Suppression of DNA Polymerase β Activity Is Synthetically Lethal in BRCA1-Deficient Cells

Abstract

People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase β (Pol β) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol β or the treatment with a Pol β pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol β as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.

Graphical Abstract

Cells that are deficient in DNA double-strand break repair face a significant increase in the risk of becoming cancerous. The breast cancer type 1 and 2 (BRCA1/2) gene products are examples of tumor suppressors involved in homologous recombination (HR), a double-strand break repair pathway, which when mutated give rise to significant increases in cancer incidence.^1,2 Although mutations in BRCA1 increase the susceptibility to breast and ovarian and to lesser extents melanoma, prostate, and pancreatic cancers, they also provide a target of opportunity for selective treatment. The inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an enzyme involved in DNA repair, is significantly more lethal in BRCA1-deficient cells than in healthy ones. This example of synthetic lethality has given rise to a new generation of anticancer agents, including Olaparib, Niraparib, and Rucaparib that are selectively cytotoxic to cancer cells that are HR-deficient (HRD).^3–5 Unfortunately, not all cancers respond to these treatments, and others develop resistance.⁶ Hence, there is a need for additional synthetically lethal targets to selectively target HRD cells.⁷ DNA polymerase θ is one such target, as is the nucleosome remodeling protein ALC1.^8,9 It has been postulated that inhibiting DNA polymerase β (Pol β) would be synthetically lethal in BRCA1-deficient cells.¹⁰ We wish to report that inhibiting Pol β or suppressing its expression using siRNA is a viable synthetically lethal approach to selectively kill BRCA1-deficient cells.

Pol β is a bifunctional polymerase that is most well-known for its roles in base excision repair (BER) within the nucleus (Scheme 1A).¹¹ Pol β removes the remnants of an abasic site (AP) following incision by apurinic endonuclease 1 (Ape1), and in short, patch BER extends the 3′-teminus of the cleaved DNA strand to fill in the resulting gap. More recently, Pol β has been shown to also be involved in double-strand break repair and mitochondrial DNA repair.^12–15 Pol β is overexpressed in many human cancers, including colon cancer where ~40% of tumors contain the mutated enzyme.¹⁶ For these reasons, Pol β is an increasingly popular inhibition target.^10,17–20 We have developed a platform for identifying mechanism-based covalent inhibitors of Pol β from chemical libraries (Scheme 1B).^21–23 This strategy was inspired by potent cytotoxic antitumor agents that produce 1,4-dicarbonyl containing DNA lesions, which inactivate Pol β.^24,25 Recently, this approach provided a covalent inhibitor (1) that inactivates Pol β by modifying a lysine in the polymerase active site, which then prevents DNA binding.²⁶ Inhibitor 1 is selective for Pol β over other polymerases, including the backup repair enzyme Pol λ, and the corresponding pro-inhibitor (pro-1) is not toxic (<10% cell death) by itself. Experiments in wild-type and Pol β −/− mouse embryonic fibroblasts establish that pro-1 selectively acts on the target polymerase.²⁶ Herein, we knocked down Pol β expression using siRNA or in separate experiments treated with pro-1 to demonstrate synthetic lethality with BRCA1.

Scheme 1.

Role of Pol β in BER and Its Inactivation

Synthetic lethality is defined by a relationship between two proteins, such that while the loss of function of either one is not cytotoxic, the loss of function of both is.^3–5 Consequently, a synthetic lethal partner for a nonfunctional, mutated protein in a cancer cell is an attractive pharmacological target. Pol β has been speculated to be a synthetic lethal partner for BRCA1.¹⁰ Furthermore, while this manuscript was in preparation, it was reported that the suppression of Pol β and BRCA2 activities is synthetically lethal.²⁷ Our goal was to determine whether deficiencies in Pol β and the tumor suppressor, BRCA1, are synthetically lethal. Pol β deficiency was introduced to the cells in one of two ways. Pol β was knocked down using siRNA, as previously reported in the literature.¹² Alternatively, a bisacetate pro-inhibitor form of 1 (pro-1) was used.²⁶ Compound 1 is a recently reported covalent inhibitor of Pol β that reacts with lysine residues in the polymerase domain to prevent DNA binding. When pro-1 is administered to cells, cytotoxicity results from the selective interaction with the target Pol β.

The isogenic ovarian cancer cell line (UWB1.289) that lacks BRCA1 (UWB1.289 (BRCA1 −/−)) or is BRCA1 complemented (UWB1.289 + BRCA1) provided an excellent environment in which to probe for a synthetic lethal interaction between Pol β and BRCA1 (Figure 1). The effect of pro-1 (2 h treatment) on cell survival, as determined via a clonogenic assay, was examined with and without the siRNA knockdown of Pol β (siPol β) or with nontargeting control siRNA (siNT). Western blotting indicated that Pol β was knocked down by ~80% following siPol β treatment (Figure S1). SiPol β had a modest cytotoxic effect (80% survival) on BRCA1-complemented ovarian cancer cells (UWB1.289 + BRCA1) but was significantly more toxic (~33% survival) in BRCA1-null cells (UWB1.289). Treatment with the Pol β inhibitor, pro-1 (5 μM), mirrored the effects of Pol β knockdown, showing some toxicity (~75% survival) in UWB1.289 + BRCA1 cells but a significantly larger effect in the BRCA1-null UWB1.289 cells (~25% survival). Importantly, the toxicity of pro-1 was experimentally indistinguishable in siNT and siPol β treated cells, consistent with selective targeting of the covalent inhibitor for Pol β, as reported in mouse embryonic fibroblasts.²⁶ Moreover, the detrimental effects of pro-1 and siPol β on BRCA1-deficient cell survival are fully consistent with a synthetically lethal interaction between Pol β and BRCA1.

