BRCA1 Depletion Research Articles

BRCA1 Deficiency Upregulates NNMT, Which Reprograms Metabolism and Sensitizes Ovarian Cancer Cells to Mitochondrial Metabolic Targeting Agents.

BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. SIGNIFICANCE: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors

Lung cancer is the leading cause of cancer death worldwide. PARP inhibitors have become a new line of cancer therapy and a successful demonstration of the synthetic lethality concept. The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer and breast cancer, yet such studies are still relatively fewer in lung cancer. Here we found that HR genes are frequently mutated in lung cancer patients, exposing a window for targeted therapies by PARP inhibitors. We depleted BRCA1 and BRCA2 in non-small cell lung cancer (NSCLC) cancer cells and found these cells are hypersensitive to the PARP inhibitor olaparib in cell viability and clonogenic survival assays. Olaparib specifically induces apoptosis in A549 cells with BRCA1 or BRCA2 depletion, as determined by positive Annexin-V staining. In addition, we show that A549 cells with ATM shRNA knockdown are also hypersensitive to Olaparib. In summary, our data support the potential use of PARP inhibitors in NSCLC with HR deficiency.

BRCA-1 depletion impairs pro-inflammatory polarization and activation of RAW 264.7 macrophages in a NF-κB-dependent mechanism.

BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-β and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.

Chromatin Remodeling in Response to BRCA2-Crisis

Individuals with a single functional copy of the BRCA2 tumor suppressor have elevated risks for breast, ovarian, and other solid tumor malignancies. The exact mechanisms of carcinogenesis due to BRCA2 haploinsufficiency remain unclear, but one possibility is that at-risk cells are subject to acute periods of decreased BRCA2 availability and function ("BRCA2-crisis"), which may contribute to disease. Here, weestablish an invitro model for BRCA2-crisis that demonstrates chromatin remodeling and activation of an NF-κB survival pathway in response to transient BRCA2 depletion. Mechanistically, we identify BRCA2 chromatin binding, histone acetylation, and associated transcriptional activity as critical determinants of the epigenetic response to BRCA2-crisis. These chromatin alterations are reflected in transcriptional profiles of pre-malignant tissues from BRCA2 carriers and, therefore, may reflect naturalsteps in human disease. By modeling BRCA2-crisis invitro, we have derived insights into pre-neoplastic molecular alterations that may enhance the development of preventative therapies.

BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells.

BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.

Brca2 abrogation engages with the alternative lengthening of telomeres via break-induced replication.

A subset of cancer cells maintains their telomeres without telomerase through the recombination-based alternative lengthening of telomeres (ALT) pathway. Currently, it is not yet clear in what context ALT is induced and how the pathway choice is made. Here, we show that abrogation of Brca2 reinforces break-induced replication (BIR) and engages with ALT pathway. Brca2 depletion in telomerase-null mouse cells alleviated the growth defect, accompanied by telomere elongation, suggesting the induction of ALT. We also found that Brca2-depleted telomerase-null cells exhibited dynamic clustering of telomeres from G2 phase in Promyelocytic Nuclear (PML) bodies. For Brca2-deficient ALT induction, Rad51 was dispensable but Mre11 and Rad52 were required. Congruently, conservative telomeric DNA synthesis was apparent in mitosis, indicating that the absence of Brca2 directed towards Rad52-mediated BIR. Collectively, we propose that Brca2 abrogation can instigate ALT tumourigenesis through the induction of BIR. This study implies that inhibitors of BIR may be useful for BRCA2-associated ALT-type cancers. Assessing ALT features may be considered for the tailored therapy of BRCA2-associated cancers.

BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II–DNA complexes

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G1 phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1 phase. We disrupted the BRCA1 gene in 53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1 phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1 phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.

