Significance One origin of cancer arises from defects in the homologous recombination (HR) DNA repair pathway, which results in oncogenic genomic instability. Normal cells prefer HR for repair of stressed replication forks because it is highly conservative. The back-up pathway for repair of stressed replication forks, alternative non-homologous end-joining (aNHEJ), can result in chromosomal instability. Normal cells use microRNA (miR) 223-3p to down-regulate aNHEJ and thus promote HR, which protects genomic stability. However, those cancers that arise from inherited or acquired defects in HR components, such as the BRCA1 breast and ovarian cancers, silence miR223-3p to enhance aNHEJ repair of stressed replication forks. Reconstituting expression of miR223-3p in these cancers causes synthetic lethality, and is a potential therapeutic modality.

Abstract Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223–3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.

DNA replication is not a smooth and continuous process, but rather prone to interruptions (1, 2). DNA damage or nucleotide depletion can induce stalling of the replication machinery (3, 4), which can result in replication stress and subsequent fork collapse, with disassociation of the replication apparatus (5, 6). As such, replication stress is a common etiology of genomic instability, resulting in either cell death or neoplastic transformation (4, 6⇓–8). HR is the most common pathway for stressed replication fork repair and restart (5, 7, 8). The rate-limiting step in HR is the nucleolytic resection of the 5′ strand at a DNA free end, initiated when BRCA1/CtIP replaces 53BP1/RIF1/Shieldin at the site of DNA damage (9⇓⇓–12). After 5′ strand resection (13⇓⇓–16), BRCA2 then loads the RAD51 recombinase onto the 3′ single-stranded DNA for invasion of the sister chromatid (17⇓–19), which results in Holliday junction formation that is later resolved by MUS81 and SLX4 (20, 21). Germline or acquired mutations in BRCA1 or its interacting partner BAP1 result in defects in the HR pathway, and subsequent replication fork instability and oncogenesis (22⇓⇓–25). HR-deficient cancers become addicted to other repair pathways to resolve replication fork stress such as aNHEJ (26⇓⇓⇓–30). This addiction is a singular weakness of these cancer cells, and can be targeted with therapeutic agents (8, 25, 29, 30), generating synthetic lethality. This synthetic lethality underlies the efficacy of PARP1 inhibitors in breast, ovarian, and prostate cancers that harbor inherited or acquired mutations in BRCA1 (31, 32).

PARP1 can initiate aNHEJ by displacing the Ku complex from the DNA double-strand break (33, 34). CtIP, Pso4, and Mre11 then mediate limited 5′ end resection, followed by DNA polymerase theta promoting microhomology annealing (27⇓–29). DNA ligase 1 or 3 with XRCC1 then promote ligation of the free DNA ends (27, 33). Pharmacologic inhibitors of PARP1 lock it onto DNA, generating obstacles that stall replication forks, which in BRCA1- or BAP1-mutant cells cannot be repaired by HR (31, 32, 35). PARP1 inhibition also prevents the use of the aNHEJ back-up repair pathway to resolve the replication forks stalled by PARP1 locked on DNA, resulting in fork collapse and cell death (8, 25, 27). While aNHEJ promotes repair of stressed replication forks in BRCA1/BAP1-defective cells, it comes at a cost; aNHEJ can mediate chromosomal translocations (33, 34). This occurs when aNHEJ erroneously anneals and then ligates free DNA ends on distinct chromosomes (26, 33, 34). The three FDA-approved PARP1 inhibitors, olaparib, rucaparib, and niraparib, have each shown impressive response rates in HR-deficient cancers (36⇓⇓⇓⇓–41). However, these agents can occasionally lead to myelodysplastic syndrome, and resistance can develop (30, 41, 42). In this study, we found that the microRNA species miR223-3p is a negative regulator of aNHEJ, likely to restrain aNHEJ from generating aberrant chromosomal translocations that would lead to genomic instability. Importantly, we discovered that cancer cells deficient in HR repress miR223-3p strand selection and instead express miR223-5p. Reconstituting expression of miR223-3p is synthetically lethal to BRCA1- and BAP1-defective cancer cells, demonstrating its potential as an effective cancer therapeutic agent.

