- Email: [email protected]
Defining and Modulating ‘BRCAness’
TICB 1525 No. of Pages 12
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Review
Defining and Modulating ‘BRCAness’ Andrea K. Byrum,1 Alessandro Vindigni,2,* and Nima Mosammaparast1,* The concept of ‘BRCAness’ defines the pathogenesis and vulnerability of multiple cancers. The canonical definition of BRCAness is a defect in homologous recombination repair, mimicking BRCA1 or BRCA2 loss. In turn, BRCA-deficient cells utilize error-prone DNA-repair pathways, causing increased genomic instability, which may be responsible for their sensitivity to DNA damaging agents and poly-(ADP)-ribose polymerase inhibitors (PARPis). However, recent work has expanded the mechanistic basis of BRCAness, to include defects in replication fork protection (RFP). Here, we broaden the definition of BRCAness to include RFP and regulatory mechanisms that cause synthetic lethality with PARPis. We highlight these recent discoveries, which include mechanisms of RFP regulation, DNA damage checkpoint proteins, as well as kinases that regulate BRCA1/2 function. Importantly, many of these emerging mechanisms may be targeted for inhibition with small molecule inhibitors, thus inducing BRCAness in a much larger subset of BRCA-proficient tumors, with significant translational potential.
Highlights BRCA1 and BRCA2 both contribute to homologous recombination repair, loss of which is a defining feature of BRCAness. Replication fork protection has emerged as a major function of these tumor suppressors. Numerous kinases involved in checkpoint function and the cell cycle impinge upon BRCA1/2 function. Targeting BRCA1/2 modulators may be an effective strategy to sensitize tumors to DNA damaging agents.
Expanding the Definition of ‘BRCAness’ The repair of DNA damage is essential to ensure faithful transmission of genetic information to subsequent generations of cells. Many genomic lesions are cytotoxic, potentially mutagenic, or lead to oncogenic translocations. These lesions are often repaired by homologous recombination repair (HRR; see Glossary), which relies upon an undamaged chromosome to be used as the template for error-free damage reversal. Not surprisingly, factors involved in promoting HRR, such as the breast cancer susceptibility genes BRCA1 and BRCA2, function as tumor suppressors. Indeed, inherited mutations in these genes confer significant lifetime risks of breast, ovarian, and other cancers. At the same time, DNA repair deficiencies associated with BRCA1/2 loss represent the Achilles’ heel of these cancer cells, which may be exploited in therapies with DNA-damaging drugs, such as platinum-based compounds, or agents that inhibit specific DNA-repair pathways, such as poly (ADP-ribose) polymerase (PARP) inhibitors. The intimate association between reduced HRR and the sensitivity of such cells to DNA-damaging agents and PARPis has been central to the original definition of BRCAness. Recent deep-sequencing studies have revealed sporadic mutations in these tumor suppressors not only in breast and ovarian cancer, but also in prostate, colon, and pancreatic adenocarcinoma [1–3]. Thus, the mechanistic role of BRCA proteins in tumorigenesis, as well as the potential use of DNA damaging agents and PARPi therapy for such tumors, may be significantly broader than initially recognized. Interestingly, certain types of cancer, such as Ewing’s sarcoma, do not harbor mutations in BRCA1 or BRCA2, yet still behave as if they are deficient in these factors and are PARPi sensitive [4]. In addition, several other factors are known to modulate BRCA1/2 protein function, and therefore have altered HRR and PARPi responses. For example, hypoxic conditions reduce BRCA1 transcript expression via repressive E2F transcription factors and changes in histone methylation, resulting in significantly reduced HRR without affecting nonhomologous end joining (NHEJ) [5,6]. The RNA polymerase II-associated kinase cyclin-dependent kinase (CDK) 12 also appears to selectively affect transcription and splicing of several HRR genes, including BRCA1 and BRCA2. Thus, modulation of BRCA1/2 expression is a straightforward mechanism that may alter HRR function. Besides their role in promoting HRR at several distinct steps (Box 1), recent discoveries have uncovered not only additional layers of BRCA1/2 regulation, but also Trends in Cell Biology, Month 2019, Vol. xx, No. xx
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Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO 63110, USA 2 Division of Oncology, Department of Internal Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA
*Correspondence: [email protected] (A. Vindigni) and [email protected] (N. Mosammaparast).
https://doi.org/10.1016/j.tcb.2019.06.005 © 2019 Published by Elsevier Ltd.
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Box 1. Established Roles of BRCA1 and BRCA2 in HRR
Glossary
When a DNA lesion such as a DSB is formed, it is initially recognized by sensors such as the Mre11–Rad50–Nbs1 (MRN) complex. In turn, this damage recognition complex is thought to activate damage signaling kinases, particularly ATM (ataxia telangiectasia mutated; Step 1 in Figure I), as well as the related kinases ATR and DNA-PK. These kinases phosphorylate hundreds of proteins, which ultimately leads to the recruitment of repair effectors, including BRCA1 (Step 2). BRCA1 promotes DNA end resection by nucleases, such as EXO1, through undefined mechanisms. BRCA1 also promotes the recruitment of BRCA2, which is necessary to assemble RAD51 onto single-stranded DNA (Step 3). BRCA1 facilitates the assembly of the D-loop intermediate (Step 4). The homologous strands are used for templated replication (Step 5), followed by Holliday junction resolution (Step 6).
Figure I. Established Roles of BRCA1 and BRCA2 in Homologous Recombination Repair. Abbreviations: ATM, ataxia telangiectasia mutated; MRN, Mre11–Rad50– Nbs1 complex.
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additional functions of these factors beyond HRR which relate to chemosensitivity. This necessitates revisiting the definition of BRCAness, which we define functionally as a DNA repair defect that phenocopies loss of BRCA1 or BRCA2, ultimately leading to PARPi sensitivity.
BRCA1/2 in RFP Aside from their well-established roles in HRR, BRCA1 and BRCA2 play key roles in the maintenance of replication fork stability, which goes beyond the repair of double-strand breaks (DSBs) at collapsed forks. Using genome-wide single-molecule DNA fiber approaches, multiple groups 2
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Cyclin-dependent kinase (CDK): type of kinase that is activated by a regulatory protein partner, called a cyclin. Homologous recombination repair (HRR): pathway of DNA DSB repair that depends upon the homologous chromosome for templated synthesis, and is thus considered error free. Meiotic recombination 11-like (MRE11): component of the MRN (Mre11–Nbs1–Rad50) complex that recognizes DNA DSBs and harbors both exonuclease and endonuclease activities. Poly(ADP-ribose) polymerase (PARP): ssDNA break binding protein that catalyzes the addition of poly(ADP-ribose) chains to itself and other proteins. Replication fork protection (RFP): mechanism of genome maintenance that is activated during replication to protect nascent DNA from nucleolytic degradation.
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have shown that BRCA proteins are essential for replication fork protection (RFP) from nucleolytic attack upon treatment with genotoxic agents. In their absence, nucleases extensively degrade the unprotected replication forks [7–11]. Following this discovery, much effort has been devoted to uncovering the nucleases and regulatory factors leading to extensive fork degradation, as well as the structure of the replication intermediates targeted by nucleases in the absence of BRCA1 or BRCA2. Four recent studies defined that the structure targeted by nucleases in a BRCA-deficient background is a reversed replication fork [12–15]. Fork reversal is a key mechanism that allows replication forks to reverse their course in order to cope with DNA lesions [16–19]. These four studies have shown that BRCA1 and BRCA2 are required to stabilize the RAD51 filament on the regressed arms of already formed reversed replication forks, thereby protecting the open double-stranded end of the regressed arm from nucleolytic attack (Figure 1) [12–15]. In the absence of BRCA, the open DNA end of the unprotected regressed arm of reversed replication forks represents the entry point for nucleases. In addition, recent studies have shown how BRCA2 is also important to prevent single-stranded (ss)DNA gap accumulation both at replication fork junctions and behind forks by stabilizing RAD51 binding [12]. Forks with persistent ssDNA gaps would be subsequently converted into reversed forks, leading to extensive degradation. Meiotic recombination 11-like (MRE11) was the first nuclease associated with fork degradation in BRCA-deficient tumors [7–11]. However, fork degradation leads to long stretches of
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Figure 1. Mechanism of Reversed Fork Protection and Degradation. Genotoxic agents lead to fork uncoupling and reversal. BRCA2 protects reversed forks from degradation by stabilizing the RAD51 filament on the regressed arm, allowing accurate fork restart and genome stability. BRCA1 shares a similar function in reversed fork stabilization. Loss of BRCA1 or BRCA2 leads to meiotic recombination 11-like (MRE11)-mediated degradation of the reversed replication forks, causing genomic instability. MLL, Pax2 transactivation domain-interacting protein (PTIP), RAD52, CHD4, sterile α motif and HD-domain containing protein 1 (SAMHD1), and poly-(ADP)-ribose polymerase (PARP) are required for MRE11 loading on stalled replication forks.