Abstract B58: Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability

Abstract Introduction: Over recent years, our knowledge of breast cancer biology has greatly improved; however, the mechanisms of tumor initiation and genomic drivers of this disease remain poorly understood. Hormone-negative breast cancers, such as triple-negative breast cancer (TNBC), are specifically challenging to treat, since they are genetically heterogeneous, often aggressive, and lack targetable driver mutation. Most TNBCs (80%) are basal-like (BLBC) and the majority of BLBCs harbor pathogenic mutations in TP53 tumor-suppressor gene (84%), indicating that TP53 plays a critical role in the pathogenesis of this cancer. Other less frequently observed alterations include mutation or epigenetic silencing of BRCA1/2 genes (36%), amplifications of oncogenes (MYC 40%, CCNE1 9%), and mutations or deletions of tumor suppressors (PTEN 35%, RB1 20%). Unfortunately, none of these genetic aberrations is currently druggable, mostly due to the lack of specific inhibitors. However, modeling these oncogenic events in precursor cells will enable us to study cancer evolution and acquisition of genomic instability. Experimental Procedures: We used basal-like mammary epithelial cells, MCF10A and MCF12A, to generate precursor models of TNBC. We overexpressed in these cell lines a hotspot p53 mutation (R175H), which demonstrates a dominant-negative phenotype. We confirmed that ectopic expression of R175H p53MUT impairs the ability of p53WT to properly respond to DNA damage induced by hydroxyurea or etoposide. As a result, cells fail to elevate the expression of p53 downstream targets, such as CDKN1A (cell cycle inhibitor), PUMA, BAX, and MDM2 (apoptosis regulators). Using CRISPR/Cas9-mediated genome editing we further performed depletion of RB1, PTEN, BRCA1, and BRCA2 genes in derived precursor models. Depletion of olfactory receptors OR10A4 and OR2W5 was performed as a control. In addition, we overexpressed S. pyogenes Cas9 endonuclease in these cell lines, in order to utilize them in CRISPR/Cas9-based gene knockout screens. Multiplexed functional analysis of CRISPR/Cas9-induced genomic alterations can be performed with lentiviral gRNA-based libraries in Cas9-expressing cells. All modified cell lines were clonally derived and gene depletion was validated with qRT-PCR and Western blot. Cas9 efficacy was confirmed with doxycycline-inducible RB1 knockout verified by T7 Endonuclease I genotyping assay. We further performed a range of phenotypic assays, such as proliferation, migration, anchorage-independent growth, and genomic instability assays, to measure the gain of tumorigenic properties upon acquisition of different genomic alterations. Summary: We established breast cancer precursor cell line models harboring genomic alterations in tumor suppressor genes commonly altered in TNBC, such as p53, RB1, PTEN, BRCA1, and BRCA2. Conclusions: Studying early transformation events leading to development of TNBC is necessary for understanding the etiology of this disease and identifying therapeutic vulnerabilities of precancerous lesions. To our knowledge, no comprehensive repertoire of breast cancer precursor models has been derived before. Also, ectopic expression of Cas9 in these cell lines makes them suitable for CRISPR/Cas9-based genomic screens to study disease evolution. CRISPR/Cas9 screens represent powerful tools to understand pathogenicity of different mutation events in disease initiation and progression. The use of genomically stable precursor cells in such studies makes the initial identification of putative drivers easier to define, in contrast to cancer cell lines, in which various compensatory pathways have evolved to cope with high genomic instability. Thus, established precursor models can be utilized in large-scale CRISPR/Cas9 screens focused on discovery of therapeutic targets, synthetic lethality, and genomic drivers of TNBC. Citation Format: Justyna Kanska, Kruttika Dabke, Simon A. Gayther. Modeling oncogenic events in breast cancer precursor cells to study cancer evolution and genomic instability [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B58.

Abstract 346: Targeting DNA repair with the combination of p97 and HDAC6 inhibitors in mantle cell lymphoma

Abstract p97 is an ATPase which is involved in remodeling multi-subunit protein complexes. It plays a pivotal role in the retrotranslocation and proteasomal degradation of misfolded ubiquitinated proteins in the endoplasmic reticulum. In association with cytosolic histone deacetylase 6 (HDAC6), p97 promotes the autophagic degradation of polyubiquitylated proteins and damaged organelles. More recently p97 has been implicated in regulating cell division and in facilitating DNA repair by promoting the recruitment of DNA repair proteins to the site of DNA damage. In this study, we determined the molecular mechanisms through which HDAC6 promotes p97 function in mantle cell lymphoma (MCL) cells. We report that treatment of MCL cells with the p97 inhibitors, DBeQ, ML240, NMS-873 and CB-5083 induces ER stress markers GRP78 and CHOP and resulted in dose-dependent apoptosis. Treatment of Z138C xenografts in NSG mice with CB-5083, the first in-class p97 inhibitor, resulted in increased accumulation of polyubiquitylated proteins and improved survival in vivo compared to control mice. Co-treatment with CB-5083 and the HDAC6-selective inhibitor ACY-1215, resulted in accumulation of polyubiquitylated proteins, impaired DNA double-stranded break repair and resulted in the partial depletion of BRCA1 and Cyclin D1 in MCL cells compared to either agent alone. We also demonstrate that shRNA-mediated knockdown of HDAC6 induced enhanced accumulation of H2AX-γ under basal conditions and following treatment with CB-5083 in cultured and primary MCL cells. Finally, treatment of MCL cells with p97 inhibitors and ACY-1215 results in synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL. Citation Format: Pratikkumar Vekaria, Anusha Vallurupalli, Dharmalingam Subramaniam, Frank Schoenen, Joseph McGuirk, Rekha M. Rao. Targeting DNA repair with the combination of p97 and HDAC6 inhibitors in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 346.

