Mapping Interindividual Variability of Toxicodynamics Using High-Throughput Transcriptomics and Primary Human Hepatocytes from Fifty Donors.

BACKGROUND: Understanding the variability across the human population with respect to toxicodynamic responses after exposure to chemicals, such as environmental toxicants or drugs, is essential to define safety factors for risk assessment to protect the entire population. Activation of cellular stress response pathways are early adverse outcome pathway (AOP) key events of chemical-induced toxicity and would elucidate the estimation of population variability of toxicodynamic responses. OBJECTIVES: We aimed to map the variability in cellular stress response activation in a large panel of primary human hepatocyte (PHH) donors to aid in the quantification of toxicodynamic interindividual variability to derive safety uncertainty factors. METHODS: High-throughput transcriptomics of over 8,000 samples in total was performed covering a panel of 50 individual PHH donors upon 8 to 24 h exposure to broad concentration ranges of four different toxicological relevant stimuli: tunicamycin for the unfolded protein response (UPR), diethyl maleate for the oxidative stress response (OSR), cisplatin for the DNA damage response (DDR), and tumor necrosis factor alpha (TNFα) for NF-κB signaling. Using a population mixed-effect framework, the distribution of benchmark concentrations (BMCs) and maximum fold change were modeled to evaluate the influence of PHH donor panel size on the correct estimation of interindividual variability for the various stimuli. RESULTS: Transcriptome mapping allowed the investigation of the interindividual variability in concentration-dependent stress response activation, where the average of BMCs had a maximum difference of 864-, 13-, 13-, and 259-fold between different PHHs for UPR, OSR, DDR, and NF-κB signaling-related genes, respectively. Population modeling revealed that small PHH panel sizes systematically underestimated the variance and gave low probabilities in estimating the correct human population variance. Estimated toxicodynamic variability factors of stress response activation in PHHs based on this dataset ranged between 1.6 and 6.3. DISCUSSION: Overall, by combining high-throughput transcriptomics and population modeling, improved understanding of interindividual variability in chemical-induced activation of toxicity relevant stress pathways across the human population using a large panel of plated cryopreserved PHHs was established, thereby contributing toward increasing the confidence of in vitro-based prediction of adverse responses, in particular hepatotoxicity. https://doi.org/10.1289/EHP11891.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Systematic screening identifies ABCG2 as critical factor underlying synergy of kinase inhibitors with transcriptional CDK inhibitors.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive. METHODS: Kinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors. RESULTS: We show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation. CONCLUSION: Overall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.

BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

Evaluation of an imaging-based in vitro screening platform for estrogenic activity with OECD reference chemicals.

Estrogen receptor alpha (ERα) is often a primary target of endocrine disrupting chemicals (EDCs) and therefore several biochemical and cell-based assays for the detection of chemicals with estrogenic properties have been developed in the past. However, the current approaches are not suitable for the monitoring of pathway activation dynamics, and they are mostly based on expression constructs that lack physiological promoter regulation. We recently developed MCF7 fluorescent reporter cell lines of 3 different green fluorescent protein (GFP)-tagged ERα target genes: GREB1, PGR and TFF1. These reporters are under control of the full physiological promoter region and allow the monitoring of dynamic pro-proliferative pathway activation on a single cell level using a live-cell imaging set-up. In this study, we systematically characterized the response of these reporters to a full reference compound set of known estrogenic and non-estrogenic chemicals as defined by the Organization for Economic Co-Operation and Development (OECD). We linked activation of the pro-proliferative ERα pathway to a potential adverse outcome by additionally monitoring cell cycle progression and proliferation. The correct classification of the OECD reference compounds showed that our reporter platform has the same sensitivity and specificity as other validated artificial ERα pathway reporters, such as the ERα CALUX and VM7 Luc ER TA assay. By monitoring several key events (i.e. ER target activation, cell cycle progression and proliferation), and subsequently determining Point-of-Departure (POD) values, our reporter panel can be used in high-throughput testing for a physiologically more relevant, quantitative temporal endocrine modulation analysis to improve human carcinogen risk assessment.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters.

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid-rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with normal DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolisation and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g. 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.

A BRCA1 Coiled-Coil Domain Variant Disrupting PALB2 Interaction Promotes the Development of Mammary Tumors and Confers a Targetable Defect in Homologous Recombination Repair.

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Integration of temporal single cell cellular stress response activity with logic-ODE modeling reveals activation of ATF4-CHOP axis as a critical predictor of drug-induced liver injury.

Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.

Physiologically Relevant Estrogen Receptor Alpha Pathway Reporters for Single-Cell Imaging-Based Carcinogenic Hazard Assessment of Estrogenic Compounds.

Estrogen receptor alpha (ERα) belongs to the nuclear hormone receptor family of ligand-inducible transcription factors and regulates gene networks in biological processes such as cell growth and proliferation. Disruption of these networks by chemical compounds with estrogenic activity can result in adverse outcomes such as unscheduled cell proliferation, ultimately culminating in tumor formation. To distinguish disruptive activation from normal physiological responses, it is essential to quantify relationships between different key events leading to a particular adverse outcome. For this purpose, we established fluorescent protein MCF7 reporter cell lines for ERα-induced proliferation by bacterial artificial chromosome-based tagging of 3 ERα target genes: GREB1, PGR, and TFF1. These target genes are inducible by the non-genotoxic carcinogen and ERα agonist 17ß-estradiol in an ERα-dependent manner and are essential for ERα-dependent cell-cycle progression and proliferation. The 3 GFP reporter cell lines were characterized in detail and showed different activation dynamics upon exposure to 17ß-estradiol. In addition, they demonstrated specific activation in response to other established reference estrogenic compounds of different potencies, with similar sensitivities as validated OECD test methods. This study shows that these fluorescent reporter cell lines can be used to monitor the spatial and temporal dynamics of ERα pathway activation at the single-cell level for more mechanistic insight, thereby allowing a detailed assessment of the potential carcinogenic activity of estrogenic compounds in humans.

Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining.

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.

Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

EZH2 Is Overexpressed in BRCA1-like Breast Tumors and Predictive for Sensitivity to High-Dose Platinum-Based Chemotherapy.

PURPOSE: BRCA1-deficient breast cancers carry a specific DNA copy-number signature ("BRCA1-like") and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors. EXPERIMENTAL DESIGN: EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1-like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model. RESULTS: The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1-like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents. CONCLUSIONS: Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.

PARP Inhibitor Efficacy Depends on CD8+ T-cell Recruitment via Intratumoral STING Pathway Activation in BRCA-Deficient Models of Triple-Negative Breast Cancer.

Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T-cell infiltration and activation in vivo, and that CD8+ T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8+ T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer.This article is highlighted in the In This Issue feature, p. 681.

Radiosensitivity Is an Acquired Vulnerability of PARPi-Resistant BRCA1-Deficient Tumors.

The defect in homologous recombination (HR) found in BRCA1-associated cancers can be therapeutically exploited by treatment with DNA-damaging agents and PARP inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identify radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo. In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquire drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. SIGNIFICANCE: These findings uncover radiosensitivity as a novel, therapeutically viable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway.

BRCA1-associated mammary tumorigenesis is dependent on estrogen rather than progesterone signaling.

Hereditary breast cancers in BRCA1 mutation carriers are mostly estrogen receptor α (ERα)-negative and progesterone receptor (PR)-negative; however, hormone depletion via bilateral oophorectomy does result in a marked reduction in breast cancer risk, suggesting that BRCA1-associated breast tumorigenesis is dependent on hormone signaling. We used geneticaly engineered mouse models to determine the individual influences of ERα and PR signaling on the development of BRCA1-deficient breast cancer. In line with the human data, BRCA1-deficient mouse mammary tumors are ERα-negative, and bilateral ovariectomy leads to abrogation of mammary tumor development. Hormonal replacement experiments in ovariectomized mice showed that BRCA1-deficient mammary tumor formation is promoted by estrogen but not by progesterone. In line with these data, mammary tumorigenesis was significantly delayed by the selective ERα downregulator fulvestrant, but not by the selective PR antagonist Org33628. Together, our results illustrate that BRCA1-associated tumorigenesis is dependent on estrogen signaling rather than on progesterone signaling, and call into question the utility of PR antagonists as a tumor prevention strategy for BRCA1 mutation carriers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity.

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.

Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.

Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death. PARP-inhibitor-induced multinucleated cells fail clonogenic outgrowth, and high percentages of multinucleated cells are found in vivo in remnants of PARP inhibitor-treated Brca2-/-;p53-/- and Brca1-/-;p53-/- mammary mouse tumours, suggesting that mitotic progression promotes PARP-inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogates PARP-inhibitor-induced cytotoxicity. These findings provide insight into the cytotoxic effects of PARP inhibition, and point at combination therapies to potentiate PARP inhibitor treatment of HR-deficient tumours.