Anti-Tumour TreatmentRestored replication fork stabilization, a mechanism of PARP inhibitor resistance, can be overcome by cell cycle checkpoint inhibition
Introduction
Poly(ADP-ribose) polymerase (PARP) inhibition is at the forefront of cancer treatment, particularly in cancers with homologous recombination (HR) repair defects such as BRCA mutations [1], [2]. PARP1 is the most abundant PARP family member and is involved in multiple DNA damage repair pathways, including base excision repair (BER), HR repair, and non-homologous end joining (NHEJ) [3], [4]. Upon sensing DNA damage, PARP1 undergoes a conformational change to increase its catalytic activity for adding poly(ADP-ribose) chains (PARylation) to various DNA repair enzymes, histones and itself [5], [6]. PARP2 is less abundant and contributes 5% to 10% of the total PARP activity [7], [8]. AutoPARylation of PARP1 and PARP2, and PARylation of chromatin proteins promotes recruitment of repair factors and releases PARP1 and PARP2 from DNA to allow repair [5], [9]. All clinically active PARP inhibitors (PARPis) are designed to compete with NAD+, a substrate of poly(ADP-ribose) chain, and inhibit the enzymatic activity of PARP1 and PARP2 [10].
Defects in HR repair offer a therapeutic opportunity in which DNA repair inhibitors, e.g. PARPis, can be used to induce lethal DNA double stranded breaks (DSBs). PARPis induce DSBs via catalytic inhibition [1], [2] and PARP-DNA trapping [11], [12], [13], by which PARPis prompt synthetic lethality in BRCA deficient cells. This synthetic lethality due to BRCA loss and PARPi has been extensively investigated in the preclinical and clinical settings, particularly in BRCA mutated ovarian cancer [14], [15], [16], [17], [18].
Ovarian cancer is the most lethal gynecologic cancer among women world wide accounting for an estimated 152,000 deaths annually[19], [20]. Molecular profiling has identified that nearly 40% of high grade serous ovarian cancer (HGSOC) have mutations in HR genes [21], [22], [23]. Results from clinical trials investigating the benefit of PARPis in ovarian cancer led to the United States Food and Drug Administration approving three PARPis, olaparib, rucaparib and niraparib. Olaparib and rucaparib are approved for the treatment of germline BRCA and both germline and somatic BRCA mutated advanced ovarian cancer patients, respectively, who have previously been treated with chemotherapy [15], [24]. Also, all three PARPis are licensed for use in maintenance treatment of recurrent ovarian cancer with complete or partial response to platinum-based therapy [25], [26], [27], [28]. Two additional PARPis, talazoparib and veliparib, are in advanced clinical trials. PARPi treatment however primarily results in partial tumor regression with rare complete responses and most overall responses are short lived (<1 year) with the emergence of resistance [29]. Work is now ongoing to optimize PARPi combination approaches to broaden the target patient population and to avoid development of resistance. Combination with cell cycle checkpoint inhibitors (hereafter described as cell cycle inhibitors) is becoming a testable therapeutic option to enhance the anti-tumor activity of PARPis.
Cells initiate a multitude of responses to protect the genome and ensure survival in response to DNA damage [30]. These responses include activation of cell cycle checkpoints, subsequent cell cycle arrest to provide the cell time to repair damaged DNA, and activation of the appropriate DNA repair mechanisms to efficiently complete repair. DSBs induced by PARPis are generated during S phase through collision of replication forks with unrepaired SSBs and PARP-DNA trapping lesions and would normally result in halting of the S phase checkpoint [13]. However, ovarian cancer, like many others, have mutant or null p53 causing dysfunction of the p53-dependent S phase checkpoint [22]. These cancers instead rely heavily on G2 checkpoint stoppage to facilitate DNA damage repair (Fig. 1) [31]. ATR (ataxia telangiectasia and Rad3-related) is a central checkpoint protein kinase that is activated by single strand DNA (ssDNA) damage, including the resected ends of DNA DSBs and stalled replication forks. ATR activation induces a global shutdown of origin firing and slows down fork speed through activation of checkpoint kinase 1 (CHK1; a critical component of G2 checkpoint arrest) and inactivation of cyclin-dependent (CDK), specifically CDK1 and CDK2 (CDK1/2) [32], [33]. WEE1 kinase, similarly integral for the G2 checkpoint, also keeps CDK1/2 inactive by phosphorylating CDK1/2 directly [34]. Therefore, the combination of cell cycle (ATR, CHK1, and WEE1) inhibitors with PARPis limits the time given to repair DNA, by restored HR, and promotes replication of damaged DNA resulting in cell death. This indication has spurred several clinical trials combining PARPis and cell cycle inhibitors (Table 1).