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ssDNA (N4 kb) and MRE11 is unlikely to be the only nuclease responsible to degrade several kb of DNA due to its poor processivity. In agreement with this model, human exonuclease (EXO)1 was later shown to contribute to fork degradation in BRCA-deficient tumors [13]. In addition, CtIP protein is required to initiate MRE11-dependent fork degradation [13], although a more recent study seems to contradict this conclusion [20]. As discussed above, these nucleases target reversed replication forks. The regressed arm of a reversed replication fork resembles by all means a one-ended DSB. Previous biochemical studies showed that the endonuclease activity of MRE11 initiates resection at DSBs and that CtIP promotes MRE11 endonuclease activity at the 5′ strand [21,22]. Resection is then continued by the 5′–3′ exonuclease activity of EXO1 [23– 25]. The discovery that MRE11, CtIP, and EXO1 cooperate in reversed fork degradation suggests that the same pathway that initiates DSB resection in the context of HRR is likely required to process the open dsDNA ends of the regressed arms in the context of DNA replication. DNA2 is also implicated in DSB resection [25]. However, whether human DNA2 contributes to reversed fork degradation in the absence of BRCA remains controversial [9,13]. Interestingly, DNA2 but not MRE11 promotes fork degradation in cells lacking the biorientation defect 1-like protein (BOD1L) [26], the human RecQ helicase RECQ1 [27], or Abro1 [28], which is a paralog of the BRCA1 interacting protein Abraxas. These results suggest that different nucleases might participate in fork degradation depending on the particular genetic background. The structure specific nuclease MUS81 has been also implicated in fork processing in the absence of BRCA [13,29,30], and its recruitment is mediated by EZH2, a histone methyltransferase [29]. Specifically, MUS81 was shown to cleave the partially resected reversed forks and promote DNA polymerase δ-dependent DNA fork rescue through a break-induced replication (BIR)-like mechanism [13]. MUS81 and EZH2 modulate fork rescue in BRCA2- but not BRCA1-deficient cells, suggesting that BRCA1-deficient cells rescue the resected forks through a different pathway [13,29]. The notion that BRCA1 and BRCA2 might have different functions during replication fork stalling and restart is also supported by a recent study showing BRCA1, but not BRCA2, is required to suppress tandem duplications at Tus/Ter replication fork barriers [31]. These tandem duplications are enriched in loci containing known oncogenes, while the breakpoints of these duplications are enriched in tumor suppressor genes, contributing to the genomic instability of BRCA1-deficient cells [32]. The extensive fork degradation phenotype observed in the absence of BRCA proteins is one of the leading causes for the increased genome instability and chemosensitivity of BRCA-deficient tumors [9]. Further studies will be however necessary to define the different contribution of the HRR and fork stability functions of BRCA proteins in genome stability and chemosensitivity [9,33]. BRCA-deficient tumors are sensitive to therapies that target DNA or that inhibit specific repair pathways, such as PARPis [34]. The efficacy of these therapies is often hampered by the development of chemoresistance. Chemoresistance has been associated with the recovered ability of BRCA-deficient cells to protect replication forks from nucleolytic degradation [9]. Aside from genetic reversion, which might occur in a limited subset of BRCA-mutant breast cancers, the pathways that rescue the ability of BRCA-deficient cells to protect forks from degradation and that mediate resistance to therapy remain largely unknown. In an effort to define these chemoresistance pathways, recent studies have focused on identifying the factors involved in nuclease recruitment at stalled/damaged replication forks. The Pax2 transactivation domaininteracting protein (PTIP) and the histone methyltransferases MLL3 and MLL4 were identified as key factors involved in MRE11 recruitment at stalled replication forks in BRCA1/2-deficient cells [9,35]. Recovered fork protection in BRCA-mutant cells was also linked to loss of the nucleosome remodeling factor CHD4 and inhibition of the poly(ADP-ribosyl)ation, suggesting that CHD4 and PARP are two other factors involved in MRE11 recruitment [9,36]. Moreover, recent studies suggested that human RAD52 and the sterile α motif and HD-domain containing 4
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protein (SAMHD)1 nuclease also regulate MRE11 loading on replication forks [14,37]. These studies open the provocative scenario that drug resistance in BRCA-deficient cells derives from deficiencies in these factors because they would limit MRE11 loading to stalled replication forks. Besides the mechanism of MRE11 recruitment to stalled forks, the mechanisms that control the recruitment of other nucleases involved in fork degradation remain unknown. We envision that defining these mechanisms will provide a major breakthrough toward the understanding of the molecular pathways that control genome integrity and chemoresistance in BRCA-deficient tumors.
Regulation of BRCA Function by Kinases ATR and CHK1 As described above, the mechanism of fork reversal may function to give the cell additional time to repair a lesion before continuing with S phase. Similarly, the DNA damage response involves additional signaling modalities to halt the cell cycle for the purposes of DNA repair. Cancer cells harboring mutations in the tumor suppressor gene p53 exhibit a defective G1 cell cycle checkpoint, creating a reliance on pathways that regulate the subsequent S, G2, and M phase checkpoints [38]. For this reason, components of the ATR/CHK1 signaling cascade, which activates the intra-S and G2/M checkpoints in response to DSBs or replication stress, have long been viewed as promising drug targets. Beyond its cell cycle functions, more recent studies have identified the ATR/CHK1 pathway as a key modulator of BRCAness by regulating the association of BRCA1/2 with their binding partners. It has long been known that ATR and its downstream effector CHK1 promote gene-conversionmediated HRR of DSBs and collapsed replication forks [39–41]. Mechanistically, ATR-mediated phosphorylation of PALB2 at S59 enhances the BRCA1–PALB2 interaction and is required for PALB2 localization to DSBs (Figure 2A). Furthermore, ATR/CHK1 inhibition of CDK activity following DNA end resection appears necessary for the BRCA1–PALB2 interaction and subsequent HRR [42]. Phosphorylation of PALB2 by ATR/CHK1 may also promote HRR by enhancing the interaction between PALB2 and RAD51 [43]. Furthermore, phosphorylation by ATR/CHK1 has been shown to stabilize the BRCA1 protein by protecting it from proteasomemediated degradation [44]. Additionally, chronic inhibition of ATR and CHK1 decreases transcription of pro-HRR factors such as BRCA1, TOPBP1, and RAD51, and this effect is augmented in cancer cell lines that overexpress HRR proteins [44]. Therefore, the role of ATR/CHK1 in promoting HRR is multifaceted and is critical for genomically unstable tumors to thrive. ATR and CHK1 are also required to stabilize stalled replication forks in response to stress on both a global and localized level. Globally, ATR/CHK1 inhibits CDK2 activity when replication forks stall in order to prevent new origins from firing, which would ultimately lead to an exhaustion of RPA, ssDNA accumulation, and DNA breakage, which would require the BRCA1/2 pathway [45] (Figure 2B). When ATR is inhibited, origin firing can still be blocked via a backup pathway in which DNA-PK activates CHK1 [46]. Likewise, inhibition of CHK1 alone increases ATR activity, suggesting that CHK1 and ATR globally promote genomic stability via both overlapping and distinct mechanisms [47]. This phenomenon is also observed locally at stalled replication forks. In response to CHK1 inhibition, stressed replication forks are processed into DSBs by the MUS81–EME1 nuclease complex, while fork collapse in ATR-inhibited cells is not dependent on MUS81–EME1 [48,49]. Additionally, two CHK1-independent mechanisms for ATRmediated replication fork protection have been proposed. First, ATR activity has been shown to mediate global replication fork reversal to prevent chromosomal breakage at sites of DNA interstrand crosslinks [50]. This contradicts previous findings suggesting that ATR phosphorylates the fork reversal enzyme SMARCAL1 in order to suppress excessive fork reversal and subsequent DNA cleavage by SLX4-dependent nucleases [51] (Figure 2C). This apparent contradiction
