Synthetic lethality, initially observed in drosophila, is defined as the result of co-alteration of individual viable genes. Synthetic lethality has become a promising anticancer drug discovery strategy, especially for some challenging tumor suppressor genes. By using the synthetic lethal mechanism, cancer cells with mutated or inactivated driver genes can be selectively targeted, potentially reducing the side effects of treatment ^[1]. The use of polyadenylation diphosphate ribose polymerase (PARP) inhibitors in tumor patients with defects of homologous recombination repair is a classic example of targeted therapy and one of the typical representatives of the successful transformation of the "synthetic lethal" effect into clinical application. To date, this treatment has been successfully approved for ovarian cancer, breast cancer, prostate cancer and pancreatic cancer, and its potential therapeutic benefits are being explored and confirmed in endometrial cancer, renal cancer, gastrointestinal cancer and other tumors ^[2]. However, the previously known mechanism of action of PARP inhibitors may be subverted by the conclusion of a latest study, which holds that in cancer cells with defects repaired by homologous recombination, the way in which PARP inhibitors kill cancer cells is due to transcription-replication conflicts (TRCs) rather than "capture".

Known action mechanism of PARP inhibitors

PARP is a ribozyme that participates in a variety of DNA repair pathways, and its effect on nucleotide excision repair (BER) has been most widely studied. To date, 17 members of the PARP family have been identified, but only PARP1, PARP2, play a role in DNA repair. PARP changes itself and/or the other protein of the poly (ADP- ribose) (PAR) moiety with a negative charge after translation, a process known as PARYLATION. PARP1 does 80% of the work in this process. The mechanism of action of PARP inhibitors has been traditionally recognized based on the "capture" theory, which includes competing with nicotinamide adenine dinucleotide at the active site of PARP to inhibit the formation of poly (ADP- ribose) polymers; nicotinamide adenine dinucleotide binding pocket bound to PARP1 and/or PARP2 causes conformational isomerization, and stabilizes the reversible dissociation of DNA-PARP, thus enabling PARP to maintain its binding to DNA. This process is called "capture" of DNA-PARP complex, resulting in the long-term existence of DNA-PARP complex to inhibit the subsequent DNA repair process ^[3].

PARP inhibitors and its possible mechanism of action ^[4]

"synthetic Fatal" Effect of HRD Tumors

Single-chain cleavage occur when cells are subject to DNA damage. PARPs, mainly PARP1, bind to DNA fragmentation sites and undergo conformational changes to increase their catalytic activity. The resulting PADPr polymer altered the function of the modified protein (e.g., reducing the affinity of PARP1 for damaged DNA to dissociate it) and recruited additional protein for non-covalent binding to the polymer. When tumor patients with HRD are treated with PARP inhibitors, the PARP inhibitors capture the PARP on DNA. The captured PARP blocks the progression of the replication fork, leading to the formation of DNA double-strand breaks. At this time, NHEJ is the only way to repair double-stranded break, which will lead to genomic instability and cell death ^[5].

Synthetic lethal interaction between PARP and HRD ^[6]

New discovery on the mechanism of PARP inhibitors

Based on the known principles outlined above, the inhibition of PARP inhibitors should in principle be proportional to the ability of PARPs to be captured. However, some studies have shown that the inhibition of PARP inhibitors on PARPs is not closely related to their ability to capture PARPs. This seems to indicate that its "synthetic lethal" effect is not as simple as it seems.

Intracellular transcription and replication require macromolecular complexes to act on DNA templates, but these functions cannot act on the same DNA sequence at the same time. Conflicts between replication and transcription mechanisms (transcription-replication conflicts, TRCs) are widespread in both prokaryotes and eukaryotes and can lead to DNA damage, genomic instability, and affect high-fidelity replication ^[7].

TIMELESS and TIPIN are two protein that can protect the replicators from transcription conflict. In vitro experiments show that the treatment of PARP inhibitors, especially in the early S phase, can lead to TRCs.

To further explore the need for PARP capture to induce TRCs-dependent DNA damage responses, the researchers removed PARP1 and PARP2 by siRNA and monitored for DNA damage markers in stage S HeLa cells. The deletion of PARP1 induced a DNA damage response in a manner that was dependent on transcriptional extension, whereas the deletion of PARP2 had no effect. This result supports the conclusion that inhibition of PARP enzyme activity is sufficient to induce TRCs leading to DNA damage independent of capture, as removed PARP cannot be captured. It also indicated that only PARP1 seemed to have the function of preventing TRCs-induced DNA damage.

The known interaction between PARP1 and TIMELESS suggests that they may block TRCs through the same molecular pathway. The investigators used siRNA to knock out either TIMELESS or TIPIN in cells treated with PARP inhibitors. The absence of TIMELESS or TIPIN resulted in the unavoidable presence of TRCs and enhanced activity of PARP1. The expression of TIMELESS and PARP1 proteins prevented TRCs and TRCs-induced DNA damage.

At the same time, we found that the absence of PARP1 accelerated the development of replication fork to a degree similar to that of TIMELESS or TIPIN, but the absence of PARP2 had no significant effect. This result was consistent with the conclusion that PARP1 transmitted TRCs signal to the replicators through TIMELESS, while PARP2 did not interact with TIMELESS.

Because PARP1, TIMLESS, and TIPIN work in the same way that TRCs are prevented, the researchers tested whether the absence of TIMLESS or TIPIN causes "synthetic death" with HRD cells. The co-deletions of BRCA2 and TIMELESS or BRCA2 and TIPIN caused a strong DNA damage response in HeLa cells and led to cell death. This may offer new targets for drug development.

In summary, PARP1 signals TIMELESS and TIPIN that TRCs are imminent, and the cells halt replication until TRCs are dissociated. If PARP1 or TIMELESS and TIPIN fail to function, TRCs cause DNA damage that requires HR to repair. The main difference between the new and old mechanisms of PARP inhibitors is the nature of the replicator conflict object: the captured PARPs or transcriptional extension complex. The new mechanism is supported by the fact that synthetic lethality to PARP inhibitors in HRD cells can be reduced by inhibiting transcriptional extension, and that siRNA depletion of TIMELESS, TIPIN, or PARP1 is synthetic lethality to HRD ^[8].

The discovery of new mechanisms facilitates the development of clinical drugs

A better understanding of how PARP inhibitors target HRD cells will help guide their future clinical development. All PARP inhibitors currently used clinically inhibit PARP1 and PARP2. However, it is possible that only PARP1 protects the replicates from TRCs. Thus, selective inhibitors of PARP1, such as saruparib, may be sufficient to clinically induce synthetic lethality in HRD. The new mechanism also raises the question of whether the regulation of PARP capture enhances the clinical accessibility of PARP inhibitors. The capture capacity of PARP inhibitors varies, so this is a parameter that can be optimized independently of inhibition. It has been concluded that reducing capture potential may reduce the toxicity of PARP inhibitors without compromising efficacy.

References

[1] J Med Chem 2024 Jul 25; 67(14):11488-11521.

[2] Nat Commun 2020 Nov 4; 11(1):5584.

[3] Guideline for clinical application of PARP inhibitors in ovarian canc

[4] Nat Rev Clin Oncol 2021 Dec; 18(12):773-791.

[5] J Clin Oncol 2015 Apr 20; 33(12):1397-406.

[6] Mol Cancer 2020 Jun 20; 19(1):107.

[7] Annu Rev Genet2023 Nov 27:57:157-179.

[8] Nature 2024 Apr; 628(8007):433-441.