Figure 1.

Synthetically lethal interactions in isogenic ovarian cancer cells that are either proficient (UWB1.289 + BRCA1) or deficient (UWB1.289) in homologous recombination. Synthetic lethality between Pol β and BRCA1 established using siRNA or pro-1. siNT = nontargeting siRNA; siPol β = siRNA for Pol β. Treatment time = 2 h.

Having established the synthetically lethal interaction between Pol β and BRCA1, we endeavored to compare the cell killing effectiveness of this pair with that involving PARP1 and BRCA1. Consequently, the cytotoxicity of pro-1 and Olaparib was compared in two cell lines. An initial comparison was made using the isogenic ovarian cancer cell line (UWB1.289). Again, pro-1 (1 h treatment) was only modestly toxic in BRCA1-complemented cells (Figure 2A), as was Olaparib (Figure 2B). However, only 40% of the BRCA1 −/− cells survived treatment with either 1 or 5 μM pro-1. The surviving fraction of the cells treated with pro-1 (1 μM) was slightly smaller than that treated with the same concentration of Olaparib. Cell death attributable to a similar covalent Pol β inhibitor was shown to increase with exposure time.^22,23 This proved to be true regarding the cytotoxicity of pro-1 (1 μM) and Olaparib (1 μM) in BRCA1-deficient cells as well (Figure 2C,D). While the cytotoxicity of both inhibitors was proportional to incubation time, the surviving fraction of the BRCA1-deficient cells after 6 h of exposure to pro-1 was more than 3-fold smaller than when Olaparib was employed. These data support synthetic lethality between homologous recombination and Pol β repair pathways. Furthermore, the data indicate that pro-1 is of comparable or even greater efficacy to FDA approved Olaparib in this cell line.

Figure 2.

Synthetically lethal interactions between Pol β or PARP1 and BRCA1 in isogenic ovarian cancer cells (UWB1.289; UWB1.289 + BRCA1). (A) Synthetic lethality between Pol β and BRCA1 established using pro-1. Treatment time, 1 h. (B) Synthetic lethality between PARP1 and BRCA1 established using Olaparib. Treatment time, 1 h. (C) Cytotoxicity of pro-1 as a function of treatment time. (D) Cytotoxicity of Olaparib as a function of treatment time.

The time course analysis (Figure 2C,D) also indicated that, upon prolonged treatment, Olaparib was more cytotoxic to BRCA1-complemented ovarian cancer cells than pro-1. As a point of comparison, the cytotoxicity of pro-1 and Olaparib was compared in a noncancerous cell line, mouse embryonic fibroblasts (MEFs) (Figure 3). The exposure of MEFs for 1 h to pro-1 at a higher concentration than Olaparib resulted in less cytotoxicity. This raises the possibility that the Pol β inhibitor is less toxic in cells in which a synthetically lethal interaction is absent. Although speculative, the lower toxicity of pro-1 than Olaparib in such cells could be due to the involvement of Pol β in fewer cellular processes than PARP1. The data also evoke the possibility that inhibiting Pol β may be a more favorable strategy than PARP1 inhibition for selectively killing HRD cancers.

Figure 3.

Cytotoxicity to mouse embryonic fibroblasts of (A) pro-1; (B) Olaparib.

The data supporting a synthetically lethal interaction between Pol β and BRCA1 were corroborated in breast cancer cell lines that are BRCA1 proficient (MCF-7) or deficient (MDA-MB-436) (Figure 4). The toxicity (~10%) of pro-1 in BRCA1 +/+ cells (MCF-7) was within the error of that observed in BRCA1-complemented UWB1.289 cells (Figure 2A), albeit under slightly different conditions in which the proinhibitor was employed at as high as 10 μM (Figure 4A) for the same amount of time (1 h). However, the survival fraction of BRCA1 −/− breast cancer cells (MDA-MB-436) was less than 60% and 50% when treated with 5 and 10 μM pro-1, respectively. Olaparib was more effective at killing BRCA1 −/− cells at a lower concentration (Figure 4B). However, it was also more toxic to the BRCA1 +/+ MCF-7 cells.

Figure 4.

Synthetically lethal interactions in breast cancer cells that are proficient (MCF-7) or deficient (MDA-MB-436) in homologous recombination due to BRCA1 deletion. (A) Synthetic lethality between Pol β and BRCA1 established using pro-1. (B) Synthetic lethality between PARP1 and BRCA1 established using Olaparib. Treatment time = 1 h.

Pol β plays a vital role in the base excision DNA repair pathway. The inhibition of the enzyme sensitizes cells to DNA damaging agents that produce nucleic acid modifications repaired by this pathway. Because Pol β is expressed constitutively in all cells, the reliance upon the synergistic cytotoxicity of a Pol β inhibitor and DNA damaging agent to kill cells may not provide high selectivity for cancer cells. Synthetic lethality provides a way to selectively kill cancer cells. This research establishes Pol β and BRCA1 as a synthetic lethal pair, which further validates the attractiveness of this polymerase as an anticancer target.