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might be linked to the different genotoxic agents used in the two studies and further work will need to be done to define the exact role of ATR signaling in fork remodeling. Second, ATR may promote loading of FANCD2 to replication forks via phosphorylation of the MCM2-7 helicase. FANCD2 then inhibits fork collapse by both slowing the DNA polymerase and by blocking Mre11 from resecting the fork intermediate [52]. Due to their critical functions in both HRR and replication fork protection, ATR and CHK1 exemplify BRCAness modulators that may be targeted to combat cancer chemoresistance. ATR inhibition sensitizes ovarian cancer cell lines to several chemotherapy agents, including the PARPi veliparib, regardless of BRCA status. Likewise, the combination of either ATRi or CHK1i with olaparib synergistically decreases viability in both BRCA-mutated and HRR-proficient cancer cells. Furthermore, treatment of an ovarian BRCA2-mutant patient-derived xenograft (PDX) model with olaparib and ATRi results in complete tumor regression, which is not observed with either single agent [53]. Additionally, ATRi treatment was recently shown to reverse PARPi resistance in cancer cells by blocking re-established HRR or fork protection pathways [54,55]. Notably, in all of these studies, inhibiting ATR was more effective than CHK1 inhibition for sensitizing cells to PARPi, which may be explained by the independent functions of ATR and CHK1 in replication fork protection. WEE1 The tyrosine kinase WEE1 has also emerged as a putative BRCAness factor that functions in both checkpoint activation and in replication fork stability. In response to DNA damage, WEE1 directly phosphorylates and inhibits CDK1/2, blocking cells from entering mitosis [56] (Figure 3A). While CHK1 has been shown to activate WEE1 in yeast and Xenopus, definitive evidence of this in mammalian cells is lacking [57,58]. However, inhibition of WEE1 during DNA damage induction in human cells results in inactivation of ATR and CHK1, suggesting that crosstalk among these kinases is required to sustain the replication stress response [59]. WEE1 and CHK1 also have roles in protecting stalled replication forks from degradation by overlapping and distinct mechanisms. Like CHK1, WEE1 depletion or inhibition induces replication fork slowing and MUS81dependent fork collapse [60,61] (Figure 3A). However, loss or inactivation of both proteins exasperates replication fork defects and significantly increases cell death when compared with individual knockdowns [61]. The synthetic lethality of combined WEE1/CHK1 inhibition was later confirmed in numerous cancer cell lines, which included ovarian, breast, colon, prostate, and melanoma, as well as xenograft models [62,63]. Dual inhibition of WEE1 and ATR has also shown promise, and such a combination may be particularly effective for combating PARPi and cisplatin resistance. Two groups found that the combination of the ATR inhibitor AZD6738 and the WEE1 inhibitor AZD1775, which are currently in clinical trials, inhibited breast cancer cell growth and reduced metastasis in PDX models [64,65]. Furthermore, combination of ATRi and WEE1i sensitized triple-negative breast cancer cells to cisplatin and the PARPi veliparib, with an increased effect in BRCA1-deficient cells [65]. WEE1 inhibition alone also sensitizes cancer cells and xenograft models to PARPi and cisplatin [55,66–68]. However, given the above evidence that ATR, CHK1, and WEE1 loss induce BRCAness via distinct mechanisms, inhibiting more than one of these kinases may be more effective. Figure 2. ATR and CHK1 Promote BRCA1/2 Function via Overlapping and Distinct Mechanisms. (A) ATR phosphorylates PALB2, promoting PALB2 association with BRCA1 (top) and RAD51 (bottom). (B) As a global response to replication stress, ATR-activated CHK1 phosphorylates CDC25 that inhibits cyclin-dependent kinase (CDK) activity. This prevents RPA exhaustion and additional DNA breaks which require repair by BRCA proteins. (C) ATR phosphorylates SMARCAL1 to inhibit replication fork remodeling and nucleolytic degradation. Additionally, ATR phosphorylation of the MCM2-7 helicase helps recruit FANCD2 to stalled replication forks where it also blocks nucleases from degrading nascent DNA.
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Figure 3. WEE1 and Aurora A Kinases Regulate BRCAness. (A) In response to DNA damage, activated WEE1 phosphorylates cyclin-dependent kinase (CDK)1/2 in order to inhibit cell cycle progression, which would amplify replication stress. WEE1 also inhibits the MUS81–EME1 nuclease from degrading stalled replication fork intermediates through a direct or indirect mechanism. (B) The TPX2/Aurora A heterodimer negatively regulates 53BP1 function during double-stranded break repair, promoting BRCA1 recruitment and subsequent repair by homologous recombination (HR). (C) In response to replication stress, Aurora A and BRCA1 protect stalled replication forks from meiotic recombination 11-like (MRE11)-mediated degradation via complementary but distinct pathways. BRCA1 prevents Pax2 transactivation domain-interacting protein (PTIP) recruitment to the fork intermediate, which thereby inhibits the recruitment of MRE11. Aurora A and its cofactor TPX2 inhibits MRE11 function by negatively regulating 53BP1 through a mechanism dependent upon Aurora A kinase activity. Abbreviations: AurA, Aurora A; NHEJ, nonhomologous end joining.
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Aurora A In contrast to checkpoint kinases ATR, CHK1, and WEE1, the Aurora A kinase promotes cell cycle progression through G2 and mitosis, and is required for numerous mitotic events including centrosome maturation, spindle assembly, and cytokinesis. Generally, these functions are also dependent upon the Aurora A cofactor TPX2, which places Aurora A into its active conformation upon binding and localizes Aurora A to the spindle [69,70]. Importantly, TPX2 is necessary for sustaining Aurora A kinase activity throughout mitosis, as it protects the catalytic T288 site from dephosphorylation and inhibits Aurora A proteasomal degradation [69,71]. In humans, the TPX2 and Aurora A genes are located on the long arm of chromosome 20, which is commonly amplified in tumors. In fact, TPX2 and Aurora A overexpression occurs in a long list of cancer types and is notably associated with those exhibiting BRCA1/2 mutations [72–74]. Furthermore, high Aurora A to BRCA2 expression ratios are associated with poor prognosis in ovarian carcinoma patients [75,76]. Several Aurora A inhibitors have been developed over the past two decades. The aforementioned clinical findings and the molecular studies discussed below suggest that Aurora A inhibitors may modulate BRCAness via multiple mechanisms to enhance tumor chemosensitivity. Recent investigations have demonstrated that prior to mitosis, TPX2 and Aurora A promote BRCA functions. Inhibition of Aurora A with the small molecule inhibitor alisertib decreases PARP activity as well as the expression of PARP1 and BRCA1/2 in cell lines and xenograft models. This leads to an increased reliance on error-prone NHEJ to repair DSBs and a reduction in HRR [77]. Similarly, we showed that loss of either TPX2 or Aurora A inhibits BRCA1 and RAD51 recruitment, causing a modest but significant decrease in HRR (Figure 3B). More notably, using DNA fiber analysis, we found that TPX2 and Aurora A are required to protect stalled replication forks from MRE11-mediated degradation in a manner dependent upon Aurora A kinase activity (Figure 3C). This function is carried out via a separate pathway from BRCA1, as loss of both TPX2 and BRCA1 has an additive effect on replication fork instability. The BRCAness phenotypes resulting from TPX2 and Aurora A depletion are rescued upon loss of 53BP1, suggesting that TPX2 and Aurora A promote HRR and replication fork protection by negatively regulating 53BP1 (Figure 3B,C). Importantly, these novel functions of TPX2 and Aurora A appear to be independent of their mitotic roles, demonstrating the multifaceted ways in which properly regulated TPX2 and Aurora A promote genome integrity [78]. In addition to these functions of TPX2 and Aurora A, there are conflicting results pointing to a functional connection between Aurora A and BRCA1/2 in cell cycle regulation [76,79–81]. These findings suggest that Aurora A is an effective target for combating chemoresistance. In combination with a PARPi or DNA-damaging agent, inhibition of Aurora A in mitosis may induce mitotic catastrophe while inhibition in S/G2 should impair HRR repair and replication fork protection. Consistent with this the combination of alisertib and the PARPi, rucaparib had a synergistic effect on cell viability in both BRCA wild type and mutant cancer cell lines [77]. Our work also showed that Aurora-A-depleted cells are sensitive to hydroxyurea treatment [78]. Additionally, BRCA2-deficient cancer cells are preferentially sensitive to alisertib treatment and to depletion of TPX2 and Aurora A [82]. Together, these results suggest that Aurora A inhibition induces a functional BRCA loss and may be used in combination with PARP inhibitors to target multiple DNA damage response pathways in tumors, thus potentially preventing chemoresistance. Polo-Like Kinase (Plk)1 Plk1 is a substrate of the Aurora A kinase during mitosis, and has been implicated as a BRCAness factor. Recent work showed that the BRCA2 N-terminal domain is phosphorylated by CDKs in G2/M phase as well in response to genotoxic stress [83,84]. Plk1 binds to phosphorylated
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BRCA2, which serves as a scaffold to bring the kinase within close proximity to Rad51 [84]. Plk1 then phosphorylates Rad51 on S14, which subsequently promotes CK2-mediated phosphorylation of the adjacent T13 residue. Phosphorylated T13 then stimulates a direct binding interaction with NBS1 and recruitment of Rad51 to sites of damage. Notably, loss of Plk1-dependent S13/T14 phosphorylation leads to defects in HR and PARPi sensitivity [85]. Plk1 also facilitates Rad51 recruitment to both active and stalled replication forks and is necessary for efficient fork restart following hydroxyurea (HU) treatment [84]. Like Aurora A, Plk1 is overexpressed in numerous cancers, and high Plk1 levels are associated with BRCA deficiencies, making it a promising drug target to combat chemoresistance [86]. In fact, a recent study demonstrated that the Plk1 inhibitor BI2536 sensitizes BRCA1-deficient castration-resistant prostate cancer cells and xenograft tumors to olaparib, making the case for testing this drug combination in clinical trials [87].
