 or -2 to be independently causative in cancer development. They are embryonically lethal in knock-out settings, like many other tumor-suppressor genes [5]; selected knock-out is complementary to second genomic hits. The data for causality come from epidemiologic studies that define a tight relationship between deleterious BRCA-1 and -2 mutations (BRCA1/2MUT+) and development of breast and ovarian cancers [6], and increasingly with other cancers [7]. The seminal advance since the cloning and recognition of the relationship between loss-of-function mutations and breast and ovarian cancers is the identification, validation, and application of new biologically important molecular targets, poly-ADP ribose polymerase (PARP)-1 and PARP family members, and other proteins involved in homologous recombination (HR) repair of DNA damage.

DNA damage repair pathways

Six primary pathways of DNA repair have been identified [8]. They are variably used to address single- and double-stranded DNA break damage (SSB; DSB) from a variety of mechanisms of injury (Figure ​(Figure1);1); current results suggest pathway interaction and interdependence. Normal functions, such as cellular metabolism with associated generation of free oxygen radicals and reactive intermediates, ultraviolet light, therapeutic and ambient radiation, chemicals, and day-to-day replication errors, are common factors in the generation of DNA errors [9]. The function of the primary DNA repair pathways begins with sensing DNA damage, followed by recruitment of proteins involved in building the repair complexes [9]. Absence, reduction, or dysfunction of proteins in these pathways can be associated with loss of function of proper DNA repair. Four of the six repair pathways sense single-strand damage. HR, a high fidelity system, and nonhomologous end-joining (NHEJ), lower fidelity, are the two DSB repair programs [8]. BRCA1/2 mediate potentially rate-limiting events in HR [10]. It is now estimated that at least 15% of HGSOC occur in women with germline BRCA1/2MUT+, and another nearly 35% may have acquired defects in the HR pathway, including silencing by methylation, mutation in other repair genes, and activation of pathway inhibitors [311].

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Figure 1.

Double-strand break repair and single-strand break repair with poly(ADP-ribose)polymerase inhibitors (PARPis).

Multiple studies suggest that the loss of p53 function cooperates with the loss of BRCA1/2 in tumorigenesis [1213]. The normal function of p53 is to recognize DNA damage and arrest cell cycle to either allow repair or to shut the cell down [14]. Incomplete or inadequate DNA repair thus triggers cell death in normal cells. TCGA [4] describes molecular similarities between HGSOC and triple-negative breast cancers (TNBCs), including dysregulation of the p53 and Rb checkpoints, leading to alterations in the expression of cell proliferation genes, DNA synthesis, DNA damage repair, cell cycle regulation, and apoptosis. p53 mutations are found in nearly 90% of HGSOC and in 80% of TNBC, both cancers with BRCA1/2 loss-of-function cohorts [3415]. Chromosome breaks caused by loss of BRCA1/2 function activate p53-dependent checkpoint controls and/or apoptosis to prevent tumor formation. Selective pressure favors loss of p53 function to allow cell proliferation [16]. Mutant p53 facilitates G2/M transition, and cells acquire and propagate unrepaired DNA damage.

Loss of HR repair caused by loss of BRCA1/2 function leaves the cell needing alternative methods for DNA damage repair. SSB base excision repair (BER) is a primary back-up system for HR loss in response to BRCA1/2MUT+ [10]. The rate-limiting enzyme in BER, PARP-1, identifies the site of DNA injury and recruits repair complexes [17]. Recently, PARP-1 has been shown also to regulate NHEJ activity by holding this poor fidelity pathway in check [18], and to guide repair by forming PARP/DNA adducts [19]. These varied actions of PARP-1 form the increasingly strong basis for development of the PARP inhibitor class of agents (PARPi).Go to:

biology and beyond: parp inhibition

PARP-1 is a highly conserved enzyme focused to assist in the maintenance of genomic integrity [20]. It collaborates with PARG, polyADPribose glycohydrolase, required for hydrolysis and release of single-ADP-ribose moieties [20]. It has numerous other functions, including its cleavage and involvement in apoptosis, gene regulation through histone modification, and DNA decondensation for higher order chromatin function [21] and DNA repair [22]. The PARP-1 enzyme has been implicated in signaling DNA damage through its ability to recognize and rapidly bind to DNA SSB [23]; it also has been shown to participate in controlling the telomere length and chromosome stability [1724].