Abstract 4749: VISTA immune checkpoint deregulation in human triple-negative breast cancer

Abstract Immunotherapy for breast cancer has lagged behind the advances in other tumor types, suggesting the need for novel immune targets. VISTA/PD-1H is a checkpoint receptor expressed on immune cells that is an effective anti-cancer target in mouse models and now in early human clinical trials. We utilized an in vitro system to examine epigenetic changes arising early after depletion of breast cancer tumor suppressor proteins. This demonstrates that epigenetic alterations occur in normal human mammary cell lines lacking BRCA2 and result in regulation of VISTA through an NF-kB-dependent pathway. VISTA is strongly induced by growth factor limitation, an effect that is blunted by BRCA2-induced NF-kB activation and binding to a kB site in the VISTA promoter. This suggests that repression of VISTA may be an early consequence of BRCA2 depletion in pre-neoplastic tissues leading to immune infiltration. In contrast, in primary, untreated human triple-negative breast cancers, VISTA is over-expressed on tumor cells, independent of its presence on immune cells. VISTA positive TNBCs (14%) have significantly increased tumor infiltrating lymphocytes (TILs) whereas VISTA strong positive TNBCs (6%) have significantly decreased TILs and tumor PD-L1 staining, suggesting a higher level of immunosuppression. Gene expression and tumor genomics in TNBCs suggests that transcriptional networks to regulate physiologic immunoreceptor levels are corrupted during malignant transformation. In summary, this work identifies a BRCA2-loss-of-function signaling pathway that regulates an important immune checkpoint molecule via NF-kB activation in pre-neoplastic tissues and triple-negative breast cancers with important consequences for the immune microenvironment. We expect these observations to enhance immunotherapeutics for breast cancer. Citation Format: Joshua J. Gruber, Marisa M. Juntilla, Soo-Ryum Yang, Benjamin Geller, Natalie Jager, Chien-Yu Lin, Andrew M. Lipchik, Justin Chen, Ashwin Ram, Shaveta Vinayak, Melinda L. Telli, Robert B. West, James M. Ford, Michael P. Snyder. VISTA immune checkpoint deregulation in human triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4749.

Abstract 4838: R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells

Abstract G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR

Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5′-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNA-PKcs and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome manipulation.

Abstract P2-08-01: Loss of function of Brca1 or Gata3 induces basal-like breast cancer

Abstract BACKGROUND: Breast cancer is mainly divided into estrogen receptor (ER)-positive luminal and ER-negative basal-like tumors. Luminal-type tumors are associated with better survival and respond to hormone therapies whereas basal-like tumors are more aggressive and associated with a poor prognosis. Mammary epithelia are mainly composed of luminal and basal cells that are maintained by luminal and basal progenitors, respectively. The maintenance of luminal cell fate is orchestrated by networks of transcription factors, including BRCA1 and GATA3. Functional loss of BRCA1 by germline or somatic mutation or by promoter methylation is associated with more than one third of basal-like breast cancers. GATA3 expression is reduced in basal-like breast cancers and cancers that metastasize. Overexpression of GATA3 in cancer cells inhibits tumor formation. Deletion of Brca1 or Gata3 in mice results in early lethality or growth defects. How BRCA1 and GATA3 suppress breast cancer remains elusive. METHODS: We generated mice lacking Brca1 or Gata3 in mammary epithelia. Due to the proliferative defects and induction of p18Ink4c (p18), an inhibitor of CDK4/6, in mammary epithelial cells of these mice, we then generated mice lacking Brca1 or Gata3 in p18 deficient mammary epithelia. We determined spontaneous mammary tumor development in mutant mice and the mechanisms underlying the role of Brca1 and Gata3 in suppressing tumorigenesis and progression. RESULTS: Depletion of Brca1 or Gata3 led to growth defects of mammary epithelial cells, which was rescued by loss of p18. Depletion of Brca1 or Gata3 in a p18 null background induced heterogeneous mammary tumors with less luminal and more basal-like features and accelerated metastasis. Deletion of Brca1 eliminated Gata3 expression in human and mouse mammary tissues and cells. How Brca1 interacts with Gata3 to control mammary tumor development and progression is currently under investigation. CONCLUSION: Our results suggest that loss of function of either Brca1 or Gata3 induces basal-like mammary tumors in p18 deficient background. Citation Format: Pei X-H, Chan HL, Zhang L, Wang C, Robbins DJ, Capobianco T, Bai F. Loss of function of Brca1 or Gata3 induces basal-like breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-01.