Outstanding Questions What is the mechanistic relationship between HRR and RFP? What are the relative contributions of HRR and RFP to tumor suppression and chemosensitivity in the BRCAdeficient state? A recent study investigating separation-of-function mutations in BRCA1 and BARD1 suggests that defects in HRR and RFP may promote distinct stages of tumorigenesis. with RFP playing a critical role earlier than HRR [1,88]. However, additional experiments are needed to directly test this model.
Concluding Remarks Recent work has significantly broadened the concept of BRCAness and the molecules involved in this phenotype. Specifically, aside from a loss of canonical HRR, several additional factors, including DNA damage checkpoint proteins and kinases, promote the BRCA1/2 pathway and contribute to BRCAness. In addition, BRCAness is characterized by defects in replication fork protection. Thus, both the conceptual understanding of the BRCA-deficient state, as well as the molecules involved in regulating BRCA1/2 function, have been significantly expanded. This reflects the growing number of strategies that exploit BRCAness in a clinical setting. We envision a number of innovative combinations of pathway inhibition to be tested to induce functional BRCA loss and combat chemotherapy resistance. As described above, many of these BRCAmodulating factors are kinases with readily available inhibitors, thus making such combination therapies testable immediately. At the same time, these recent discoveries raise several additional questions that need to be addressed (see Outstanding questions). By addressing these fundamental questions on BRCA function we envision that more effective and novel and strategies will be developed to increase cancer chemosensitivity and overcome chemoresistance. Acknowledgments We wish to thank the numerous scientists who have contributed significantly to our understanding BRCA function, and we apologize that we could not cite all pertinent papers due to space limitations. A.K.B. was supported by the Washington University Department of Pathology Hu and Zheng Scholarship. This work was supported by the National Institutes of Health (NIH) (R01 CA237263 to A.V. and R01 CA227001 to N.M.), the Department of Defense [BCRP (Breast Cancer Research Program) Breakthrough Award BC151728 to A.V.], the Structural Biology of DNA Repair program project (P01 CA092584), and an American Cancer Society Research Scholar Award (to N.M.). The authors have no conflicts of interest to declare.
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What are the relative contributions of HRR and RFP to chemosensitivity in BRCA-deficient cells? To date, the prevailing assumption has been that the loss of these same functions (namely HRR and RFP) are also responsible for increased chemosensitivity, yet this notion has not been definitively established. Are there functional differences between BRCA1 and BRCA2 in tumor suppressive and chemosensitivity contexts? What are the substrates of the cell cycle and checkpoint kinases in regulating BRCAness? What additional factors and signaling pathways contribute to BRCAness?
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12. Kolinjivadi, A.M. et al. (2017) Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Mol. Cell 67, 867–881.e7 13. Lemacon, D. et al. (2017) MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat. Commun. 8, 860 14. Mijic, S. et al. (2017) Replication fork reversal triggers fork degradation in BRCA2-defective cells. Nat. Commun. 8, 859 15. Taglialatela, A. et al. (2017) Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2family fork remodelers. Mol. Cell 68, 414–430.e8 16. Berti, M. et al. (2013) Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nat. Struct. Mol. Biol. 20, 347–354 17. Neelsen, K.J. and Lopes, M. (2015) Replication fork reversal in eukaryotes: from dead end to dynamic response. Nat. Rev. Mol. Cell Biol. 16, 207–220 18. Ray Chaudhuri, A. et al. (2012) Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat. Struct. Mol. Biol. 19, 417–423 19. Zellweger, R. et al. (2015) Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J. Cell Biol. 208, 563–579 20. Przetocka, S. et al. (2018) CtIP-mediated fork protection synergizes with BRCA1 to suppress genomic instability upon DNA replication stress. Mol. Cell 72, 568–582 21. Anand, R. et al. (2016) Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection. Mol. Cell 64, 940–950 22. Symington, L.S. (2014) End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect. Biol. 6 Published online August 1, 2014. https://doi.org/10.1101/ cshperspect.a016436 23. Cannavo, E. et al. (2013) Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. Proc. Natl. Acad. Sci. U. S. A. 110, E1661–E1668 24. Nicolette, M.L. et al. (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat. Struct. Mol. Biol. 17, 1478–1485 25. Nimonkar, A.V. et al. (2011) BLM-DNA2-RPA-MRN and EXO1BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev. 25, 350–362 26. Higgs, M.R. et al. (2015) BOD1L is required to suppress deleterious resection of stressed replication forks. Mol. Cell 59, 462–477 27. Thangavel, S. et al. (2015) DNA2 drives processing and restart of reversed replication forks in human cells. J. Cell Biol. 208, 545–562 28. Xu, S. et al. (2017) Abro1 maintains genome stability and limits replication stress by protecting replication fork stability. Genes Dev. 31, 1469–1482 29. Rondinelli, B. et al. (2017) EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation. Nat. Cell Biol. 19, 1371–1378 30. Lai, X.N. et al. (2017) MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells. Nat. Commun. 8, 5983 31. Willis, N.A. et al. (2017) Mechanism of tandem duplication formation in BRCA1-mutant cells. Nature 551, 590–595 32. Menghi, F. et al. (2016) The tandem duplicator phenotype as a distinct genomic configuration in cancer. Proc. Natl. Acad. Sci. U. S. A. 113, E2373–E2382 33. Feng, W. and Jasin, M. (2017) BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination. Nat. Commun. 8, 525 34. Lord, C.J. and Ashworth, A. (2017) PARP inhibitors: synthetic lethality in the clinic. Science 355, 1152–1158 35. Ding, X. et al. (2016) Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies. Nat. Commun. 7, 12425 36. Guillemette, S. et al. (2015) Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4. Genes Dev. 29, 489–494
37. Coquel, F. et al. (2018) SAMHD1 acts at stalled replication forks to prevent interferon induction. Nature 557, 57–61 38. Qiu, Z. et al. (2018) ATR/CHK1 inhibitors and cancer therapy. Radiother. Oncol. 126, 450–464 39. Sorensen, C.S. et al. (2005) The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat. Cell Biol. 7, 195–U121 40. Wang, H.Y. et al. (2004) ATR affecting cell radiosensitivity is dependent on homologous recombination repair but independent of nonhomologous end joining. Cancer Res. 64, 7139–7143 41. Adamson, B. et al. (2012) A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat. Cell Biol. 14, 318–328 42. Buisson, R. et al. (2017) Coupling of homologous recombination and the checkpoint by ATR. Mol. Cell 65, 336–346 43. Ahlskog, J.K. et al. (2016) ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function. EMBO Rep. 17, 671–681 44. Kim, D. et al. (2018) ATR-mediated proteome remodeling is a major determinant of homologous recombination capacity in cancer cells. Nucleic Acids Res. 46, 8311–8325 45. Toledo, L.I. et al. (2013) ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 46. Buisson, R. et al. (2015) Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase. Mol. Cell 59, 1011–1024 47. Sanjiv, K. et al. (2016) Cancer-specific synthetic lethality between ATR and CHK1 kinase activities. Cell Rep. 14, 298–309 48. Techer, H. et al. (2016) Signaling from Mus81-Eme2-dependent DNA damage elicited by Chk1 deficiency modulates replication fork speed and origin usage. Cell Rep. 14, 1114–1127 49. Forment, J.V. et al. (2011) Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation. PLoS One 6, e23517 50. Mutreja, K. et al. (2018) ATR-mediated global fork slowing and reversal assist fork traverse and prevent chromosomal breakage at DNA interstrand