PARP-1 mediates BER by recruiting the scaffolding proteins XRCC1, DNA ligase III, and DNA polymerase ß [22]. The importance of PARP-1 in HR was shown in knock-out studies by a spontaneous increase in nuclear RAD51 focus formation [25], an event that signals active DSB repair. DNA-bound activated PARP-1 uses nicotinamide adenine dinucleotide (NAD+) to polyADPribosylate nuclear target proteins, the site of DNA damage, including topoisomerases, histones, and PARP-1 itself, to signal the need for both DNA SSB and DSB repair [26]. This observation suggests loss of PARP-1 activity where HR is compromised would lead to adverse consequences for the tumor cell.

New findings implicate PARP-1 as a negative regulator of NHEJ. Patel et al. [18] reported that PARP inhibition induces phosphorylation of DNA-dependent protein kinase cs (DNA-PKcs), a rate-limiting step in NHEJ activation. PARP-1-directed NHEJ may occur more selectively in HR-deficient cells where there is a default to secondary pathways. Implications of this include reversal of the genomic instability reported in HR-deficient cells after PARP inhibition. Murai et al. [19] showed PARP inhibitors trap PARP-1 and -2 at damaged DNA where the PARP–DNA complexes were more cytotoxic than unrepaired SSB, implicating PARPi as direct DNA poisons.

BRCA-like behavior and HR dysfunction

Understanding DNA repair biology has allowed us to identify patient subsets with high potential for response PARPi treatment. The marked susceptibility of patients with BRCA1/2MUT+-associated cancers has validated BRCA1/2MUT+ as a predictive biomarker for PARPi response [27]. Tumors in patients with germline BRCA1/2MUT+ contain a second, somatic loss of BRCA1/2, following the Knudson Hypothesis [28]; this occurs as a result of genomic injury and generally incorporates part or all of the second BRCA allele. This leaves the tumor tissue homozygous null for functional BRCA1/2, with impaired HR function. Fong et al. [27] were the first to confirm this link clinically, demonstrating that BRCA1/2MUT+-associated breast, ovarian, and prostate cancer patients receiving the olaparib had a 63% likelihood of clinical benefit. This led to the broad recognition of HR dysfunction (HRD) as a functional biomarker, and opened the door to examine phenocopy susceptibility. Phenocopy patients, those with HRD not caused by BRCA1/2MUT+, are those described as having BRCA-like behavior [29].

BRCA-like behavior has both molecular and clinical characteristics. Many mechanisms reducing BRCA1/2 function and resulting in BRCA-like behavior have been identified. Examples include BRCA1 promoter methylation [11–35% of epithelial ovarian cancers (EOCs)], Fanconi F (FANCF) methylation (5∼20%), and loss or reduction in FANCD2 [30], or other proteins necessary for HR [3132]. Nearly always associated with this level of HRD is an obligate mutation in p53 and frequent c-myc amplification. Loss of function of the suppressor gene, PTEN, has been shown to yield BRCA-like behavior, more common in breast and prostate cancers [3334]. Coexpression of BRCA1MUT+ and loss of PTEN protein expression were reported to occur in 82.4% of 34 breast tumor biopsies, suggesting that PTEN loss may be a common contributing event causing HRD [33]. Increased PARPi susceptibility was shown in a series of cell lines with PTEN mutation or haploinsufficiency, confirmed in xenograft experiments using the PARPi, olaparib. There is also clinical evidence that olaparib may have a therapeutic utility in PTEN-deficient endometrioid endometrial cancer [35]. These studies provide evidence that PTEN loss of function is a potential predictive biomarker of PARPi responsiveness.