A comprehensive analysis of BRCA2 gene: focus on mechanistic aspects of its functions, spectrum of deleterious mutations, and therapeutic strategies targeting BRCA2-deficient tumors.

BRCA2is the main susceptibility gene known to be involved in the pathogenesis of breast cancer. It plays an important role in maintaining the genome stability by homologous recombination through DNA double-strand breaks repairing, by interacting with various other proteins including RAD51, DSS1, RPA, MRE11, PALB2, and p53. BRCA2-deficient cells show the abnormalities of chromosome number. BRCA2 is also found to be involved in centrosome duplication specifically in the metaphase to anaphase transition. Inactivation or depletion of BRCA2 leads to centrosome amplification that results in unequal separation of chromosomes. BRCA2 localizes with central spindle and midbody during telophase and cytokinesis. Inactivation or depletion of BRCA2 leads to multinucleation of cell. Around 2000 mutations have been reported in BRCA2 gene. BRCA2-deficient tumors are being taking into consideration for targeted cancer therapy by using different inhibitors like poly ADP-ribose polymerase and thymidylate synthase. The present review focusses on the role of BRCA2 in various critical cellular processes based on the mechanistic approaches. Mutations reported in the BRCA2 gene in various ethnic groups till date have also been compiled with an insight into the functional aspects of these alterations. The therapeutic strategies for targeting BRCA2-deficient tumors have also been targeted.

DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells

Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs.

Abstract 2488: Tumor necrosis factor-alpha signaling determines cytotoxicity induced by BRCA2 deficiency

Abstract Introduction Breast cancer 2, early onset (BRCA2) is involved in DNA-damage repair via homologous recombination (HR). Defective HR leads to severe genomic instability and homozygous deletion of BRCA2 is therefore deleterious for survival of normal cells. In apparent contrast, inherited heterozygous mutations in BRCA2 increase the risk to develop cancer, which goes along with somatic loss of the wild type allele. So paradoxically, BRCA2 loss is lethal in normal cells, but can be tolerated in cancer cells. It is currently unclear how tumor cells can survive without BRCA2. To address this question, we performed a genome-wide functional genetic screen and identify Tumor Necrosis Factor-alpha (TNFα) signaling, via TNF receptor 1 (TNF-R1) and Src-associated-substrate-during-mitosis-of-68kDa (Sam68), as an important determinant of cell death induction upon BRCA2 inactivation. Material & Methods A genetic screen was performed in human near-haploid KMB-7 cells. Two monoclonal KMB-7 lines were engineered to express independent doxycycline-inducible BRCA2 short hairpin RNAs (shRNAs). Cells were randomly mutagenized using retrovirus carrying a strong splice-acceptor, resulting in gene knock-outs. Deep sequencing of surviving colonies revealed gene mutations that prevented shBRCA2-mediated cell death. Validation of TNF-R1 and Sam68 was done by shRNAs in multiple breast- and leukemic cell lines. Cell viability was determined by MTT and clonogenic assays. Competition assays with vectors expressing shRNA including IRES-driven mCherry were analyzed with flow cytometry. Biochemical analyses were assessed by western blotting and ELISA. Immunoprecipitation with FLAG-tagged TNFα was performed to study TNF-R1 complex formation. Results From our genetic screens, loss-of-function mutations involved in TNFα signaling were found to significantly rescue cell death in BRCA2-depleted cells. To validate these findings, BRCA2 was stably depleted in a panel of cancer cell lines, which decreased viability. Subsequent co-depletion of TNF-R1 or Sam68 rescued survival. Notably, BRCA2 depletion resulted in the production of TNFα. In line with this finding, the majority of tested cancer cell lines were found to be sensitive to recombinant TNFα upon BRCA2 depletion. This phenomenon was not restricted to BRCA2 inactivation, since depletion of related DNA repair genes (BRCA1, FANCD2) or hydroxyurea-induced replication stress also sensitized cells to recombinant TNFα. Mechanistically, BRCA2 depletion was induced to observe differential complex formation of TNF-R1 with downstream effector proteins upon TNFα activation. Conclusion Our data reveal that down-regulation of TNF-R1 or its downstream signaling component Sam68 rescued cytotoxicity upon BRCA2 inactivation. These findings and mechanistic follow-up experiments describe a novel mechanism by which autocrine TNFα signaling, induced by loss of BRCA2, limits tumor cell viability. Citation Format: Anne M. Heijink, Francien G. Talens, Anouk Baars, Marcel A. van Vugt. Tumor necrosis factor-alpha signaling determines cytotoxicity induced by BRCA2 deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2017-2488