Discussion Faithful repair of damaged replication forks is critical for maintaining genomic stability and suppressing oncogenesis (4, 6). The proper choice of which DNA repair pathway to use to repair and restart damaged replication forks is crucial to conserve sequence integrity (27, 52). HR repair and restart of damaged replication forks is conservative, while aNHEJ repair has risk of aberrant ligation of free DNA ends at such forks, resulting in chromosomal translocations (33, 34). Such chromosomal abnormalities lead to mitotic catastrophe and cell death, or neoplastic transformation and cancer (51, 53). Thus, it is not surprising that cells negatively regulate aNHEJ to promote the choice of HR to repair stressed replication forks. In this study, we identified miR223-3p as a repressor of aNHEJ pathway repair efficiency by down-regulating expression of aNHEJ components. MiR223-3p is highly expressed in most normal tissues, especially in hematopoietic cells which have a high proliferative rate, and thus face a higher risk of replication stress (44). This heightened risk of replication stress can lead to genomic instability if such stress is not properly managed by efficient and conservative fork repair pathways such as HR. We have previously demonstrated reduced aNHEJ and decreased chromosomal translocation when PARP1 is inhibited (34). We demonstrate here that miR223-3p functions to maintain genomic stability by decreasing use of aNHEJ. However, when there is extensive replication stress requiring full deployment of all relevant repair pathways, levels of miR223-3p decrease. This increases PARP1 levels and promotes aNHEJ activity to address the repair of damaged replication forks. In miR223−/− mice, hematopoietic cells accumulate unprovoked chromosomal translocations, indicating that miR223-3p maintains steady state genomic stability in vivo. Conversely, provoked chromosomal translocations are decreased after over-expression of miR223-3p in cell lines. Interestingly, miR223-3p−/− mice develop dysplastic hematopoiesis (44), similar to myelodysplasia in humans, which could be the result of genomic instability from unregulated use of aNHEJ in stressed replication fork repair (33, 34). Interestingly, BRCA1- and BAP1-deficient cancer cells have greatly decreased expression of the miR223-3p strand, and instead process premiR223 to miR223-5p (54, 55). Since miR223-3p inhibits aNHEJ, and aNHEJ replaces HR in processing stressed replication forks in BRCA1-deficient cancers, it is unlikely that such cancers could originate in the first place without repressing miR223-3p. A single transfection of miR223-3p induced synthetic lethality in BRCA1- and BAP1-deficient malignancies, likely by triggering the destruction of required aNHEJ components CtIP and Pso4 (Fig. 4). SiRNA reduction of PARP1 levels did not result in BRCA1-deficient cell cytotoxicity (Fig. 4D), consistent with the finding that PARP1 inhibitors trap PARP1 on DNA, and one mechanism of cancer resistance to such inhibitors is reducing PARP1 levels (35). Reconstituting miR223-3p in BRCA1- and BAP1-resistant cells led to slowed repair of stressed replication forks, but ultimately the stressed forks recovered and restarted. However, we and others have shown that even a short delay in stressed replication fork repair results in cell death (3, 9). In cells lacking both HR and aNHEJ, cleaved replication forks can be fused via 53BP1-dependent classical NHEJ to give rise to the micronuclei and chromosomal bridges as seen here in the BRCA1- and BAP1-deficient miR223-3p-reconstituted cells (50, 56). Because BRCA1- and BAP1-deficient malignancies are addicted to aNHEJ to cope with replication stress, miR223-3p holds promise as a therapeutic agent for such cancers. Indeed, reconstituting miR223-3p led to synthetic lethality even in BRCA1-mutant cells that were resistant to the PARP1 inhibitor olaparib (Fig. 4). Thus, MiR223-3p could be an important therapy in BRCA1- or BAP1-deficient cancers, especially those that develop resistance to PARP1 inhibitors (57, 58). In addition, since miR223-3p is normally expressed in most tissues, it would be well tolerated by normal cells compared with the clinical PARP1 inhibitors, which have some gastrointestinal and hematologic toxicities (35⇓⇓⇓⇓–40). Notably, BAP1 mutations occur in difficult to treat malignancies, such as MPM, renal cell carcinoma, uveal melanoma, and cholangiocarcinoma (24, 45, 46). In summary, miR223-3p negatively regulates aNHEJ, and is repressed in BRCA1- and BAP1-deficient cancers. Its reconstitution is synthetically lethal to these cancers, and it would therefore be an anti-neoplastic therapeutic agent.