Common clinical manifestations complement the molecular characteristics of BRCA-like behavior. The first BRCA-like behavior identified is susceptibility to platinum and other DNA damaging agents. This was initially inferred from studies demonstrating improved long-term survival of women with BRCA1/2MUT+-associated EOC receiving platinum-based combination chemotherapy [36]. Intra- and inter-strand platinum-DNA crosslinks can create torsion on the double helix and lead to DSBs [31], requiring HR for proper and successful correction. Without repair, further genomic injury is sustained, leading to cell death. Reports also describe increased overall survival and progression-free survival (PFS) for mutation carriers receiving other DNA-damaging agents, such as pegylated liposomal doxorubicin (PLD) [3738]. Overall survival with PLD alone was nearly double that expected from large trials in a non-selected (general) population (median PFS 7.1 months; 95% CI 3.7–10.7), and similar findings were reported in a retrospective analysis of outcome following PLD in women who were BRCA1/2 germline mutation carriers and those considered not to harbor a germline mutation [38]. Subsequently, these characterizations have led to population evaluations, now suggesting that HRD occurs in up to 50% of HGSOC [113940] and 20% of TNBC [41]. Dissection of these clinical and molecular data will inform further study design and improve therapeutic application of PARPi.Go to:

updating clinical applications of PARP inhibitors

Multiple PARPis are in clinical development as single agents and/or in combination therapy (Table ​(Table1).1). The most common PARPi chemistry is that of reversible NAD mimetics, with differences in bioavailability and molar equivalence of PARP enzyme inhibition. There are at least six agents under study in this class; iniparib (BSI-201) is another compound that is not a true PARPi [42]. The loss of BER capacity produced by PARPi has prompted evaluation of these drugs as potential enhancers of DNA damaging cytotoxic agents, such as alkylating agents or radiation therapy, leading to new directions for combination therapies [1819].


Active PARP is under development

PARPiTreatmentCancer typesPhase
Olaparib (AstraZeneca)-Monotherapy
-Combinations with cytotoxic chemotherapy
-Combinations with targeted agents
-Combinations with RT
BRCA1/2MUT+ associated
BRCA-like tumors,
Advanced hematologic malignancies and solid tumors,
Maintenance study following remission in platinum sensitive OvCa (pending)
Veliparib (Abbott)-Monotherapy
-Combinations with cytotoxic chemotherapy
-Combinations with targeted agents
-Combinations with RT
BRCA1/2MUT+ associated BrCa/OvCa,
BRCA-like tumors,
Advanced hematologic malignancies and solid tumors
BMN 673 (BioMarin)– MonotherapyAdvanced hematologic malignancies and solid tumorsI
Rucaparib (Clovis)-Monotherapy
-Combinations (carboplatin)
Advanced solid tumors,
Recurrent OvCa,
BRCA1/2MUT+ associated BrCa/OvCa
CEP-9722 (Cephalon)-Monotherapy
-Combinations with cytotoxic chemotherapy
Advanced solid tumorsI
Niraparib (MK-4827) (TesaroBio)-Monotherapy
-Combinations (temazolomide)
Advanced hematologic malignancies and solid tumors,
BRCA1/2MUT+ associated and HER2 negative BrCa,
Maintenance study following remission in platinum sensitive OvCa (pending)

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*OvCa, ovarian cancer; BrCa, breast cancer; RT, radiation therapy.