cross-links. Cell Rep. 24, 2629–2642 51. Couch, F.B. et al. (2013) ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Genes Dev. 27, 1610–1623 52. Lossaint, G. et al. (2013) FANCD2 binds MCM proteins and controls replisome function upon activation of S phase checkpoint signaling. Mol. Cell 51, 678–690 53. Kim, H. et al. (2017) Targeting the ATR/CHK1 axis with PARP inhibition results in tumor regression in BRCA-mutant ovarian cancer models. Clin. Cancer Res. 23, 3097–3108 54. Murai, J. et al. (2016) Resistance to PARP inhibitors by SLFN11 inactivation can be overcome by ATR inhibition. Oncotarget 7, 76534–76550 55. Yazinski, S.A. et al. (2017) ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev. 31, 318–332 56. Watanabe, N. et al. (1995) Regulation of the human Wee1hu Cdk tyrosine 15-kinase during the cell-cycle. EMBO J. 14, 1878–1891 57. OConnell, M.J. et al. (1997) Chk1 is a wee1 kinase in the G (2) DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 16, 545–554 58. Lee, J. et al. (2001) Positive regulation of Wee1 by Chk1 and 14-3-3 proteins. Mol. Biol. Cell 12, 551–563 59. Saini, P. et al. (2015) Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress. Oncotarget 6, 13072–13087 60. Beck, H. et al. (2012) Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption. Mol. Cell. Biol. 32, 4226–4236 61. Dominguez-Kelly, R. et al. (2011) Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease. J. Cell Biol. 194, 567–579 62. Guertin, A.D. et al. (2012) Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition. Cancer Cell Int. 12, 45 63. Carrassa, L. et al. (2012) Combined inhibition of Chk1 and Wee1 in vitro synergistic effect translates to tumor growth inhibition in vivo. Cell Cycle 11, 2507–2517
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78. Byrum, A.K. et al. (2019) Mitotic regulators TPX2 and Aurora A protect DNA forks during replication stress by counteracting 53BP1 function. J. Cell Biol. 218, 422–432 79. Sun, H.Z. et al. (2014) Aurora-A controls cancer cell radio- and chemoresistance via ATM/Chk2-mediated DNA repair networks. Biochim. Biophys. Acta Mol. Cell Res. 1843, 934–944 80. Sun, J.M. et al. (2015) Inhibition of Aurora A promotes chemosensitivity via inducing cell cycle arrest and apoptosis in cervical cancer cells. Am. J. Cancer Res. 5, 1133–1145 81. Wang, Y. et al. (2014) The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy. Mol. Cancer 13, 94 82. van Gijn, S.E. et al. (2019) TPX2/Aurora kinase A signaling as a potential therapeutic target in genomically unstable cancer cells. Oncogene 38, 852–867 83. Esashi, F. et al. (2005) CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 434, 598–604 84. Yata, K. et al. (2014) BRCA2 coordinates the activities of cellcycle kinases to promote genome stability. Cell Rep. 7, 1547–1559 85. Yata, K. et al. (2012) Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair. Mol. Cell 45, 371–383 86. Carbajosa, S. et al. (2019) Polo-like kinase 1 inhibition as a therapeutic approach to selectively target BRCA1-deficient cancer cells by synthetic lethality induction. Clin. Cancer Res. Published online March 19, 2019. https://doi.org/10.1158/1078-0432. CCR-18-3516 87. Li, J. et al. (2017) Targeting Plk1 to enhance efficacy of olaparib in castration-resistant prostate cancer. Mol. Cancer Ther. 16, 469–479 88. Billing, D. et al. (2018) The BRCT domains of the BRCA1 and BARD1 tumor suppressors differentially regulate homologydirected repair and stalled fork protection. Mol Cell 72, 127–139.e8
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Review
Defining and Modulating ‘BRCAness’ Andrea K. Byrum,1 Alessandro Vindigni,2,* and Nima Mosammaparast1,* The concept of ‘BRCAness’ defines the pathogenesis and vulnerability of multiple cancers. The canonical definition of BRCAness is a defect in homologous recombination repair, mimicking BRCA1 or BRCA2 loss. In turn, BRCA-deficient cells utilize error-prone DNA-repair pathways, causing increased genomic instability, which may be responsible for their sensitivity to DNA damaging agents and poly-(ADP)-ribose polymerase inhibitors (PARPis). However, recent work has expanded the mechanistic basis of BRCAness, to include defects in replication fork protection (RFP). Here, we broaden the definition of BRCAness to include RFP and regulatory mechanisms that cause synthetic lethality with PARPis. We highlight these recent discoveries, which include mechanisms of RFP regulation, DNA damage checkpoint proteins, as well as kinases that regulate BRCA1/2 function. Importantly, many of these emerging mechanisms may be targeted for inhibition with small molecule inhibitors, thus inducing BRCAness in a much larger subset of BRCA-proficient tumors, with significant translational potential.
Highlights BRCA1 and BRCA2 both contribute to homologous recombination repair, loss of which is a defining feature of BRCAness. Replication fork protection has emerged as a major function of these tumor suppressors. Numerous kinases involved in checkpoint function and the cell cycle impinge upon BRCA1/2 function. Targeting BRCA1/2 modulators may be an effective strategy to sensitize tumors to DNA damaging agents.
Expanding the Definition of ‘BRCAness’ The repair of DNA damage is essential to ensure faithful transmission of genetic information to subsequent generations of cells. Many genomic lesions are cytotoxic, potentially mutagenic, or lead to oncogenic translocations. These lesions are often repaired by homologous recombination repair (HRR; see Glossary), which relies upon an undamaged chromosome to be used as the template for error-free damage reversal. Not surprisingly, factors involved in promoting HRR, such as the breast cancer susceptibility genes BRCA1 and BRCA2, function as tumor suppressors. Indeed, inherited mutations in these genes confer significant lifetime risks of breast, ovarian, and other cancers. At the same time, DNA repair deficiencies associated with BRCA1/2 loss represent the Achilles’ heel of these cancer cells, which may be exploited in therapies with DNA-damaging drugs, such as platinum-based compounds, or agents that inhibit specific DNA-repair pathways, such as poly (ADP-ribose) polymerase (PARP) inhibitors. The intimate association between reduced HRR and the sensitivity of such cells to DNA-damaging agents and PARPis has been central to the original definition of BRCAness. Recent deep-sequencing studies have revealed sporadic mutations in these tumor suppressors not only in breast and ovarian cancer, but also in prostate, colon, and pancreatic adenocarcinoma [1–3]. Thus, the mechanistic role of BRCA proteins in tumorigenesis, as well as the potential use of DNA damaging agents and PARPi therapy for such tumors, may be significantly broader than initially recognized. Interestingly, certain types of cancer, such as Ewing’s sarcoma, do not harbor mutations in BRCA1 or BRCA2, yet still behave as if they are deficient in these factors and are PARPi sensitive [4]. In addition, several other factors are known to modulate BRCA1/2 protein function, and therefore have altered HRR and PARPi responses. For example, hypoxic conditions reduce BRCA1 transcript expression via repressive E2F transcription factors and changes in histone methylation, resulting in significantly reduced HRR without affecting nonhomologous end joining (NHEJ) [5,6]. The RNA polymerase II-associated kinase cyclin-dependent kinase (CDK) 12 also appears to selectively affect transcription and splicing of several HRR genes, including BRCA1 and BRCA2. Thus, modulation of BRCA1/2 expression is a straightforward mechanism that may alter HRR function. Besides their role in promoting HRR at several distinct steps (Box 1), recent discoveries have uncovered not only additional layers of BRCA1/2 regulation, but also Trends in Cell Biology, Month 2019, Vol. xx, No. xx
1
Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO 63110, USA 2 Division of Oncology, Department of Internal Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA
*Correspondence: [email protected] (A. Vindigni) and [email protected] (N. Mosammaparast).
https://doi.org/10.1016/j.tcb.2019.06.005 © 2019 Published by Elsevier Ltd.
