Summary
Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.
Introduction
Our DNA is subject to damage from endogenous sources, including reactive oxygen species or replication stress, or from external threats, such as UV light, environmental chemicals, or cosmic radiation. To cope with DNA damage and maintain genome stability, organisms have evolved an array of mechanisms, collectively termed the DNA damage response (DDR), that sense and repair DNA damage. Inaccurate DNA repair results in the accumulation of mutations or genomic rearrangements, which are early hallmarks of cancers. Furthermore, germline mutations affecting DNA repair pathways are associated with an increased risk of developing different types of cancer.1,2,3 Similarly, acquired genetic or epigenetic alterations that compromise DDR promote cellular transformation and are found in a large proportion of cancers.4,5
DNA double-strand breaks (DSBs) are the most toxic type of lesion that occurs in DNA and can be repaired by multiple pathways. Two-ended DSBs, which are created by radiation or nucleases, can be repaired by one of three DSB repair pathways: non-homologous end joining (NHEJ), homologous recombination (HR), or microhomology-mediated end joining (MMEJ, also referred to as alternative end joining, or altEJ). MMEJ relies on the annealing of small microhomologies (on average ∼3–20 bp) that flank the break site6,7 and that are exposed by short-range resection of the DSB, a step that is shared with HR.8 In mammals, MMEJ depends on polymerase theta (Polθ, encoded by POLQ), which promotes annealing of the microhomologies and is responsible for the polymerization step.9,10,11,12,13,14 In addition to Polθ, PARP1 (Poly[ADP-ribose] polymerase 1), FEN1 (Flap endonuclease 1), XRCC1 (X-ray repair cross complementing 1), and ligase 3 have been implicated in MMEJ.15,16,17,18,19 MMEJ is intrinsically error-prone, as microhomology annealing results in deletions, while Polθ is an inaccurate polymerase that frequently introduces mutations and can produce templated insertions.20,21,22 MMEJ was first described as a backup pathway because cells that are deficient in either NHEJ or HR become critically dependent on MMEJ for survival.23 However, low levels of MMEJ activity are detected at Cas9-induced DSBs in cells that have functional NHEJ and HR,24 and MMEJ repair scars are found in normal and cancer cells.5,22,25
Although it is not fully understood why HR and MMEJ are synthetically lethal, a widespread hypothesis is the role of both pathways at single-ended DSBs (seDSBs), which occur when a replication fork converts an unrepaired single-strand break and collapses. In unchallenged cells, they constitute the vast majority of unscheduled DSBs.26 Notably, repair of seDSBs by NHEJ is expected to induce aberrant chromosomal structures toxic to the cells and is repressed.27,28 The main repair pathway that is active at seDSBs is therefore HR,28 with MMEJ as a possible alternative.29 Upon loss of HR, MMEJ likely becomes critical to the repair of these seDSBs, explaining the synthetic lethal interaction between HR and MMEJ. Supporting this model, MMEJ has been shown to repair seDSBs that are mediated by Cas9D10A or that arise from replication fork collapse.29 Recent work has also implicated Polθ in post-replicative single-stranded DNA (ssDNA) gap filling, which is essential in HR-deficient (HRD) cells or following PARP inhibitor (PARPi) treatment.30,31,32
HR deficiency results in high levels of genomic instability, which can in part be explained by the upregulation of the intrinsically mutagenic MMEJ.12,22,33,34,35,36 It is therefore not surprising that, among all the DNA repair genes that are frequently mutated in cancer, somatic mutations or epigenetic repression of HR genes are the most commonly found.37 The most prominent example is high-grade serous epithelial ovarian cancers (HGS-EOCs), in which HR is deficient in over 50% of tumors.38 Germline mutations in BRCA (breast cancer) genes, PALB2 (partner and localizer of BRCA2) and RAD51 paralogs, all critical for the HR pathway, are also observed in ovarian, breast, prostate, and pancreatic cancers.39
Seminal studies have found that small molecule inhibitors of PARP confer selective killing of HRD tumors.40,41 This led to the clinical development of PARPis, which are now commonly used to treat patients with HRD HGS-EOC as well as metastatic breast, prostate, and pancreatic cancers.42 However, only 50% of HRD tumors respond to PARPi treatment due to innate resistance, and of those that respond, most tumors eventually relapse due to acquired drug resistance.43 Encouragingly, Polθ inhibitors have been developed that could be used to treat PARPi-resistant tumors, at least those that have lost the 53BP1-Shieldin pathway.44 Because POLQ expression is very low or absent in normal cells but is over-expressed in many cancers, including (but not limited to) those with HRD, it has emerged as a powerful therapeutic target complementary to PARPis.45 However, our knowledge of MMEJ remains limited and lacks a complete understanding of the factors that cooperate with Polθ to promote MMEJ repair. Filling this knowledge gap is critical, as the identification of unknown MMEJ proteins could provide alternative targets for treatment of HRD cancers.
Through genome-wide CRISPR-Cas9 screens46 that exploit the synthetic lethal interaction between MMEJ and HR, we have identified the APE2 nuclease as an MMEJ effector.
Section snippets
Results
To discover potentially uncharacterized effectors of MMEJ, we exploited the synthetic lethality observed when cells lose both HR and MMEJ.12,13 To identify genes that are synthetically lethal with HRD in general, rather than with one gene in particular, we compared the genetic interactions of two HR genes, BRCA1 and PALB2.47,48,49 We created isogenic cell lines by knocking out BRCA1 or PALB2 with transient CRISPR-Cas9 expression in HT1080 fibrosarcoma HR-proficient cells and established clonal
Discussion
We demonstrate here that APE2 is recruited to DSBs and that its nuclease activity is essential for DSB repair by MMEJ, establishing APE2 as a critical player for the MMEJ-driven repair mechanism. We consistently found that APE2 or Polθ suppression leads only to partial repression of MMEJ, suggesting the existence of a Polθ-independent MMEJ mechanism. Indeed, Polλ and HELQ were recently shown to mediate some MMEJ activity….