Initial dose-finding trials have demonstrated significant clinical activity of PARPi especially in BRCA1/2MUT+ breast and ovarian cancers [4346]. This suggests that BRCA 1/2MUT+ is a genetic marker for targeted therapy, similar to other therapies targeted against loss-of-suppressor function mutations that have been shown to have clinical benefit. Angiogenesis inhibition provided benefit in germline Von Hippel Landau mutation-related renal clear cell cancer, shown to have a VHL-mediated hypoxia-inducing factor 1α-VEGF drive [47]. Similarly, activating mutations of RET are associated with the pathogenesis and vandetanib-sensitivity of medullary thyroid cancer [48]. Current clinical development for PARPi builds upon these observations. The patient populations targeted in PARPi clinical trials include patients with BRCA1/2MUT+ cancers, BRCA-like cancers, and those with recognized susceptibility to DNA-damaging agents, but without BRCA-like association, such as lung or pancreas cancers (Table ​(Table22).

Table 2.

Ongoing clinical trials of PARPis for other malignancies, except breast and ovarian cancers

Cancer typeSubtypesPARP inhibitorPhase
GI malignanciesColorectal cancerVeliparib + TMZ
Olaparib + irinotecan
Pretreated colorectal cancer stratified by Microsatellite Instability (MSI)Olaparib monotherapyI/II
Gastric cancerVeliparib + FOLFIRII/II
Gastric cancer with low ATM protein levelPaclitaxel +/− olaparibII
Esophageal cancerOlaparib + RTI
Metastatic pancreatic cancerOlaparib + Gemcitabine
Veliparib + modified FOLFOX6
    + gemcitabine
    + gemcitabine/IMRT
Veliparib for BRCA or PALB2 mutated pancreatic cancer
Gem/cis +/− veliparib
Advanced liver cancerVeliparib + TMZII
Lung cancerSmall cell lung cancerTMZ +/− veliparib
Cisplatin/etoposide +/− veliparib
Stage III surgically unresectable NSCLCVeliparib + RT
   + carbo/taxol,
   + cis/gem
Olaparib + RT +/− cisplatin
EGFR mutation positive advanced NSCLCGefitinib +/− olaparibI/II
Advanced NSCLCOlaparib
Carbo/taxol +/− veliparib
Head and Neck (H&N) cancerLocally advanced H&N cancerOlaparib and cetuximab/RTI
Gynecologic cancerRecurrent or persistent cervix cancerVeliparib + cisplatin/paclitaxel
   + topotecan
Hematologic malignanciesRefractory multiple myelomaVeliparib + bortezomib/dexamethasoneI
Acute leukemiaVeliparib + TMZ
Veliparib + topotecan +/− carboplatin
Refractory lymphomaVeliparib + topotecanI
Advanced MCLCEP-9722 + cis/gemI
Advanced hematologic malignanciesBMN673 monotherapy
E7449 versus E7449 + TMZ versus E7449+ carbo/taxol
Prostate cancerMetastatic castration resistant prostate cancerVeliparib + TMZ
Abiraterone +/− veliparib Olaparib
Glioblastoma multiformeRelapsed GlioblastomaOlaparib + TMZI
MelanomaMetastatic melanomaVeliparib + TMZ
Olaparib + dacarbazine
E7449 versus E7449 + TMZ versus E7449+ carboplatin/paclitaxel
SarcomaRecurrent Ewing’s sarcomaOlaparibII
Advanced solid tumorsVeliparib + low dose cyclophosphamide
   + capecitabine/oxaliplatin
   + mitomycin C
   + carbo/taxol
   + gemcitabine
   + carbo/gem
Olaparib + topotecan
   + cis/gem
   + PLD
BMN 673
E7449 versus E7449 + TMZ versus E7449+ carbo/taxol

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*TMZ, temozolomide; carbo/taxol, carboplatin/paclitaxel; cis/gem, cisplatin/gemcitabine.

