Murine Brca2 Research Articles

Abstract 3234: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3234.

Abstract A079: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez-Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A079.

Sustained delivery of PARP inhibitor Talazoparib for the treatment of BRCA-deficient ovarian cancer.

Ovarian cancer has long been known to be the deadliest cancer associated with the female reproductive system. More than 15% of ovarian cancer patients have a defective BRCA-mediated homologous recombination repair pathway that can be therapeutically targeted with PARP inhibitors (PARPi), such as Talazoparib (TLZ). The expansion of TLZ clinical approval beyond breast cancer has been hindered due to the highly potent systemic side effects resembling chemotherapeutics. Here we report the development of a novel TLZ-loaded PLGA implant (InCeT-TLZ) that sustainedly releases TLZ directly into the peritoneal (i.p.) cavity to treat patient-mimicking BRCA-mutated metastatic ovarian cancer (mOC). InCeT-TLZ was fabricated by dissolving TLZ and PLGA in chloroform, followed by extrusion and evaporation. Drug loading and release were confirmed by HPLC. The in vivo therapeutic efficacy of InCeT-TLZ was carried out in a murine Brca2-/-p53R172H/-Pten-/- genetically engineered peritoneally mOC model. Mice with tumors were divided into four groups: PBS i.p. injection, empty implant i.p. implantation, TLZ i.p. injection, and InCeT-TLZ i.p. implantation. Body weight was recorded three times weekly as an indicator of treatment tolerance and efficacy. Mice were sacrificed when the body weight increased by 50% of the initial weight. Biodegradable InCeT-TLZ administered intraperitoneally releases 66 μg of TLZ over 25 days. In vivo experimentation shows doubled survival in the InCeT-TLZ treated group compared to control, and no significant signs of toxicity were visible histologically in the surrounding peritoneal organs, indicating that the sustained and local delivery of TLZ greatly maximized therapeutic efficacy and minimized severe clinical side effects. The treated animals eventually developed resistance to PARPi therapy and were sacrificed. To explore treatments to overcome resistance, in vitro studies with TLZ sensitive and resistant ascites-derived murine cell lines were carried out and demonstrated that ATR inhibitor and PI3K inhibitor could be used in combination with the InCeT-TLZ to overcome acquired PARPi resistance. Compared to intraperitoneal PARPi injection, the InCeT-TLZ better inhibits tumor growth, delays the ascites formation, and prolongs the overall survival of treated mice, which could be a promising therapy option that benefits thousands of women diagnosed with ovarian cancer.

Abstract 1994: Sustained delivery of PARP inhibitor Talazoparib for the treatment of BRCA deficient ovarian cancer

Abstract Ovarian cancer has long been known to be the deadliest cancer associated with the female reproductive system. More than 15% of ovarian cancer patients have a defective BRCA-mediated homologous recombination repair pathway that can be therapeutically targeted with PARP inhibitors, such as Talazoparib (TLZ). The expansion of TLZ clinical approval beyond breast cancer has been hindered due to the highly potent systemic side effects resembling chemotherapeutics. Here we report the development of a novel TLZ-loaded PLGA implant (InCeT-TLZ) that sustainedly releases TLZ over 25 days directly into the peritoneal cavity (i.p.) to treat BRCA-mutated metastatic ovarian cancer (mOC). Methods: InCeT-TLZ was fabricated by dissolving TLZ and PLGA in chloroform followed by extrusion and evaporation. Drug loading and release were confirmed by HPLC. The detailed structure of InCeT-TLZ was checked by scanning electron microscopy. The in vivo therapeutic efficacy of InCeT-TLZ was carried out in a murine Brca2-/-p53R172H/-Pten-/- genetically engineered peritoneally metastatic ovarian cancer model. Treatment started 7 days post tumor inoculation. All mice were monitored by IVIS imaging once a week to confirm and track tumor growth. On day 8, mice with tumors (4-5 mm in diameter) were divided into 4 groups: PBS i.p. injection (50 μl, three times a week), empty implant i.p. implantation (2.5 mm, every 25 days), TLZ i.p. injection (0.33 mg/kg of TLZ by i.p. injection three times a week), and InCeT-TLZ i.p. implantation (2.5 mm, equal to a total amount of 10 i.p. injection doses, every 25 days). Body weight was recorded three times a week as an indicator of treatment tolerance and efficacy. Mice were sacrificed when the body weight increased by 50% of the initial weight. Results and Conclusion: The drug-loaded PLGA implant was developed for the sustained and local delivery of TLZ to treat high-grade serous ovarian cancer with a loading efficiency of 26.75 μg/mm. Implants can be easily placed into the peritoneal cavity of patients or animals with an 18-gauge clinical needle. The extended survival in InCeT-TLZ treated mice compared to the TLZ injection group indicated that the slow release of TLZ maximizes PARP inhibitor therapy in the peritoneal cavity for disseminated cancer treatment. Decrease in ascites formation within the intraperitoneal cavity of the InCeT-TLZ group suggests a promising treatment for ovarian cancer-associated ascites and disease progression. Histopathology following peritoneal InCeT-TLZ implantation showed no toxicity in any surrounding organs at risk. This approach would offer new sustained therapy options for substantially enhancing the therapeutic window, increasing cure rates, reducing mortality and suffering, and offering major enhancements in quality of life for the thousands of women diagnosed with OC yearly. Citation Format: Shicheng Yang, Needa Brown, Abigail L. Campbell, Kushi Aggarwal, Daniela M. Dinulescu, Srinivas Sridhar. Sustained delivery of PARP inhibitor Talazoparib for the treatment of BRCA deficient ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1994.

BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile-induced mesothelioma via ferroptosis resistance.

Malignant mesothelioma (MM) is still a social burden associated with asbestos exposure. Local iron accumulation thereby represents the major pathogenesis, followed by oxidative DNA strand breaks and genomic alterations in the mesothelium. BRCA1 is a critical component of homologous recombination repair directed to DNA double-stranded breaks, whereas BRCA1 germline mutation is an established risk for breast/ovarian cancer, its role in MM development remains to be elucidated. Murine Brca1 mutant models so far have not reproduced human phenotypes. However, a rat Brca1 mutant model (Mut; L63X/+ ) recently reproduced them at least partially. Here we describe the differential induction of MM in Brca1 mutant rats by intraperitoneal injection of chrysotile or crocidolite. Only Mut males injected with chrysotile revealed a promotional effect on mesothelial carcinogenesis in comparison with wild-type and/or females, with all the MMs Brca1 haploinsufficient. Array-based comparative genomic hybridization of MMs disclosed a greater extent of chromosomal deletions in Brca1 mutants, including Cdkn2a/2b accompanied by Tfr2 amplification, in comparison with wild-type tumors. Mutant MMs indicated iron metabolism dysregulation, such as an increase in catalytic Fe(II) and Ki67-index as well as a decrease in Fe(III) and ferritin expression. Simultaneously, mutant MMs revealed ferroptosis resistance by upregulation of Slc7A11 and Gpx4. At an early carcinogenic stage of 4 weeks, induced Brca1 expression in mesothelial cells was significantly suppressed in chrysotile/Mut in comparison with crocidolite/Mut, whereas significant preference to iron with a decrease in Fe(III) has been already established. In conclusion, chrysotile exposure can be a higher risk for MM in BRCA1 mutant males, considering the rat results.

Abstract NT-110: EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER

Abstract Camptothecin derivatives topotecan and irinotecan are currently the only FDA-approved topoisomerase I (Top1) inhibitors. Despite their wide usage they have many drawbacks: chemical instability, short half-life, dose-limiting bone marrow and gastrointestinal toxicity, and they are drug efflux substrates. The novel non-camptothecin Top1 inhibitors, indenoisoquinolines (LMP400, LMP776 and LMP744), have been developed to overcome these limitations. We explored whether treatment with LMPs could induce “synthetic lethality” in cells with impaired homologous recombination (HR) repair mechanism and whether it could be enhanced by combining them with PARP inhibitor, olaparib. Using the DT40 cell line, we assessed in vitro the role of HR in the cellular responses to the LMPs. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the LMP compounds than wild type cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib. To confirm the differential response of Brca1 wild type or Brca1 deficient cells to LMP treatment we assessed compound potency in primary ovarian cancer cell lines derived from a genetically engineered mouse (GEM) model for serous epithelial ovarian cancer (SEOC). As in DT40 cells, we observed that murine Brca1 deficient cells were more sensitive to all 3 LMP compounds than Brca1 wild type cells. Our results suggest that Brca1 deficiency renders cells more sensitive to treatment with LMPs or combination of LMPs and olaparib, potentially directing selection of patients with impaired HR repair mechanism in the phase 2 clinical trials that are in preparation. To confirm this observation in vivo, we are performing efficacy studies using LMP compounds alone or in combination with olaparib in orthotopic allograft mouse models for ovarian cancer derived from the GEM model. In Brca1 deficient tumors, treatment with each of the 3 LMP compounds led to significant increase of survival compared to vehicle treated group. LMP400 had the greatest beneficial impact on survival and tumor regression. To confirm synergy between LMPs and olaparib we selected LMP400 to represent the LMPs and repeated the efficacy study using Brca1 deficient tumor model. We observed that suboptimal doses of LMP400 or olaparib used as single treatments resulted in some increase in survival, however, when combined they significantly increased survival due to tumor regression. Our data demonstrate that the LMPs synergize with olaparib and provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient ovarian cancers. Citation Format: Ludmila Szabova, Melanie Gordon, Laetitia Matzi, Yves Pommier, Zoe Weaver Ohler. EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-110.

