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Information on FANCJ mutation library, including source of mutation

Sensitivity data from validated FANCJ LOF mutations

Supplementary Figure 1 from An MLH1 Mutation Links BACH1/FANCJ to Colon Cancer, Signaling, and Insight toward Directed Therapy

Supplementary Figure 2 from An MLH1 Mutation Links BACH1/FANCJ to Colon Cancer, Signaling, and Insight toward Directed Therapy

Perspective on This Article from An MLH1 Mutation Links BACH1/FANCJ to Colon Cancer, Signaling, and Insight toward Directed Therapy

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.

Figure Legends

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

Supplemental Figure Legends

Supplemental Figures 1-6. Figure S1: BRCA-deficient cancer cells fail to restrain replication in the presence of stress and ssDNA gaps develop. Figure S2: Replication restraint, depletion, and PDX controls. Figure S3: Depletion of SMARCAL1 or inhibition of MRE11 restores FP, but do not predict patient response and do not suppress ssDNA gaps, which are distinct from fork degradation. Figure S4: Fanconi Anemia Patient Fibroblasts with a RAD51 T131P Mutant Allele (HR Proficient, FP Deficient) Generate ssDNA Gaps, and FP is restored by RADX depletion. Figure S5: Apoptosis and Z-VAD-FMK Controls. Figure S6: Prediction Table of BRCAness and Chemoresponse.

PDF file - 106K, Supplemental Figure 6. FANCJ deficient cells are sensitive to DNA interstrand crosslinking agents and FANCJ suppresses UV-induced mutations

PDF file - 57K, Supplemental Figure 7. Mutations in FANCJ and MMR loci occur in melanoma genomes

PDF file - 29K, Supplemental Figure 5. FANCJ precipitates with UV modified PCNA and CPD from chromatin extracts.

PDF file - 228K, Supplemental Figure 3. FANCJ localizes to UV induced LUDs predominantly in S-Phase and contributes to the UV induced checkpoint

PDF file - 88K, Supplemental Figure 9. FANCJ expression promotes UV induced GFP-polh foci formation in U2OS cells.

PDF file - 151K, Supplemental Figure 1. FANCJ is recruited to sites of UV-induced damage in an NER dependent manner

PDF file - 260K, Supplemental Figure 2. MMR localizes FANCJ to UV induced LUDs

PDF file - 68K, Supplemental Figure 4. FANCJ and MMR function in a common pathway for RPA phosphorylation and 6-4 Photoproduct elimination, but not for gap repair.

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia–associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability. Cancer Res; 74(3); 932–44. ©2013 AACR.

PDF file - 103K

PDF file - 43K, Supplemental Table 1. FANCJ suppresses UV-induced mutations at the HPRT locus.

SummaryThe tumor suppressorBRCA1encodes multiple protein products including the canonical BRCA1-p220 (p220), which plays important roles in repair of diverse types of DNA damage. However, contributions of otherBRCA1-encoded protein isoforms to DNA damage repair are less clear. Here, we report that the BRCA1-IRIS (IRIS) isoform has critical functions in the Fanconi anemia (FA) pathway and in repair of DNA interstrand crosslinks (ICLs). Loss of IRIS expression sensitizes cells to ICLs and impairs ICL repair. ICL formation stimulates association of IRIS with both FANCD2 and the FA core complex, which promotes FANCD2 recruitment to damage sites. The unique,BRCA1intron 11-encoded C-terminal tail of IRIS is required for complex formation with FANCD2 and for ICL-inducible FANCD2 mono-ubiquitylation. Collectively, our findings reveal that IRIS plays an essential role, upstream of the p220-directed HR, in the FA pathway through a previously unrecognized mechanism that depends on the IRIS-FANCA-FANCD2 interaction.HighlightsBRCA1splicing isoform BRCA1-IRIS is required for interstrand crosslink (ICL) repair.BRCA1-IRIS interacts with FANCD2 and promotes its recruitment to sites of ICL damage.BRCA1-IRIS, but not BRCA1-p220, promotes ICL-inducible FANCD2 mono-ubiquitylation.The unique C-terminal tail of BRCA1-IRIS is essential for its function in ICL repair.

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

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.

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.