Initial phase I/II clinical trials demonstrated single-agent activity of olaparib in BRCA1/2MUT+ breast, ovarian, and prostate cancers, and recurrent HGSOC; [274449], no single agent response data have yet been reported for CEP-9722 (Table ​(Table3).3). The study by Gelmon et al. [48] clearly showed that patients with platinum-sensitive HGSOC responded to olaparib without a BRCA1/2 germline mutation. Ledermann et al. [50] recently reported maintenance olaparib significantly improved PFS in a randomized, placebo-controlled, phase II trial in platinum-sensitive HGSOC following a response to two or more lines of platinum-based therapy [50]. They demonstrated a nearly doubling of median PFS post chemotherapy (8.4 versus 4.8 months) and a 65% reduction in risk of disease progression. An interim survival analysis [51] with 58% maturity showed difference between olaparib and placebo, notably in the BRCA1/2MUT+ with a hazard ratio (HR) of 0.18 (95% CI 0.11–0.31) and with a median PFS of 11.2 versus 4.3 months, respectively. Overall survival did not show difference in this group, (HR = 0.74; median: 34.9 versus 31.9 months) probably due to 22.6% of patients on placebo switched to olaparib. As a result of these findings registration trials are being developed with olaparib and other PARPi as maintenance therapy following treatment of platinum-sensitive relapsed ovarian cancer. These types of maintenance study may even be taken into front-line therapy for selected patients.

Table 3.

Single-agent activity with PARPi in phase I/II studiesa

PhasePatients population (number)Dose and scheduleObjective Response (OR) rateSurvival
II [50]Platinum-sensitive-relapsed
OvCa (265 patients)
Olaparib 400 mg bid versus placebo12 versus 4%PFS:
8.4 versus 4.8 months
OS: (interim analysis)
29.7 versus 29.9months
II [37]Recurrent OvCa/ BRCA1/2mut (97 patients)Olaparib 200 mg bid versus 400 mg bid versus PLD 50 mg/m2 IV every 28 days31 versus 25% versus 18%PFS:
6.5 months versus
8.8 months versus
7.1 months
II [49]OvCa (63 patients; 17/63 BRCA1/2mut+)Olaparib 400 mg bdOverall 29% (18/63);
41% in BRCAmut (7/17)/24% in non-BRCAmut (11/46)
II [45]Recurrent OvCa/BRCA1/2mut(57 patients)Olaparib 400 mg bd (33 patients) versus 100 mg bd (24 patients)33% versus 13%PFS:
5.8 months versus 1.9 months
II [44]Advanced BrCa/BRCA1/2mut (54 patients)Olaparib 400 mg bd (27pts) versus 100 mg bd (27pts)41% versus 22%PFS:
5.7 months versus 3.8 months
I [43]Recurrent OvCa/BRCA1/2mut (50 patients)Olaparib
40–600 mg bd (dose escalation) and 200 mg bd (dose expansion)
Overall 40%;
CBR 46%;
CBR 69% in platinum-sensitive (13 patients),
CBR 45% in platinum resistant (24 patients),
CBR 23% in platinum refractory (13 patients)
Response duration: 28 weeks
II [52]Recurrent OvCa/BRCA1/2mut (51 patients)Veliparib
400 mg bd
I [46]Recurrent solid tumors (100 patients; 29/100 patients BRCA1/2mut+)Niraparib 30 mg–400 mg qd (dose escalation)40% in
OvCa (8/20 patients)
50% in
BrCa (2/4 patients)
CBR 43% in CRPC (9/21 patients: 5PTEN loss and 1BRCAmut+)
Response duration: 387 days (range 159–518) in BRCAmut+
OvCa; 132 and 133 days in BRCAmut+
254 days(range 124–375) in CRPC
I [53]Recurrent solid tumors (29 patients; 11/29 patients BRCA1/2mut+))Rucaparib
40 mg–500 mg qd (dose escalation)
2 PR (2 patients with BRCAmut+)
10 SD (9/10 patients with BRCAmut+)
I [54]Advanced solid tumors (39 patients; 25/39 patients BRCA1/2mut+)BMN673
25 µg–1100 µg qd (dose escalation)
RECIST and/or CA-125 responses in 11/17 patients with BRCA1/2mut+ OvCa;
ORR: 2/6 patients with BRCA1/2mut+ BrCa
I [55]Advanced solid tumors (27 patients)CEP-9722
150–1000 mg qd (dose escalation)
Only safety data reportedNA

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aNA, not applicable; CBR, clinical benefit rate; CRPC, castrate-resistant prostate cancer.