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Box 1. Established Roles of BRCA1 and BRCA2 in HRR
Glossary
When a DNA lesion such as a DSB is formed, it is initially recognized by sensors such as the Mre11–Rad50–Nbs1 (MRN) complex. In turn, this damage recognition complex is thought to activate damage signaling kinases, particularly ATM (ataxia telangiectasia mutated; Step 1 in Figure I), as well as the related kinases ATR and DNA-PK. These kinases phosphorylate hundreds of proteins, which ultimately leads to the recruitment of repair effectors, including BRCA1 (Step 2). BRCA1 promotes DNA end resection by nucleases, such as EXO1, through undefined mechanisms. BRCA1 also promotes the recruitment of BRCA2, which is necessary to assemble RAD51 onto single-stranded DNA (Step 3). BRCA1 facilitates the assembly of the D-loop intermediate (Step 4). The homologous strands are used for templated replication (Step 5), followed by Holliday junction resolution (Step 6).
Figure I. Established Roles of BRCA1 and BRCA2 in Homologous Recombination Repair. Abbreviations: ATM, ataxia telangiectasia mutated; MRN, Mre11–Rad50– Nbs1 complex.
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additional functions of these factors beyond HRR which relate to chemosensitivity. This necessitates revisiting the definition of BRCAness, which we define functionally as a DNA repair defect that phenocopies loss of BRCA1 or BRCA2, ultimately leading to PARPi sensitivity.
BRCA1/2 in RFP Aside from their well-established roles in HRR, BRCA1 and BRCA2 play key roles in the maintenance of replication fork stability, which goes beyond the repair of double-strand breaks (DSBs) at collapsed forks. Using genome-wide single-molecule DNA fiber approaches, multiple groups 2
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Cyclin-dependent kinase (CDK): type of kinase that is activated by a regulatory protein partner, called a cyclin. Homologous recombination repair (HRR): pathway of DNA DSB repair that depends upon the homologous chromosome for templated synthesis, and is thus considered error free. Meiotic recombination 11-like (MRE11): component of the MRN (Mre11–Nbs1–Rad50) complex that recognizes DNA DSBs and harbors both exonuclease and endonuclease activities. Poly(ADP-ribose) polymerase (PARP): ssDNA break binding protein that catalyzes the addition of poly(ADP-ribose) chains to itself and other proteins. Replication fork protection (RFP): mechanism of genome maintenance that is activated during replication to protect nascent DNA from nucleolytic degradation.
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have shown that BRCA proteins are essential for replication fork protection (RFP) from nucleolytic attack upon treatment with genotoxic agents. In their absence, nucleases extensively degrade the unprotected replication forks [7–11]. Following this discovery, much effort has been devoted to uncovering the nucleases and regulatory factors leading to extensive fork degradation, as well as the structure of the replication intermediates targeted by nucleases in the absence of BRCA1 or BRCA2. Four recent studies defined that the structure targeted by nucleases in a BRCA-deficient background is a reversed replication fork [12–15]. Fork reversal is a key mechanism that allows replication forks to reverse their course in order to cope with DNA lesions [16–19]. These four studies have shown that BRCA1 and BRCA2 are required to stabilize the RAD51 filament on the regressed arms of already formed reversed replication forks, thereby protecting the open double-stranded end of the regressed arm from nucleolytic attack (Figure 1) [12–15]. In the absence of BRCA, the open DNA end of the unprotected regressed arm of reversed replication forks represents the entry point for nucleases. In addition, recent studies have shown how BRCA2 is also important to prevent single-stranded (ss)DNA gap accumulation both at replication fork junctions and behind forks by stabilizing RAD51 binding [12]. Forks with persistent ssDNA gaps would be subsequently converted into reversed forks, leading to extensive degradation. Meiotic recombination 11-like (MRE11) was the first nuclease associated with fork degradation in BRCA-deficient tumors [7–11]. However, fork degradation leads to long stretches of
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Figure 1. Mechanism of Reversed Fork Protection and Degradation. Genotoxic agents lead to fork uncoupling and reversal. BRCA2 protects reversed forks from degradation by stabilizing the RAD51 filament on the regressed arm, allowing accurate fork restart and genome stability. BRCA1 shares a similar function in reversed fork stabilization. Loss of BRCA1 or BRCA2 leads to meiotic recombination 11-like (MRE11)-mediated degradation of the reversed replication forks, causing genomic instability. MLL, Pax2 transactivation domain-interacting protein (PTIP), RAD52, CHD4, sterile α motif and HD-domain containing protein 1 (SAMHD1), and poly-(ADP)-ribose polymerase (PARP) are required for MRE11 loading on stalled replication forks.
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ssDNA (N4 kb) and MRE11 is unlikely to be the only nuclease responsible to degrade several kb of DNA due to its poor processivity. In agreement with this model, human exonuclease (EXO)1 was later shown to contribute to fork degradation in BRCA-deficient tumors [13]. In addition, CtIP protein is required to initiate MRE11-dependent fork degradation [13], although a more recent study seems to contradict this conclusion [20]. As discussed above, these nucleases target reversed replication forks. The regressed arm of a reversed replication fork resembles by all means a one-ended DSB. Previous biochemical studies showed that the endonuclease activity of MRE11 initiates resection at DSBs and that CtIP promotes MRE11 endonuclease activity at the 5′ strand [21,22]. Resection is then continued by the 5′–3′ exonuclease activity of EXO1 [23– 25]. The discovery that MRE11, CtIP, and EXO1 cooperate in reversed fork degradation suggests that the same pathway that initiates DSB resection in the context of HRR is likely required to process the open dsDNA ends of the regressed arms in the context of DNA replication. DNA2 is also implicated in DSB resection [25]. However, whether human DNA2 contributes to reversed fork degradation in the absence of BRCA remains controversial [9,13]. Interestingly, DNA2 but not MRE11 promotes fork degradation in cells lacking the biorientation defect 1-like protein (BOD1L) [26], the human RecQ helicase RECQ1 [27], or Abro1 [28], which is a paralog of the BRCA1 interacting protein Abraxas. These results suggest that different nucleases might participate in fork degradation depending on the particular genetic background. The structure specific nuclease MUS81 has been also implicated in fork processing in the absence of BRCA [13,29,30], and its recruitment is mediated by EZH2, a histone methyltransferase [29]. Specifically, MUS81 was shown to cleave the partially resected reversed forks and promote DNA polymerase δ-dependent DNA fork rescue through a break-induced replication (BIR)-like mechanism [13]. MUS81 and EZH2 modulate fork rescue in BRCA2- but not BRCA1-deficient cells, suggesting that BRCA1-deficient cells rescue the resected forks through a different pathway [13,29]. The notion that BRCA1 and BRCA2 might have different functions during replication fork stalling and restart is also supported by a recent study showing BRCA1, but not BRCA2, is required to suppress tandem duplications at Tus/Ter replication fork barriers [31]. These tandem duplications are enriched in loci containing known oncogenes, while the breakpoints of these duplications are enriched in tumor suppressor genes, contributing to the genomic instability of BRCA1-deficient cells [32]. The extensive fork degradation phenotype observed in the absence of BRCA proteins is one of the leading causes for the increased genome instability and chemosensitivity of BRCA-deficient tumors [9]. Further studies will be however necessary to define the different contribution of the HRR and fork stability functions of BRCA proteins in genome stability and chemosensitivity [9,33]. BRCA-deficient tumors are sensitive to therapies that target DNA or that inhibit specific repair pathways, such as PARPis [34]. The efficacy of these therapies is often hampered by the development of chemoresistance. Chemoresistance has been associated with the recovered ability of BRCA-deficient cells to protect replication forks from nucleolytic degradation [9]. Aside from genetic reversion, which might occur in a limited subset of BRCA-mutant breast cancers, the pathways that rescue the ability of BRCA-deficient cells to protect forks from degradation and that mediate resistance to therapy remain largely unknown. In an effort to define these chemoresistance pathways, recent studies have focused on identifying the factors involved in nuclease recruitment at stalled/damaged replication forks. The Pax2 transactivation domaininteracting protein (PTIP) and the histone methyltransferases MLL3 and MLL4 were identified as key factors involved in MRE11 recruitment at stalled replication forks in BRCA1/2-deficient cells [9,35]. Recovered fork protection in BRCA-mutant cells was also linked to loss of the nucleosome remodeling factor CHD4 and inhibition of the poly(ADP-ribosyl)ation, suggesting that CHD4 and PARP are two other factors involved in MRE11 recruitment [9,36]. Moreover, recent studies suggested that human RAD52 and the sterile α motif and HD-domain containing 4
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protein (SAMHD)1 nuclease also regulate MRE11 loading on replication forks [14,37]. These studies open the provocative scenario that drug resistance in BRCA-deficient cells derives from deficiencies in these factors because they would limit MRE11 loading to stalled replication forks. Besides the mechanism of MRE11 recruitment to stalled forks, the mechanisms that control the recruitment of other nucleases involved in fork degradation remain unknown. We envision that defining these mechanisms will provide a major breakthrough toward the understanding of the molecular pathways that control genome integrity and chemoresistance in BRCA-deficient tumors.