Defects in DNA double-strand break repair are thought to render BRCA1 or BRCA2 (BRCA) mutant tumors selectively sensitive to PARP inhibitors (PARPis). Challenging this framework, BRCA and PARP1 share functions in DNA synthesis on the lagging strand. Thus, BRCA deficiency or "BRCAness" could reflect an inherent lagging strand problem that is vulnerable to drugs such as PARPi that also target the lagging strand, a combination that generates a toxic accumulation of replication gaps.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability., In the genome, repetitive guanine-rich sequences have the potential to spontaneously fold into non-canonical DNA secondary structures known as G-quadruplex (G4). Using novel single-molecule imaging approaches, the authors reveal that G4 formation within active replication forks locally perturb replisome dynamics and damage response signaling, which require RPA and FANCJ for regulation.

The toxic lesion emanating from chemotherapy that targets the DNA was initially debated, but eventually the DNA double strand break (DSB) ultimately prevailed. The reasoning was in part based on the perception that repairing a fractured chromosome necessitated intricate processing or condemned the cell to death. Genetic evidence for the DSB model was also provided by the extreme sensitivity of cells that were deficient in DSB repair. In particular, sensitivity characterized cells harboring mutations in the hereditary breast/ovarian cancer genes, BRCA1 or BRCA2, that function in the repair of DSBs by homologous recombination (HR). Along with functions in HR, BRCA proteins were found to prevent DSBs by protecting stalled replication forks from nuclease degradation. Coming full-circle, BRCA mutant cancer cells that gained resistance to genotoxic chemotherapy often displayed restored DNA repair by HR and/or restored fork protection (FP) implicating that the therapy was tolerated when DSB repair was intact or DSBs were prevented. Despite this well-supported paradigm that has been the impetus for targeted cancer therapy, here we argue that the toxic DNA lesion conferring response is instead single stranded DNA (ssDNA) gaps. We discuss the evidence that persistent ssDNA gaps formed in the wake of DNA replication rather than DSBs are responsible for cell killing following treatment with genotoxic chemotherapeutic agents. We also highlight that proteins, such as BRCA1, BRCA2, and RAD51 known for canonical DSB repair also have critical roles in normal replication as well as replication gap suppression (RGS) and repair. We review the literature that supports the idea that widespread gap induction proximal to treatment triggers apoptosis in a process that does not need or stem from DSB induction. Lastly, we discuss the clinical evidence for gaps and how to exploit them to enhance genotoxic chemotherapy response.

SUMMARYInhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1, identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 is required for PARPi-induced fork acceleration in BRCA1-deficient cells and that LIG3 loss increases fork asymmetry. Furthermore, LIG3 depletion in BRCA1-deficient cells results in an increase in ssDNA gaps behind the replication forks, resulting in accumulation of chromosomal abnormalities. We also report that high expression of LIG3 in patients with invasive breast cancer correlates in with poorer overall survival, rendering LIG3 as a potential therapeutic target for enhancing PARPi sensitivity.

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.

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.

SUMMARYThe replication stress response which serves as an anti-cancer barrier is activated not only by DNA damage and replication obstacles, but also oncogenes, mystifying how cancer evolves. Here, we identify that oncogene expression, similar to cancer therapies, induces single stranded DNA (ssDNA) gaps that reduce cell fitness, unless suppressed by translesion synthesis (TLS). DNA fiber analysis and electron microscopy reveal that TLS restricts replication fork slowing, reversal, and fork degradation without inducing replication fork gaps. Evidence that TLS gap suppression is fundamental to cancer, 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. This work illuminates that gap suppression during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

Defects in DNA repair and/or 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, recent findings indicate that chemotherapies such as cisplatin used to treat BRCA-deficient tumors do not initially cause DNA double-strand-breaks (DSBs). Here, we show that single stranded DNA (ssDNA) replication gaps underlie the hypersensitivity of BRCA-deficient cancer, and we propose that defects in homologous recombination (HR) or fork protection (FP) do not. Specifically, in BRCA-deficient cells, ssDNA gaps develop because replication is not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling confers therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR can be uncoupled from therapy resistance when gaps are present. Moreover, we find that DSBs are not detected after therapy when apoptosis is inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather from cell death nucleases. Together, this data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapies. Citation Format: Sharon B. Cantor, Nicholas Panzarino. Gaps alone kill BRCA deficient cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-026.