Recent studies implicated replication fork stabilization (hereafter described as fork protection) as a compensatory PARPi resistance mechanism in the absence of HR competency [35], [36], [37]. Clinically, fork protection may pose a challenge as this process can continue to promote the development of PARPi resistance even though HR is abrogated by ATR, CHK1, and WEE1 inhibitors. Therefore, it is essential to target both restored HR and fork protection to fully combat PARPi resistance with combination therapy. In this review, we address the differential roles of ATR, CHK1, and WEE1 in fork protection and the therapeutic benefit of combining PARPis with cell cycle inhibitors. Additionally, we discuss the potential for the continued development of PARPi resistance if both restored HR and fork protection are not addressed.
Section snippets
Cooperative fork destabilization and synthetic lethality
Stabilization of stalled DNA replication forks, which includes the process of replication fork reversal, protection and restart, is gaining attention in the field of DNA damage response (DDR; Fig. 2) [38], [39]. Following replication perturbation, replication forks remodel into four-way junctions, known as fork reversal, to allow more time for repair of DNA lesions, avoid conversion of single stranded breaks (SSBs) into DSBs, and enable excision repair by re-positioning a lesion in
Restored fork protection as a mechanism PARPi resistance
Stabilization of stalled DNA replication forks is recently identified as mechanism of PARPi resistance [37], [44], [48], [49]. Reversed forks are extensively degraded in cells with defective BRCA2, RAD51, FANCD2 or FANCA, through uncontrolled resection by the MRE11 and EXO1 nucleases [50], [51], [52], [53]. Defects in BRCA2, RAD51, FANCD2, and FANCA are also known to sensitize cancer cells to PARPis [15], [54], [55]. Fork degradation by MRE11 in BRCA1 and/or BRCA2 deficient cells is promoted by
Mechanisms of resistance to PARPis by HR restoration
Whereas fork protection is a burgeoning mechanism of PARPi resistance, restoration of HR is a well-known PARPi resistance mechanism [56], [57]. HR function is restored by secondary reversion mutations of BRCA1, BRCA2 and RAD51 isoforms [56], [57]. Clinically, secondary mutations restoring BRCA function were found in patients with germline BRCA mutation associated ovarian and breast cancer upon acquired resistance to PARPis and/or platinums [58], [59]. Additionally, four RAD51C secondary
Therapeutic potential of cell cycle inhibitors in combination with PARPis
Combinations of PARPis with different ATP-competive cell cycle inhibitors are being investigated in clinical trials (Table 1). The rationale of these combinations is to limit the time the cell has to repair damaged DNA and to dysregulate replication, resulting in cell death.
Recent data indicate that ATR may function independently of CHK1 to protect replication forks [64], [65]. When cells were analyzed for replication induced DSBs in response to UV irradiation, only ATR inhibition with caffeine
Conclusion
The use of PARPis are gradually evolving, including combination strategies with cell cycle checkpoint inhibitors in ovarian cancers with and without BRCA mutations. An improved understanding of the mechanisms underlying PARPis and cell cycle inhibitors clinical resistance will be important to enable the development of new approaches to increase efficacy. In particular, developing biomarkers to identify tumors with heightened HR and fork protection ability may classify a subgroup of patients who
Conflict of interest
The authors declare no potential conflicts of interest.
Acknowledgements
We thank Dr. Elise C. Kohn, Cancer Therapy Evaluation Program, National Cancer Institute for her careful review.
Financial support
This research was supported by the Intramural Research Program of the National Cancer Institute (NCI), Center for Cancer Research (CCR; Grant No. ZIA BC011525 [JML]).
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