Regulation of BRCA Function by Kinases ATR and CHK1 As described above, the mechanism of fork reversal may function to give the cell additional time to repair a lesion before continuing with S phase. Similarly, the DNA damage response involves additional signaling modalities to halt the cell cycle for the purposes of DNA repair. Cancer cells harboring mutations in the tumor suppressor gene p53 exhibit a defective G1 cell cycle checkpoint, creating a reliance on pathways that regulate the subsequent S, G2, and M phase checkpoints [38]. For this reason, components of the ATR/CHK1 signaling cascade, which activates the intra-S and G2/M checkpoints in response to DSBs or replication stress, have long been viewed as promising drug targets. Beyond its cell cycle functions, more recent studies have identified the ATR/CHK1 pathway as a key modulator of BRCAness by regulating the association of BRCA1/2 with their binding partners. It has long been known that ATR and its downstream effector CHK1 promote gene-conversionmediated HRR of DSBs and collapsed replication forks [39–41]. Mechanistically, ATR-mediated phosphorylation of PALB2 at S59 enhances the BRCA1–PALB2 interaction and is required for PALB2 localization to DSBs (Figure 2A). Furthermore, ATR/CHK1 inhibition of CDK activity following DNA end resection appears necessary for the BRCA1–PALB2 interaction and subsequent HRR [42]. Phosphorylation of PALB2 by ATR/CHK1 may also promote HRR by enhancing the interaction between PALB2 and RAD51 [43]. Furthermore, phosphorylation by ATR/CHK1 has been shown to stabilize the BRCA1 protein by protecting it from proteasomemediated degradation [44]. Additionally, chronic inhibition of ATR and CHK1 decreases transcription of pro-HRR factors such as BRCA1, TOPBP1, and RAD51, and this effect is augmented in cancer cell lines that overexpress HRR proteins [44]. Therefore, the role of ATR/CHK1 in promoting HRR is multifaceted and is critical for genomically unstable tumors to thrive. ATR and CHK1 are also required to stabilize stalled replication forks in response to stress on both a global and localized level. Globally, ATR/CHK1 inhibits CDK2 activity when replication forks stall in order to prevent new origins from firing, which would ultimately lead to an exhaustion of RPA, ssDNA accumulation, and DNA breakage, which would require the BRCA1/2 pathway [45] (Figure 2B). When ATR is inhibited, origin firing can still be blocked via a backup pathway in which DNA-PK activates CHK1 [46]. Likewise, inhibition of CHK1 alone increases ATR activity, suggesting that CHK1 and ATR globally promote genomic stability via both overlapping and distinct mechanisms [47]. This phenomenon is also observed locally at stalled replication forks. In response to CHK1 inhibition, stressed replication forks are processed into DSBs by the MUS81–EME1 nuclease complex, while fork collapse in ATR-inhibited cells is not dependent on MUS81–EME1 [48,49]. Additionally, two CHK1-independent mechanisms for ATRmediated replication fork protection have been proposed. First, ATR activity has been shown to mediate global replication fork reversal to prevent chromosomal breakage at sites of DNA interstrand crosslinks [50]. This contradicts previous findings suggesting that ATR phosphorylates the fork reversal enzyme SMARCAL1 in order to suppress excessive fork reversal and subsequent DNA cleavage by SLX4-dependent nucleases [51] (Figure 2C). This apparent contradiction
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might be linked to the different genotoxic agents used in the two studies and further work will need to be done to define the exact role of ATR signaling in fork remodeling. Second, ATR may promote loading of FANCD2 to replication forks via phosphorylation of the MCM2-7 helicase. FANCD2 then inhibits fork collapse by both slowing the DNA polymerase and by blocking Mre11 from resecting the fork intermediate [52]. Due to their critical functions in both HRR and replication fork protection, ATR and CHK1 exemplify BRCAness modulators that may be targeted to combat cancer chemoresistance. ATR inhibition sensitizes ovarian cancer cell lines to several chemotherapy agents, including the PARPi veliparib, regardless of BRCA status. Likewise, the combination of either ATRi or CHK1i with olaparib synergistically decreases viability in both BRCA-mutated and HRR-proficient cancer cells. Furthermore, treatment of an ovarian BRCA2-mutant patient-derived xenograft (PDX) model with olaparib and ATRi results in complete tumor regression, which is not observed with either single agent [53]. Additionally, ATRi treatment was recently shown to reverse PARPi resistance in cancer cells by blocking re-established HRR or fork protection pathways [54,55]. Notably, in all of these studies, inhibiting ATR was more effective than CHK1 inhibition for sensitizing cells to PARPi, which may be explained by the independent functions of ATR and CHK1 in replication fork protection. WEE1 The tyrosine kinase WEE1 has also emerged as a putative BRCAness factor that functions in both checkpoint activation and in replication fork stability. In response to DNA damage, WEE1 directly phosphorylates and inhibits CDK1/2, blocking cells from entering mitosis [56] (Figure 3A). While CHK1 has been shown to activate WEE1 in yeast and Xenopus, definitive evidence of this in mammalian cells is lacking [57,58]. However, inhibition of WEE1 during DNA damage induction in human cells results in inactivation of ATR and CHK1, suggesting that crosstalk among these kinases is required to sustain the replication stress response [59]. WEE1 and CHK1 also have roles in protecting stalled replication forks from degradation by overlapping and distinct mechanisms. Like CHK1, WEE1 depletion or inhibition induces replication fork slowing and MUS81dependent fork collapse [60,61] (Figure 3A). However, loss or inactivation of both proteins exasperates replication fork defects and significantly increases cell death when compared with individual knockdowns [61]. The synthetic lethality of combined WEE1/CHK1 inhibition was later confirmed in numerous cancer cell lines, which included ovarian, breast, colon, prostate, and melanoma, as well as xenograft models [62,63]. Dual inhibition of WEE1 and ATR has also shown promise, and such a combination may be particularly effective for combating PARPi and cisplatin resistance. Two groups found that the combination of the ATR inhibitor AZD6738 and the WEE1 inhibitor AZD1775, which are currently in clinical trials, inhibited breast cancer cell growth and reduced metastasis in PDX models [64,65]. Furthermore, combination of ATRi and WEE1i sensitized triple-negative breast cancer cells to cisplatin and the PARPi veliparib, with an increased effect in BRCA1-deficient cells [65]. WEE1 inhibition alone also sensitizes cancer cells and xenograft models to PARPi and cisplatin [55,66–68]. However, given the above evidence that ATR, CHK1, and WEE1 loss induce BRCAness via distinct mechanisms, inhibiting more than one of these kinases may be more effective. Figure 2. ATR and CHK1 Promote BRCA1/2 Function via Overlapping and Distinct Mechanisms. (A) ATR phosphorylates PALB2, promoting PALB2 association with BRCA1 (top) and RAD51 (bottom). (B) As a global response to replication stress, ATR-activated CHK1 phosphorylates CDC25 that inhibits cyclin-dependent kinase (CDK) activity. This prevents RPA exhaustion and additional DNA breaks which require repair by BRCA proteins. (C) ATR phosphorylates SMARCAL1 to inhibit replication fork remodeling and nucleolytic degradation. Additionally, ATR phosphorylation of the MCM2-7 helicase helps recruit FANCD2 to stalled replication forks where it also blocks nucleases from degrading nascent DNA.