SUMMARY 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., In Brief Peng et al. find that loss of FANCJ enhances the replisome association of helicase-like transcription factor (HLTF). HLTF depletion suppresses fork degradation in FANCJ-deficient cells, and FANCJ depletion suppresses aberrant fork elongation in HLTF-deficient cells. However, the combined loss of HLTF and FANCJ causes severe replication stress., Graphical Abstract

In this issue, Byrum et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201803003) surprisingly identify an interaction between 53BP1 and the mitotic regulators TPX2 and Aurora A that lead them to novel mechanistic insights about DNA double-stranded break repair regulation and a new fork protection pathway during replication stress.

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.

BRCAness has important implications in the management and treatment of patients with breast and ovarian cancer. In this study, we propose a computational framework to measure the BRCAness of breast and ovarian tumor samples based on their gene expression profiles. We define a characteristic profile for BRCAness by comparing gene expression differences between BRCA1/2 mutant familial tumors and sporadic breast cancer tumors while adjusting for relevant clinical factors. With this BRCAness profile, our framework calculates sample-specific BRCA scores, which indicates homologous recombination (HR)-mediated DNA repair pathway activity of samples. We found that in sporadic breast cancer high BRCAness score is associated with aberrant copy number of HR genes rather than somatic mutation and other genomic features. Moreover, we observed significant correlations of BRCA score with genome instability and neoadjuvant chemotherapy. More importantly, BRCA score provides significant prognostic value in both breast and ovarian cancers after considering established clinical variables. In summary, the inferred BRCAness from our framework can be used as a robust biomarker for the prediction of prognosis and treatment response in breast and ovarian cancers.

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia–associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability. Cancer Res; 74(3); 932–44. ©2013 AACR.

Fanconi anemia (FA) results from mutations in the genes necessary for DNA damage repair and often leads to progressive bone marrow failure. Although the exhaustion of the bone marrow leads to cytopenias in FA patients as they age, evidence from human FA and mouse model fetal livers suggests that hematopoietic defects originate in utero, which may lead to deficient seeding of the bone marrow. To address this possibility, we examined the consequences of loss of Fancd2, a central component of the FA pathway. Examination of embryonic day 14.5 (E14.5) Fancd2 knockout (KO) fetal livers showed a decrease in total cellularity and specific declines in long-term and short-term hematopoietic stem cell (LT-HSC and ST-HSC, respectively) numbers. Fancd2 KO fetal liver cells display similar functional defects to Fancd2 adult bone marrow cells, including reduced colony-forming units, increased mitomycin C sensitivity, increased LT-HSC apoptosis, and heavily impaired competitive repopulation, implying that these defects are intrinsic to the fetal liver and are not dependent on the accumulation of DNA damage during aging. Telomere shortening, an aging-related mechanism proposed to contribute to HSC apoptosis and bone marrow failure in FA, was not observed in Fancd2 KO fetal livers. In summary, loss of Fancd2 yields significant defects to fetal liver hematopoiesis, particularly the HSC population, which mimics key phenotypes from adult Fancd2 KO bone marrow independently of aging-accrued DNA damage.

FANCJ mutations are linked to Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double-strand breaks (DSBs) and interstrand cross-links (ICLs), but its mechanism of action is not well understood. Here we show with live-cell imaging that FANCJ recruitment to laser-induced DSBs but not psoralen-induced ICLs is dependent on nuclease-active MRE11. FANCJ interacts directly with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ regulates the MRE11 nuclease to facilitate DSB processing and appropriate end resection. Cells deficient in FANCJ and MRE11 show increased ionizing radiation (IR) resistance, reduced numbers of γH2AX and RAD51 foci, and elevated numbers of DNA-dependent protein kinase catalytic subunit foci, suggesting that HR is compromised and the nonhomologous end-joining (NHEJ) pathway is elicited to help cells cope with IR-induced strand breaks. Interplay between FANCJ and MRE11 ensures a normal response to IR-induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and the FA pathway. Our findings are discussed in light of the current model for HR repair.