Abstract 2709: Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model

Abstract Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. With first approval in 2014 of Olaparib the concept of tumor-specific synthetic lethality was added to the treatment portfolio of cancer patients. Although the effects of PARPi have shown promising results in multiple cancer types, how and whether patients might benefit from combination with compounds modulating the immune landscape of a tumor is largely unknown. In the current study, we investigate the cross-talk between PARPi and immune checkpoint inhibition, in particular, anti PD-1 and anti CTLA-4, as the most advanced targets in the field. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in vivo using the murine EMT6/BRCA1-/- model in monotherapy as well as in combination with anti-CTLA-4 or anti-PD1 treatment. The four PARPi showed distinct activity profiles in the two breast cancer models. Talazoparib was the most active compound in the BRCA1-/- model (optimal T/C (test/control) of 60%), followed by Niraparib and Rucaparib (65% and 67%, respectively). Olaparib was considered inactive with a T/C value of 80% in monotherapy. The EMT6/BRCA1-/- turned out to be sensitive towards anti CTLA-4 treatment (optimal T/C of 35% - 38% in three independent experiments). Anti PD-1 treatment in monotherapy induced no significant reduction in tumor growth (optimal T/C of 80% - 82%, in two independent experiments). The combination of PARPi and anti CTLA-4 induced a transient but significant reduction of tumor load early in the treatment phase (p< 0.002, one-way ANOVA, day 8). However, anti PD-1 treatment significantly prolonged overall survival in combination with Talazoparib (p< 0.011 log-rank test). The analysis of tumor infiltrating lymphocytes (TILs) by flow cytometry revealed that Talazoparib as monotherapy and in combination with checkpoint (CP) inhibitors enhanced the number of gMDSC as well as the number of CD3/CD11b double positive T cells. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. First results indicate that PARPi as well as CPi induce unique cytokine profiles correlating well with the modified TIL composition of the respective treatment groups. Further mechanistic studies as well as comparative studies with the EMT6/BRCAwt model will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer. Citation Format: Anya Avrutskaya, Cordula Tschuch, Astrid Jensen, William Durham, Maycee Robinson, Charles Krause, Emily O’Koren, Gerhard Kelter, Anne-Lise Peille, Armin Maier, Anne-Marie Zuurmond, Julia Schüler. Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2709.

Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to amesenchymal state of cultured, human basal-likeMECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB,or HES1 or chemically induced ICL damage in primarymurine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. Invivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation.

The emergence of resistance to poly-ADP-ribose polymerase inhibitors (PARPi) poses a threat to the treatment of BRCA1 and BRCA2 (BRCA1/2)-deficient tumours. Stabilization of stalled DNA replication forks is a recently identified PARPi-resistance mechanism that promotes genomic stability in BRCA1/2-deficient cancers. Dissecting the molecular pathways controlling genomic stability at stalled forks is critical. Here we show that EZH2 localizes at stalled forks where it methylates Lys27 on histone 3 (H3K27me3), mediating recruitment of the MUS81 nuclease. Low EZH2 levels reduce H3K27 methylation, prevent MUS81 recruitment at stalled forks and cause fork stabilization. As a consequence, loss of function of the EZH2/MUS81 axis promotes PARPi resistance in BRCA2-deficient cells. Accordingly, low EZH2 or MUS81 expression levels predict chemoresistance and poor outcome in patients with BRCA2-mutated tumours. Moreover, inhibition of Ezh2 in a murine Brca2-/- breast tumour model is associated with acquired PARPi resistance. Our findings identify EZH2 as a critical regulator of genomic stability at stalled forks that couples histone modifications to nuclease recruitment. Our data identify EZH2 expression as a biomarker of BRCA2-deficient tumour response tochemotherapy.

RANKL/RANK control Brca1 mutation- .