The greatest clinical experience to date is with olaparib monotherapy. It generally well tolerated at doses of 400 mg twice daily in capsule formulation with many patients able to take the drug for several years. A new tablet formulation [56 ], reducing the number of pills that need to be taken is being assessed. PK data including AUC0−T and Cmin from 300 mg and 400 mg tablet doses matched or exceeded the 400 mg capsule dose, and 300 mg tablet is expected to be incorporated into further studies in mid-2013. PARPis have been tested in combination with various DNA damaging agents. Studies have shown clinical benefit and interactive adverse events, including bone marrow toxicity and fatigue [274357]. Class-based adverse events also include fatigue, headache, nausea, and reflux in 25–40% of patients. Early reports also suggest a possible increased clinical benefit in combination therapy, that may out balance the toxicities [5758]. Continued follow-up and diligence are needed to define the risk of long term PARPi therapy.

Current therapeutic directions for PARPi are focused at designing combinations, determining optimal timing of therapy and breadth of application of this key class of agents to and beyond mutation carriers. Agents selected for the combination study include those likely to cause replication fork injury or further DNA damage, and anti-angiogeneic agents. Hypoxia was shown to cause DNA damage when a second DNA hit was included in a mouse model [59]. We exposed microvascular endothelial cells in vitro to the VEGF receptor antagonist, cediranib (AZD2171), in combination with olaparib, demonstrating a cooperative inhibition of angiogenesis (Kim and Kohn, unpublished data). Surprisingly, interactive anti-invasive activity was observed with this combination against a p53-mutant HGSOC cell line, OVCAR8. A phase I study of olaparib and cediranib showed clinical promise [60], and a multi-institutional randomized phase II study is in progress (NCT01116648). Additionally, a phase I study of continuous daily olaparib with bevacizumab was generally well tolerated in patients with advanced solid tumors [61].

Phase I/II studies are ongoing with PARPi and a variety of agents (Table ​(Table1).1). A phase I study of olaparib with carboplatin (AUC4/5) showed clinical benefit in 85% of 27 women with BRCA1/2MUT+-associated recurrent breast and ovarian cancers [58]. A randomized, phase II study of olaparib with paclitaxel (Taxol) and carboplatin (AUC4) followed by olaparib maintenance resulted in a significant improvement in PFS compared with paclitaxel, Bristol-Myers Squibb (New York) and carboplatin, Bristol-Myers Squibb (New York) (AUC6) alone in women with platinum-sensitive recurrent HGSOC (HR = 0.51; median PFS 12.2 versus 9.6 months) [62]. This suggests that combining olaparib with carboplatin required a dose modification of both drugs, illustrated the potential for toxicity interaction with DNA active agents. There was no difference in PFS during the period of chemotherapy in this trial; differences emerged in the maintenance phase. The optimal dosage, scheduling, and sequencing of PARPis and cytotoxic agents require carefully designed clinical trials linked to preclinical studies that specifically address the above issues.

This promising therapeutic potential has elicited considerable interest in clinical development of the PARPi class. Early clinical data also suggest that a BRCA-like gene expression profile may correlate with clinical responses to the platinum drugs in patients with sporadic EOC [6364]. Prospective validation and optimization of these signatures in a broad array of cancers, and appropriate selection of a patient population are imperative to achieve the full potential of PARPis.

