Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Replication fork stability confers chemoresistance in BRCA-deficient cells.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney.

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag(-/-) mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag(-/-) mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag(-/-) liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic ß-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

Single-stranded DNA gaps are postulated to be fundamental to the mechanism of anti-cancer drugs. Gaining insights into their induction could therefore be pivotal for advancing therapeutic strategies. For poly (ADP-ribose) polymerase inhibitors (PARPi) to be effective, the presence of FANCJ helicase is required. However, the relationship between FANCJ dependent gaps and PARP1 catalytic inhibition or trapping-both linked to PARPi toxicity in BRCA deficient cells-is yet to be elucidated. Here, we find that the efficacy of PARPi is contingent on S-phase PARP1 activity, which is compromised in FANCJ deficient cells because PARP1, along with MSH2, is "sequestered" by G-quadruplexes. PARP1's replication activity is also diminished in cells missing a FANCJ-MLH1 interaction, but in such cells, depleting MSH2 can release sequestered PARP1, restoring PARPi-induced gaps and sensitivity. Our observations indicate that sequestered and trapped PARP1 are different chromatin-bound forms, with FANCJ loss increasing PARPi resistance in cells susceptible to canonical PARP1 trapping. However, in BRCA1 null cells, the loss of FANCJ mirrors the effects of PARP1 loss or inhibition, with the common detrimental factor being the loss of PARP1 activity during DNA replication, not trapping. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA deficient cells and emphasize the importance of understanding drug mechanisms for enhancing precision medicine.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.

O6-methylguanine-induced cell death involves exonuclease 1 as well as DNA mismatch recognition in vivo.

Alkylation-induced O(6)-methylguanine (O(6)MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O(6)MeG-DNA methyltransferase (Mgmt) protein. O(6)MeG pairs with T during DNA replication, and if the O(6)MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O(6)MeG:T mismatch detection by MutSalpha and MutLalpha leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O(6)MeG-dependent apoptosis, we used an Mgmt-null mouse model combined with either the Msh6-null mutant (defective in mismatch recognition) or the Exo1-null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt-null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt-null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt-null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O(6)MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt-null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O(6)MeGs.

Aag-initiated base excision repair drives alkylation-induced retinal degeneration in mice.

Vision loss affects >3 million Americans and many more people worldwide. Although predisposing genes have been identified their link to known environmental factors is unclear. In wild-type animals DNA alkylating agents induce photoreceptor apoptosis and severe retinal degeneration. Alkylation-induced retinal degeneration is totally suppressed in the absence of the DNA repair protein alkyladenine DNA glycosylase (Aag) in both differentiating and postmitotic retinas. Moreover, transgenic expression of Aag activity restores the alkylation sensitivity of photoreceptors in Aag null animals. Aag heterozygotes display an intermediate level of retinal degeneration, demonstrating haploinsufficiency and underscoring that Aag expression confers a dominant retinal degeneration phenotype.

Estrogen-related receptor gamma is a key regulator of muscle mitochondrial activity and oxidative capacity.

Estrogen-related receptor gamma (ERRgamma) regulates the perinatal switch to oxidative metabolism in the myocardium. We wanted to understand the significance of induction of ERRgamma expression in skeletal muscle by exercise. Muscle-specific VP16ERRgamma transgenic mice demonstrated an increase in exercise capacity, mitochondrial enzyme activity, and enlarged mitochondria despite lower muscle weights. Furthermore, peak oxidative capacity was higher in the transgenics as compared with control littermates. In contrast, mice lacking one copy of ERRgamma exhibited decreased exercise capacity and muscle mitochondrial function. Interestingly, we observed that increased ERRgamma in muscle generates a gene expression profile that closely overlays that of red oxidative fiber-type muscle. We further demonstrated that a small molecule agonist of ERRbeta/gamma can increase mitochondrial function in mouse myotubes. Our data indicate that ERRgamma plays an important role in causing a shift toward slow twitch muscle type and, concomitantly, a greater capacity for endurance exercise. Thus, the activation of this nuclear receptor provides a potential node for therapeutic intervention for diseases such as obesity, which is associated with reduced oxidative metabolism and a lower type I fiber content in skeletal muscle.

