Restored replication fork stabilization, a mechanism of PARP inhibitor resistance, can be overcome by cell cycle checkpoint inhibition

Abstract

Poly(ADP-ribose) polymerase (PARP) inhibition serves as a potent therapeutic option eliciting synthetic lethality in cancers harboring homologous recombination (HR) repair defects, such as BRCA mutations. However, the development of resistance to PARP inhibitors (PARPis) poses a clinical challenge. Restoration of HR competency is one of the many molecular factors contributing to PARPi resistance. Combination therapy with cell cycle checkpoint (ATR, CHK1, and WEE1) inhibitors is being investigated clinically in many cancers, particularly in ovarian cancer, to enhance the efficacy and circumvent resistance to PARPis. Ideally, inhibition of ATR, CHK1 and WEE1 proteins will abrogate G2 arrest and subsequent DNA repair via restored HR in PARPi-treated cells. Replication fork stabilization has recently been identified as a potential compensatory PARPi resistance mechanism, found in the absence of restored HR. ATR, CHK1, and WEE1 each possess different roles in replication fork stabilization, providing different mechanisms to consider when developing combination therapies to avoid continued development of drug resistance. This review examines the impact of ATR, CHK1, and WEE1 on replication fork stabilization. We also address the therapeutic potential for combining PARPis with cell cycle inhibitors and the possible consequence of combination therapies which do not adequately address both restored HR and replication fork stabilization as PARPi resistance mechanisms.

Keywords: Cell cycle checkpoint inhibitors; Drug resistance; PARP inhibitor resistance; Replication fork protection; Replication fork stabilization.

Fig. 1.

G2/M checkpoint signaling pathway. ATR (ataxia telangiectasia and Rad3-related) is activated by single stranded DNA damages or replication stress and phosphorylates CHK1. Upon phosphorylation, CHK1 is activated and subsequently activates WEE1 via phosphorylation. Simultaneously, CHK1 phosphorylates and inactivates the CDC25A and CDC25C phosphatases. Activated WEE1 then phosphorylates CDK1 and CDK2 to promote G1/S and G2/M cell cycle arrest.

Fig. 2.

Consequence of replication fork stalling. In response to replication stress, replication forks stall at the site of damage (). Stalled forks can then be reversed by SMARCAL1 and these reversed forks can undergo fork protection by replication protein A (RPA; recruited by ATR), BRCA2, and/or RAD51 (recruited by PARP1 and BRCA2) and be restarted by RECQ1. Alternatively, these reversed forks can be degraded by EXO1, MRE11 (recruited by PTIP, CHD4, RAD52), and/or MUS81 (recruited by EZH2) mediated fork degradation and subsequent fork collapse and cell death. Notably, PARP1 and BRCA2 inhibits MRE11 mediated fork degradation and miR-493–5p blocks both MRE11 and EXO1 activity, supporting the role of PARP1, BRCA2, and miR-493–5p in fork protection and PARP inhibitor resistance.

Fig. 3.

Development of PARP inhibitor resistance. PARP inhibitor (PARPi) resistance develops mainly through restoration of homologous recombination (HR) with fork protection (FP) potentially playing a compensatory role in this process (left box). Combination therapies with cell cycle inhibitors that only disrupt HR, such as CHK1 inhibitors (CHK1i), may result in continued resistance mediated by fork protection (middle box). In contrast, combination therapies utilizing cell cycle inhibitors that target both restoration of HR and FP (e.g. ATR inhibitors; ATRi or WEE1 inhibitors; WEE1i) may prevent PARPi resistance and enhance cell death (right box).:PARP-DNA lesion :RPA.