Methods and Materials Cell Culture. Cells and the EJ2-GFP and MMEJ-GFP aNHEJ reporter cells were cultured as described (8, 9, 41). For inducing DNA damage, cells were treated with VP16 from Sigma Aldrich (Cat # E1383-25MG) and Cytosine B-d-arabinofuranoside (Ara-C) from Sigma Aldrich (Cat # C6645). Animal Care. MiR223 knock-out (KO) mice (Cat # 013198), wild-type (WT) mice (Cat # 002014), and NSG mice (Stock # 005557) were purchased from the Jackson Laboratory. Housing of the animals and all experimental procedures were approved by the Institutional Animal Care and Research Advisory Committee of the University of Florida, Gainesville and University of Texas Health Science Center at San Antonio. Antibodies. These are defined in the SI Appendix in the supplemental materials section. MiRNA and SiRNA. MirVana miR223-3p microRNA mimic was purchased from Ambion Life Technologies (Cat # 4464067). Small interfering RNA (siRNA) against BRCA1 (Cat # l-003461-00-0020), PARP1 (Cat # l-006656-03-0005), PSO4 (Cat # l-004668-00-0005), and CtIP (Cat # M-011376-00-0005) were purchased from Dharmacon. All were transfected using Lipofectamine RNAiMAX from Life Technologies (Cat # 13778–150) at 25 nM. Luciferase Assay for Assessing PARP1 3′ UTR mRNA Stability. PARP1 3′ UTR plasmid was transfected using Lipofectamine, and cells were collected and assessed for luciferase activity using Luc-Pair Dual Luciferase assay kit from GeneCopoeia (Cat # LPFR-P010). Firefly luciferase activity is normalized against Renilla luciferase. Survival Assays. Clonogenic survival assays were performed as described (9, 49). Cytogenetics. Structural aberrations in Giemsa-stained metaphase chromosomes were scored as described (25, 34). Immunohistology of Mouse Marrow. Femurs from WT and KO mice were dissected and fixed in 10% neutral buffered formalin for at least 4 d. The samples were then processed for H&E and immunohistochemical staining with PARP1 antibody (Santa Cruz Biotechnology, cat# sc-8007). Nuclear Structure Assays and DNA Damage Foci. Confocal analysis of DAPI-stained nuclear structural abnormalities such as micronuclei and nuclear bridging were assessed as described (9). Confocal immunofluorescence γH2Ax foci assays were performed as described (9). Replication Fork Analysis. Active replication forks were measured using immunofluorescent detection of BrdU foci after DNA denaturation as described (9). DNA fiber analysis was performed to measure replication fork arrest and restart in cells transfected with either scramble control or 25 nM miR223-3p mimic as described (9, 49). Quantitative Real-Time PCR. CDNA conversion was performed using the first-strand cDNA synthesis kit for MiRNA from Origene (Cat # HP100042). QRT-PCR for miR223 was performed on a 7900HT Fast Real37-Time PCR system (ABI) according to Origene protocol, and ΔCT values were calculated. The primers used had the following sequences: MiR223-3p - forward (5′TGTCAGTTTGTCAAATACC) and reverse (5′GAACATGTCTGCGTATCTC), MiR223-5p - forward (5′CGTGTATTTGACAAGCTG) and reverse (5′GAACATGTCTGCGTATCTC). U6 RNA was used as an endogenous control. Murine s.c. Xenograft Detection. One million BRCA1-deficient MDA-MB-436-luciferase breast cancer cells were transfected with either miR-Control or miR223-3p mimic at a concentration of 100 nM and then injected s.c. into the flank of mice (n = 6/group). Bioluminescent tumor signals were obtained by a Xenogen IVIS Spectrum imaging system (Perkin-Elmer) once every week after isoflurane anesthesia as per IACUC protocol.

Acknowledgments This work was supported by NIH (RO1 CA205224 and R01 GM109645 to R.H., RO1 ES007061 and RO1 CA220123 to P.S., and RO1 CA197796 to S.B.); the Korean Institute for Basic Science (IBS-R022-A1-2017 to O.S.); and the Cancer Prevention and Research Institute of Texas Research Training Award (RP17035 to G.S.).

Footnotes Author contributions: G.S., H.-S.K., O.S., S.B., P.S., and R.H. designed research; G.S., K.K., A.S.J., G.H., and H.-S.K. performed research; E.A.W., K.K., A.S.J., G.H., H.-S.K., and W.Z. contributed new reagents/analytic tools; G.S., E.A.W., K.K., A.S.J., H.-S.K., O.S., W.Z., S.B., P.S., and R.H. analyzed data; and G.S., O.S., W.Z., S.B., P.S., and R.H. wrote the paper.

Conflict of interest statement: R.H. has equity in Dialectic Therapeutics, which holds a licensing agreement for use of miR223-3p as a cancer therapeutic agent.

This article is a PNAS Direct Submission.

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