Targeting translesion synthesis (TLS) to expose replication gaps, a unique cancer vulnerability.

Introduction: Translesion synthesis (TLS) is a DNA damage tolerance (DDT) mechanism that employs error-prone polymerases to bypass replication blocking DNA lesions, contributing to a gain in mutagenesis and chemo-resistance. However, recent findings illustrate an emerging role for TLS in replication gap suppression (RGS), distinct from its role in post-replication gap filling. Here, TLS protects cells from replication stress (RS)-induced toxic single-stranded DNA (ssDNA) gaps that accumulate in the wake of active replication. Intriguingly, TLS-mediated RGS is specifically observed in several cancer cell lines and contributes to their survival. Thus, targeting TLS has the potential to uniquely eradicate tumors without harming non-cancer tissues. Areas Covered: This review provides an innovative perspective on the role of TLS beyond its canonical function of lesion bypass or post-replicative gap filling. We provide a comprehensive analysis that underscores the emerging role of TLS as a cancer adaptation necessary to overcome the replication stress response (RSR), an anti-cancer barrier. Expert Opinion: TLS RGS is critical for tumorigenesis and is a new hallmark of cancer. Although the exact mechanism and extent of TLS dependency in cancer is still emerging, TLS inhibitors have shown promise as an anti-cancer therapy in selectively targeting this unique cancer vulnerability.

Comprehensive Mutational Analysis of the BRCA1-Associated DNA Helicase and Tumor-Suppressor FANCJ/BACH1/BRIP1.

FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient-derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. IMPLICATIONS: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients.

Replication Gaps Underlie BRCA Deficiency and Therapy Response.

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.

Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability.

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Muscle-specific expression of PPARgamma coactivator-1alpha improves exercise performance and increases peak oxygen uptake.

The induction of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a key regulator of mitochondriogenesis, is well-established under multiple physical exercise regimens, including, endurance, resistance, and sprint training. We wanted to determine if increased expression of PGC-1alpha in muscle is sufficient to improve performance during exercise in vivo. We demonstrate that muscle-specific expression of PGC-1alpha improves the performance during voluntary as well as forced exercise challenges. Additionally, PGC-1alpha transgenic mice exhibit an enhanced performance during a peak oxygen uptake exercise test, demonstrating an increased peak oxidative capacity, or whole body oxygen uptake. This increased ability to perform in multiple exercise paradigms is supported by enhanced mitochondrial function as suggested by increased mitochondrial gene expression, mitochondrial DNA, and mitochondrial enzyme activity. Thus this study demonstrates that upregulation of PGC-1alpha in muscle in vivo is sufficient to greatly improve exercise performance under various exercise paradigms as well as increase peak oxygen uptake.

Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress.

The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.

Fork Protection and Therapy Resistance in Hereditary Breast Cancer.

The BRCA-Fanconi anemia (FA) pathway preserves the genome and suppresses cancer and is a main determinant of chemotherapeutic efficacy. The hereditary breast cancer genes BRCA1 and BRCA2 function in DNA double-strand break repair mediating distinct steps of homologous recombination (HR). More recently, independent of DNA repair, functions in the replication stress response have come to light, providing insight as to how the BRCA-FA pathway also balances genome preservation with proliferation. The BRCA-FA proteins associate with the replisome and contribute to the efficiency and recovery of replication following perturbations that slow or arrest DNA replication. Although the full repertoire of functions in the replication stress response remains to be elucidated, the function of BRCA1 and BRCA2 in protecting stalled replication forks contributes along with HR to the sensitivity of BRCA-associated tumors to chemotherapy. Moreover, chemoresistance evolves from restoration of either HR and/or fork protection. Although mechanisms underlying the restoration of HR have been characterized, it remains less clear how restoration of fork protection is achieved. Here, we outline mechanisms of "rewired" fork protection and chemotherapy resistance in BRCA cancer. We propose that mechanisms are linked to permissive replication that limits fork remodeling and therefore opportunities for fork degradation. Combating this chemoresistance mechanism will require drugs that inactivate replication bypass mechanisms.

PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag-transgenic (AagTg) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.