Abstract 3453: Progression through mitosis promotes PARP inhibitor induced cytotoxicity in homologous recombination deficient cancer cells

Abstract Mutations in homologous recombination (HR) DNA repair 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. The mechanisms underlying PARP inhibitor resistance are only partially understood. This lack of knowledge in part stems from incomplete understanding of how PARP inhibitors induce cytotoxicity in HR-deficient cancers. The aim of this study was to investigate the contribution of mitotic failure to PARP inhibitor-induced cytotoxicity. Using the DNA fiber assay we observed compromised replication fork protection upon BRCA2 depletion, which was aggravated upon PARP inhibition. In accordance with the loss of replication fork stability, we found increased FANCD2 foci in interphase cells upon BRCA2 depletion, and a significant further increase when BRCA2-depleted cells were treated with PARP inhibitor as assessed by immunofluorescence. Importantly, these replication lesions were not resolved prior to mitotic entry, as increased numbers of FANCD2 foci were observed in mitotic cells of BRCA2-depleted cells, which were further increased upon PARP inhibitor treatment. Subsequently we found that in mitosis, BRCA2 or BRCA1 and Brca2 or Brca1 deletion resulted in elevated levels of chromatin-bridges and lagging chromosomes which were greatly enhanced after PARP inhibition. Live-cell microscopy showed that chromatin bridges in anaphase often lead to cytokinesis failure with subsequent multinucleated progeny cells or cell death. Furthermore, PARP inhibitor-induced multinucleated cells failed clonogenic outgrowth and high percentages of multinucleated cells were found in vivo in the remnants of Brca2-/-;p53-/- and Brca1-/-;p53-/- mammary mouse tumors upon PARP inhibitor treatment, suggesting that mitotic progression promotes PARP inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogated the cytotoxic potential of PARP inhibitor sensitivity and indicates that progression through mitosis promotes cell death in response to PARP inhibitors in HR-defective cells. These findings add to our understanding how PARP inhibition induces cytotoxicity. Furthermore, these results provide starting points in tackling tumor resistance to PARP inhibitors, and point at combination therapies which could potentiate the effects of PARP inhibitors for the treatment of HR-deficient tumors. Citation Format: Pepijn M. Schoonen, Peter Bouwman, Floris Foijer, Madalena Tarsounas, Sohvi Blatter, Jos Jonkers, Sven Rottenberg, Marcel A. Vugt, van. Progression through mitosis promotes PARP inhibitor induced cytotoxicity in homologous recombination deficient cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3453. doi:10.1158/1538-7445.AM2017-3453