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(A)
(B)
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Figure 3. WEE1 and Aurora A Kinases Regulate BRCAness. (A) In response to DNA damage, activated WEE1 phosphorylates cyclin-dependent kinase (CDK)1/2 in order to inhibit cell cycle progression, which would amplify replication stress. WEE1 also inhibits the MUS81–EME1 nuclease from degrading stalled replication fork intermediates through a direct or indirect mechanism. (B) The TPX2/Aurora A heterodimer negatively regulates 53BP1 function during double-stranded break repair, promoting BRCA1 recruitment and subsequent repair by homologous recombination (HR). (C) In response to replication stress, Aurora A and BRCA1 protect stalled replication forks from meiotic recombination 11-like (MRE11)-mediated degradation via complementary but distinct pathways. BRCA1 prevents Pax2 transactivation domain-interacting protein (PTIP) recruitment to the fork intermediate, which thereby inhibits the recruitment of MRE11. Aurora A and its cofactor TPX2 inhibits MRE11 function by negatively regulating 53BP1 through a mechanism dependent upon Aurora A kinase activity. Abbreviations: AurA, Aurora A; NHEJ, nonhomologous end joining.
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Aurora A In contrast to checkpoint kinases ATR, CHK1, and WEE1, the Aurora A kinase promotes cell cycle progression through G2 and mitosis, and is required for numerous mitotic events including centrosome maturation, spindle assembly, and cytokinesis. Generally, these functions are also dependent upon the Aurora A cofactor TPX2, which places Aurora A into its active conformation upon binding and localizes Aurora A to the spindle [69,70]. Importantly, TPX2 is necessary for sustaining Aurora A kinase activity throughout mitosis, as it protects the catalytic T288 site from dephosphorylation and inhibits Aurora A proteasomal degradation [69,71]. In humans, the TPX2 and Aurora A genes are located on the long arm of chromosome 20, which is commonly amplified in tumors. In fact, TPX2 and Aurora A overexpression occurs in a long list of cancer types and is notably associated with those exhibiting BRCA1/2 mutations [72–74]. Furthermore, high Aurora A to BRCA2 expression ratios are associated with poor prognosis in ovarian carcinoma patients [75,76]. Several Aurora A inhibitors have been developed over the past two decades. The aforementioned clinical findings and the molecular studies discussed below suggest that Aurora A inhibitors may modulate BRCAness via multiple mechanisms to enhance tumor chemosensitivity. Recent investigations have demonstrated that prior to mitosis, TPX2 and Aurora A promote BRCA functions. Inhibition of Aurora A with the small molecule inhibitor alisertib decreases PARP activity as well as the expression of PARP1 and BRCA1/2 in cell lines and xenograft models. This leads to an increased reliance on error-prone NHEJ to repair DSBs and a reduction in HRR [77]. Similarly, we showed that loss of either TPX2 or Aurora A inhibits BRCA1 and RAD51 recruitment, causing a modest but significant decrease in HRR (Figure 3B). More notably, using DNA fiber analysis, we found that TPX2 and Aurora A are required to protect stalled replication forks from MRE11-mediated degradation in a manner dependent upon Aurora A kinase activity (Figure 3C). This function is carried out via a separate pathway from BRCA1, as loss of both TPX2 and BRCA1 has an additive effect on replication fork instability. The BRCAness phenotypes resulting from TPX2 and Aurora A depletion are rescued upon loss of 53BP1, suggesting that TPX2 and Aurora A promote HRR and replication fork protection by negatively regulating 53BP1 (Figure 3B,C). Importantly, these novel functions of TPX2 and Aurora A appear to be independent of their mitotic roles, demonstrating the multifaceted ways in which properly regulated TPX2 and Aurora A promote genome integrity [78]. In addition to these functions of TPX2 and Aurora A, there are conflicting results pointing to a functional connection between Aurora A and BRCA1/2 in cell cycle regulation [76,79–81]. These findings suggest that Aurora A is an effective target for combating chemoresistance. In combination with a PARPi or DNA-damaging agent, inhibition of Aurora A in mitosis may induce mitotic catastrophe while inhibition in S/G2 should impair HRR repair and replication fork protection. Consistent with this the combination of alisertib and the PARPi, rucaparib had a synergistic effect on cell viability in both BRCA wild type and mutant cancer cell lines [77]. Our work also showed that Aurora-A-depleted cells are sensitive to hydroxyurea treatment [78]. Additionally, BRCA2-deficient cancer cells are preferentially sensitive to alisertib treatment and to depletion of TPX2 and Aurora A [82]. Together, these results suggest that Aurora A inhibition induces a functional BRCA loss and may be used in combination with PARP inhibitors to target multiple DNA damage response pathways in tumors, thus potentially preventing chemoresistance. Polo-Like Kinase (Plk)1 Plk1 is a substrate of the Aurora A kinase during mitosis, and has been implicated as a BRCAness factor. Recent work showed that the BRCA2 N-terminal domain is phosphorylated by CDKs in G2/M phase as well in response to genotoxic stress [83,84]. Plk1 binds to phosphorylated
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BRCA2, which serves as a scaffold to bring the kinase within close proximity to Rad51 [84]. Plk1 then phosphorylates Rad51 on S14, which subsequently promotes CK2-mediated phosphorylation of the adjacent T13 residue. Phosphorylated T13 then stimulates a direct binding interaction with NBS1 and recruitment of Rad51 to sites of damage. Notably, loss of Plk1-dependent S13/T14 phosphorylation leads to defects in HR and PARPi sensitivity [85]. Plk1 also facilitates Rad51 recruitment to both active and stalled replication forks and is necessary for efficient fork restart following hydroxyurea (HU) treatment [84]. Like Aurora A, Plk1 is overexpressed in numerous cancers, and high Plk1 levels are associated with BRCA deficiencies, making it a promising drug target to combat chemoresistance [86]. In fact, a recent study demonstrated that the Plk1 inhibitor BI2536 sensitizes BRCA1-deficient castration-resistant prostate cancer cells and xenograft tumors to olaparib, making the case for testing this drug combination in clinical trials [87].
Outstanding Questions What is the mechanistic relationship between HRR and RFP? What are the relative contributions of HRR and RFP to tumor suppression and chemosensitivity in the BRCAdeficient state? A recent study investigating separation-of-function mutations in BRCA1 and BARD1 suggests that defects in HRR and RFP may promote distinct stages of tumorigenesis. with RFP playing a critical role earlier than HRR [1,88]. However, additional experiments are needed to directly test this model.
Concluding Remarks Recent work has significantly broadened the concept of BRCAness and the molecules involved in this phenotype. Specifically, aside from a loss of canonical HRR, several additional factors, including DNA damage checkpoint proteins and kinases, promote the BRCA1/2 pathway and contribute to BRCAness. In addition, BRCAness is characterized by defects in replication fork protection. Thus, both the conceptual understanding of the BRCA-deficient state, as well as the molecules involved in regulating BRCA1/2 function, have been significantly expanded. This reflects the growing number of strategies that exploit BRCAness in a clinical setting. We envision a number of innovative combinations of pathway inhibition to be tested to induce functional BRCA loss and combat chemotherapy resistance. As described above, many of these BRCAmodulating factors are kinases with readily available inhibitors, thus making such combination therapies testable immediately. At the same time, these recent discoveries raise several additional questions that need to be addressed (see Outstanding questions). By addressing these fundamental questions on BRCA function we envision that more effective and novel and strategies will be developed to increase cancer chemosensitivity and overcome chemoresistance. Acknowledgments We wish to thank the numerous scientists who have contributed significantly to our understanding BRCA function, and we apologize that we could not cite all pertinent papers due to space limitations. A.K.B. was supported by the Washington University Department of Pathology Hu and Zheng Scholarship. This work was supported by the National Institutes of Health (NIH) (R01 CA237263 to A.V. and R01 CA227001 to N.M.), the Department of Defense [BCRP (Breast Cancer Research Program) Breakthrough Award BC151728 to A.V.], the Structural Biology of DNA Repair program project (P01 CA092584), and an American Cancer Society Research Scholar Award (to N.M.). The authors have no conflicts of interest to declare.
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