Draining the FEN1s for cancer therapy

Breast and ovarian cancer are among the most frequent causes of cancer-related deaths in women worldwide. Genomic instability is prevalent in these cancer types, and it has been associated with mutations in DNA repair genes, including the BRCA1 and BRCA2 tumor suppressor genes.

FEN1 and Homologous Recombination Defects

A synthetic lethal interaction occurs when the concurrent perturbation of two nonessential genes leads to cell death. Synthetic lethality (SL) is of great interest in the context of cancer therapies, as a cancer harboring a mutation in a nonessential gene could be treated by pharmacological inhibition of a second nonessential gene. Along these lines, in 2005, Farmer et al. (1) and Bryant et al. (2) showed that BRCA1- and BRCA2-deficient cells are sensitive to PARP1 inhibition. PARP1 recognizes specific types of DNA damage, notably single-strand and double-strand DNA breaks. Recognition of DNA damage turns on the catalytic activity of PARP1, resulting in auto-poly(ADP-ribosyl)ation and induction of DNA repair processes (3). PARP1 inhibitors specifically “trap” PARP1 on DNA, inducing DNA damage, which is normally repaired by BRCA1/BRCA2/RAD51-dependent homologous recombination (HR). In cancer cells with defective HR, due to mutations in BRCA1, BRCA2, or other HR genes, inhibition of PARP1 leads to synthetically lethal accumulation of collapsed replication forks, DNA damage, and cell death (4).

The success of PARP inhibitors in the clinic has triggered the hunt for new therapeutic targets based on the SL approach (5). RNA interference and CRISPR screens have been used to identify druggable SL interactions in mammalian cells. Nevertheless, yeast-based genetic screens still represent a valuable tool to identify cancer-relevant SL vulnerabilities. In PNAS, Guo et al. (6) use known Saccharomyces cerevisiae SL interactions to predict therapeutic targets for cancers harboring mutations in genome instability suppressing (GIS) genes. The yeast radiation sensitive 27 (RAD27) gene, the ortholog of human FEN1, shows the greatest number of SL interactions, involving more than 20% of the S. cerevisiae GIS genes (59 SL partners, many of which function in HR).

The sensitivity of cancers with HR defects to FEN1 inactivation is demonstrated by Guo et al. (6) by inhibiting FEN1 in BRCA1- or BRCA2-deficient human cancer cell lines. The authors selected four chemical compounds (C2, C8, C16, and C20) from an N-hydroxyurea series of potent FEN1 inhibitors that had previously been reported to sensitize a bladder cancer cell line (T24) to DNA damage caused by methyl methanesulfonate or temozolomide (7). The BRCA2-deficient PEO1 ovarian cancer cell line was more sensitive to the selected FEN1 inhibitors, in particular to the most potent compound C8, than the matched BRCA2-proficient PEO4 cells. The PEO1 cells displayed increased DNA damage and double-strand breaks (DSBs) after FEN1 inhibition, as indicated by the higher levels of γH2AX and 53BP1 foci compared to PEO4 cells, as well as loss of viability (6).

The ability of the FEN1 inhibitor C8 to specifically impair the survival of BRCA-deficient cells was confirmed in BRCA1-knockout retinal pigmental epithelial cells as well as in BRCA2-knockout DLD1 colorectal cancer cells (6). Clonogenic survival of a panel of breast, ovarian, colorectal, and lung cancer cell lines was also evaluated upon treatment with the C8 inhibitor. In this panel, the cancer cell lines with reported BRCA1/BRCA2 mutations were more sensitive to C8-induced FEN1 inhibition. Interestingly, the sensitive cell lines included PARP1 inhibitor-resistant cell lines (6), envisioning the possibility that FEN1 inhibition may be effective in cancers that develop resistance to PARP1 inhibitors.

The SL between loss of FEN1 and BRCA1/BRCA2 deficiency was validated by small interfering RNA targeting FEN1 in vitro and was also demonstrated in a mouse xenograft model, in which the C8 FEN1 inhibitor was shown to have good pharmacokinetic properties (6). Overall, these results build upon previous observations suggesting that FEN1 might be a viable target for SL interactions in human cancers (8, 9).

Given the role of FEN1 in ensuring maturation of the Okazaki fragments during DNA replication, Guo et al. (6) investigated the effect of FEN1 inhibition on DNA replication. They observed that, upon treatment of BRCA1/BRCA2-deficient cells with the C8 inhibitor, the levels of 5-bromo-2'-deoxyuridine (BrdU) incorporation were markedly reduced. Remarkably, even 3 d after drug removal, up to 90% of the cells were still unable to incorporate BrdU. On the other hand, in BRCA1/BRCA2-proficient cells, the FEN1 inhibitor induced only a small, transient decrease in BrdU incorporation that was fully reversible upon withdrawal of the drug.

Exploiting Postreplication Gaps for Synthetic Lethalities

Our understanding of the molecular function of FEN1 can provide mechanistic insights that help explain the SL interactions with BRCA deficiency. Inhibition of FEN1 leads to delayed processing and ligation of the ends of the Okazaki fragments and consequently to single-strand nicks and gaps on the lagging strand. These nicks and gaps can then be repaired by an HR-based pathway that, in mammalian cells, utilizes BRCA1 and BRCA2 (10, 11). Specifically, BRCA1 together with BRCA2 and Rad51 are recruited at the single-strand gaps that remain behind the fork; there, these proteins are involved in HR-based repair of the gap using the intact daughter strand as template. In the absence of functional BRCA1 and BRCA2, Rad51-dependent HR is not feasible, and cells attempt to repair the DSBs with error-prone DNA repair mechanisms that can involve formation of DNA DSBs and lead to genomic rearrangements and/or cell lethality.

One repair pathway that could be involved when DNA DSBs are induced under the scenario mentioned above is break-induced replication (BIR), a pathway that repairs single-ended DSBs. Normally, BIR will not be utilized behind the fork for repair of single-strand nicks and gaps, but, in the absence of BRCA1/2, it may become a repair pathway of choice. This may explain why inactivation of Rad52, a protein required for efficient BIR, is synthetically lethal with BRCA1/2 inactivation in cancer cell lines (12, 13).

Interestingly, the SL observed between PARP1 inhibition and BRCA1/2 inactivation may be explained by the model described above. PARP1 serves as a sensor of unligated Okazaki fragments (14). Therefore, PARP1 inhibitors may result in a large number of single-strand gaps behind the replication fork, similar to FEN1 inhibitors, and to SL with BRCA1/2 inactivation.

Remarkably, the CTF18, CTF8, and DCC1 S. cerevisiae genes also have a very high number of SL interactions with mutations in GIS genes, scoring just below FEN1 in the analysis performed by Guo et al. (6). The CTF18−CTF8−DCC1 complex prevents formation of single-strand gaps on the leading strand (15). Thus, mechanistically, the SL interactions observed in the context of mutations targeting the CTF18−CTF8−DCC1 complex may be very similar to those targeting FEN1.

Although further studies are needed to elucidate FEN1’s critical role in BRCA1/BRCA2-mutated cancer cells, the Guo et al. (6) study supports FEN1 as a potential drug target in cancer therapy and underpins the need for the discovery and development of second-generation FEN1 inhibitors with better pharmacokinetic and pharmacodynamic profiles.