challenges to PARP inhibitor development

The incorporation of targeted agents into therapy of BRCA1/2MUT+ and BRCA-like cancers presents challenges. First is development of a mechanism with which to identify patients who are most likely to benefit. Discovery and validation of predictive biomarkers is an active area of ongoing research. Biomarkers for patient selection or stratification are recommended by the US Food and Drug Administration for approval of new targeted drugs. Loss of BRCA1/2 expression, generally by demonstration of a deleterious germline mutation, is a validated predictive biomarker. Routine testing of patients is being increasingly adopted as up to 17% of patients with HGSOC, the most common form of ovarian cancer, have germline mutations [65]. However, BRCA1/2 mutation testing does not identify the full range of potentially susceptible patients, and it requires a validated predictive BRCA1/2 mutational testing tool. BRCA1/2 loss in the tumor by mutation or methylation may also be inferable by loss of BRCA1/2 protein expression demonstrated by immunohistochemical staining, leaving reduction in BRCA1/2 protein expression as a potential predictive tool [39].

The histone protein H2AX becomes rapidly phosphorylated and concatemerizes at nascent DNA DSBs [66]. This creates a focus for accumulation of DNA repair and chromatin remodeling proteins. DSBs can be labeled with an antibody to the phosphorylated form, γH2AX, and extent of DSB estimated from the number of labeled foci (Figure ​(Figure2)2) [66]. RAD51 is instrumental in initiation of assembly of HR repair proteins at the site of DNA injury [67]. Formation of nuclear RAD51 foci can be assessed by immunofluorescence and is a marker of HR competence. Formation of γH2AX and/or RAD51 foci after DNA damage has been suggested as pharmacodynamic biomarkers of PARPi activity; demonstrating that a change in these parameters early in treatment may be examined as potential predictive biomarkers. A phase 1 study of veliparib and topotecan showed an increase in γH2AX focus formation by immunofluorescence in circulating tumor cells from seven of nine patients [68], with no correlation to clinical outcomes. Inhibition of RAD51 focus formation by PARPi was shown in vitro in EOC ascites primary cultures and correlated with response to PARPi [69]. This suggests that the lack of RAD51 foci may indicate potential drug response [70].Open in a separate windowFigure 2.

γH2AX binds to DNA DSBs and RAD51 initiates repair protein assembly in the homologous recombination (HR) pathway.

Predictive biomarkers applied to readily available bioresources, such as archival tissue or non-tumor tissue, have been proposed. Changes in PAR (poly ADP Ribose) incorporation into peripheral blood mononuclear cell DNA were evaluated as a putative early on-treatment pharmacodynamic measure; while present, there was no relationship to clinical outcomes [57]. Basal levels of PAR vary in different cells, reflecting their relative capacity for DNA repair, and requiring demonstration of change in PAR concentrations over time. Hence, identifying an accurate measure of HR potential for application as a predictive biomarker remains necessary to guide administration of PARPi.

Dissecting and defining mechanisms of development of resistance to PARPis, and whether this portends potential collateral resistance to other DNA damaging agents is the second challenge. Acquisition of a secondary mutation in BRCA1/2 that allows BRCA1/2 gene read-through and yields a functional protein has been demonstrated in cell lines and some patients; this was correlated with loss of susceptibility to PARPi treatment [71]. A second, preclinically defined method of resistance is loss of function of 53bp1 [72], a key protein in the NHEJ pathway. Whether or not 53bp1 expression can be used as a selective or predictive biomarker is yet to be determined. Understanding the mechanism(s) of resistance to PARPi will lead to optimal application and sequencing of PARPi and platinum compounds. Studies are needed to evaluate outcomes to subsequent chemotherapies in patients who have received PARPis [73].Go to:


Several PARPis are under investigation and it is anticipated that this novel and exciting new class of compounds will ultimately receive regulatory approval in select subsets of cancers. This class of agents has tolerable toxicity profiles and has been given to patients for long periods. Clinical benefit has been observed in patients with BRCA1/2MUT+-associated cancers and BRCA-like phenotypes in germline mutation-negative patients. It is for these patients, in particular, that predictive markers for HR deficiency and response to PARPi are needed, so that patients can be selected for therapy. Understanding more about the molecular abnormalities involved in BRCA-like tumors will be critical to advance the field of PARP inhibition therapy and in improving patient selection and consequent clinical outcomes.

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