Abstract 1409: Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells

Abstract Mutations in homologous recombination (HR) genes, including BRCA1 and BRCA2, compromise DNA repair and lead to genomic instability (GI). GI is lethal to normal cells but is a characteristic of many cancers. Apparently, these cancers are somehow re-wired to survive high levels of GI. Identification of the genetic alterations that allow viability of genomically instable tumor cells may uncover novel therapeutic targets. To elucidate how these tumor cells are rewired, we analyzed publically available mRNA expression data of 16,172 human cancer samples. Functional genomic mRNA profiling (FGmRNA-profiling) was applied on these samples to infer levels of GI and to capture the downstream effects of somatic copy number alterations on gene expression. A genome-wide association analysis was subsequently performed to assess the correlation between FGmRNA signals of individual genes with the degree of GI. From the top 250 genes with strong positive correlation with GI, 11 genes were prioritized based on a co-functionality network in which genes are co-regulated and share similar predicted biological function. The 11 genes that were identified in this cluster were: BIRC5, UBE2C, CENPA, CDCA3, DEK, SKP2, TPX2, KIF2C, RAD21, MYBL2 and WDR67. To validate these findings in genetically-defined models, we engineered a panel of 5 triple negative breast cancer (TNBC) cell lines with doxycycline-inducible shRNAs targeting BRCA2. BRCA2 depletion resulted in a failure of RAD51 foci to localize to DNA double strand breaks which generated isogenic cell line pairs proficient and deficient of HR repair. First, we depleted each of the identified 11 genes using RNA interference in BT-549 cells and observed that depletion of TPX2, a microtubule-associated protein, led to largest differential levels of cell death when comparing the BRCA2-deficient with the BRCA2-proficient context (86.6% vs 32.9% cell death in BRCA2-depleted vs controlled depleted cells respectively). Subsequently, we could replicate this decreased survival with TPX2 depletion in a BRCA2-deficient context in an additional 2 out of 4 other TNBC cell lines. Furthermore, we investigated whether BRCA2-depleted cells were also more sensitive to depletion of Aurora kinase A, a substrate of TPX2. For this purpose, mouse mammary tumor cell lines, derived from Tp53-/- or Brca2-/-;Tp53-/- mice, or a Brca2-reconstituted version thereof were treated with an Aurora A inhibitor, Alisertib. Again, we found that the BRCA2-deficient cell line was more sensitive to Aurora A inhibition than the two BRCA2-proficient cell lines. In conclusion, FGmRNA-profiling of mRNA expression data of human cancer samples identified TPX2 as an essential gene for survival of BRCA2-deficient breast cancer cells, when compared to BRCA2-proficient cells. Thus, targeting the TPX2/AURKA axis could potentially act as a novel therapeutic target in the treatment of genomically instable cancers. Citation Format: Stephanie E. van Gijn, Elles Wierenga, Anne Margriet Heijink, Rudolf S. Fehrmann, Marcel A. van Vugt. Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2017-1409

Abstract 1112: Forced reduction of DSS1, a member of TREX2 complex, highly sensitizes chemotherapy to breast cancer cells in a BRCA2-independent manner

Abstract [Introduction] DSS1 (deleted in split-hand/split-foot malformation 1) was originally identified as a BRCA2-associated/stabilizing protein. By comparison of DSS1 mRNA level, we reported that the high DSS1 expression groups in breast cancer patients showed worse prognosis in relapse-free survival (RFS); however, DSS1 expression per se was not correlated with other clinical parameters including cellular proliferation or tumor grade, suggesting that the level of DSS1 affected the sensitivity of DNA-damage agents in breast cancer cells (Rezano et al., 2013). It still remains unclear why the expression level of DSS1 is associated with chemosensitivity, because DSS1 has multifunctional properties regulating homologous recombination and RNA-mediated genomic stability. In this study, we investigated if the effect of DSS1 in chemosensitivity is influenced by two representative DSS1-associated molecules, BRCA2 and PCID2. [Methods] We established MCF7 overexpressing DSS1 (MCF7/DSS1) by retroviral transfection. DSS1, BRCA2, or PCID2 knockdown in MCF7 was performed using siRNA transfection. The susceptibility to the cytotoxic chemotherapy such as doxorubicin and paclitaxel in breast cancer cells was evaluated by flow cytometry to detect apoptosis and colony assay. In addition, we compared patient survival by dividing patients into high or low expression group of BRCA2 or PCID2 using cohort studies. [Results] BRCA2 depletion in MCF7/DSS1 did not alter the chemoresistance. Although DSS1 knockdown induced the downregulation of BRCA2, direct depletion of BRCA2 did not show marginal effect to chemosensitivity, suggesting that the enhancement of chemosensitivity by DSS1 knockdown was not related to BRCA2 expression. Importantly, over- and under-expression of PCID2 induced similar effect to chemosensitivity compared to those of DSS1. There was a trend that high PCID2 expression group in breast cancer patients showed worse prognosis in RFS. [Conclusion] DSS1 could be a molecular target to increase chemosensitivity, which is independent of BRCA2 expression. Rather, the transcription-coupled DNA damage induced by impaired TREX2 complex might be associated with induction of chemosensitivity. Citation Format: Kazuhiko Kuwahara, Naomi Gondo, Andri Rezano, Zhenhuan Zhang, Yukari Hato, Kiyotaka Kuzushima, Hiroji Iwata, Tatsuya Toyama, Eisaku Kondo. Forced reduction of DSS1, a member of TREX2 complex, highly sensitizes chemotherapy to breast cancer cells in a BRCA2-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1112. doi:10.1158/1538-7445.AM2017-1112