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

Abstract 5109: Proteomics of murine BRCA1 deficient breast tumors identifies DNA repair proteins with prognostic value in human breast cancer

Abstract Background: Breast cancer is the most common malignancy in women in the western world. The outcome of breast cancer would be strongly improved if patients could be diagnosed and treated early. This especially holds for patients with hereditary breast cancer. Aim: Our aim is to identify proteins that are associated with BRCA1 deficient hereditary breast cancer. These proteins could have potential use as screening, prognostic or predictive biomarkers. Approach: In-depth proteome profiling of tumor tissues of mouse breast cancer models was employed to identify BRCA1-associated proteins. To this end, tumor tissue lysates of five BRCA1 deficient and five proficient mouse models (Liu et al., 2007) were fractionated using SDS-PAGE followed by tryptic in-gel digestion, nanoLC-MS/MS and database searching. Normalized spectral counting was used for protein quantification and beta binomial statistics to discover significantly regulated proteins. Ingenuity Pathway Analysis and COFECO protein complex analysis was used to support data interpretation. The prognostic power was assessed by the use of publicly available gene expression human breast cancer data sets. Results: We identified a total of 3614 proteins, of which 804 were differential between the genomic instable BRCA1-deficient and the genomic stable BRCA1-proficient breast tumors. Pathway analysis and protein complex identified DNA-repair and associated function like chromatin modeling and RNA processing as the major functions associated with the upregulated proteins of the BRCA1 deficient tumors. We created a core DNA-repair protein signature using pathway and protein Complex analysis. When the signature proteins were mapped to human breast cancer gene-expression data sets, they were shown to have prognostic power. Conclusion: Proteomics of genetic mouse models for familial breast cancer is a powerful strategy to discover novel candidate BRCA1 deficiency proteins with human relevance. The major upregulation of DNA-repair complexes may indicate a compensatory mechanism for loss of BRCA1 DNA-repair. Further validation studies are required to investigate prognostic candidates in tissue microarrays. Integration with data on tumor and cancer cell-secreted proteins will pinpoint which of the BRCA1 deficiency associated proteins may have potential for screening of BRCA1 tumors and whether they have predictive value for sensitivity to PARP1 inhibition and Cisplatin treatment in BRCA1 as well as non-BRCA1-deficient tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2011-5109

BRCA2 is required for neurogenesis and suppression of medulloblastoma

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.

Homozygous germ line mutation in exon 27 of murine Brca2 disrupts the Fancd2–Brca2 pathway in the homologous recombination‐mediated DNA interstrand cross‐links' repair but does not affect meiosis

The role of the region encoded by exon 27 of the Brca2 gene in DNA repair was studied in cells and tissues from Brca2Delta27/Delta27 mice. The COOH-terminal truncated Brca2 localized to the nucleus in primary mouse embryo fibroblasts from Brca2Delta27/Delta27 mice. Fluorescence-activated cell sorting (FACS) analysis demonstrated that these fibroblasts were hypersensitive to mitomycin C-induced cross-links, but not to double-strand breaks (DSBs) induced by irradiation. The gammaH2AX appearance kinetics and comet assay showed that DSBs were repaired through non-homologous end joining pathways, while interstrand cross-links were not repaired due to deficient homologous recombination pathways. Immunoprecipitation experiments showed that Fancd2 did not coprecipitate with the mutated Brca2. There were also no detectable Rad51-positive foci formed in these cells after damage. On the other hand, we did not find any difference during gametogenesis in mice harboring exon 27 truncating mutation of the Brca2 gene and control mice, and in both cases, Rad51 localized to the recombination foci. Our results suggest that exon 27 of murine Brca2 is crucial for the interaction of Brca2 and Fancd2 in Rad51-mediated recombination in response to DNA damage, but that this interaction is not taking place in the homologous recombination during meiosis.

Brca1 expression is regulated by a bidirectional promoter that is shared by the Nbr1 gene in mouse.

The lack of functionally disrupting mutations of BRCA1 in sporadic breast tumours has suggested that other mechanisms, including dysregulation of gene expression, might be important in tumour development. We have analysed the control of expression of murine Brca1 and the adjacent gene, Nbr1, which lie head-to-head and are separated by less than 300 bp. Our results show that the expression of these two genes is under complex regulation, through a bidirectional promoter. Brca1 expression is driven by this single promoter, whereas Nbr1 expression is driven by this and one additional promoter, which generate two distinct transcripts, differing by the alternate use of the first exons. By comparison of mRNA transcription in adult murine tissues and also in the mammary gland during pregnancy and lactation, we show that Brca1 and Nbr1 expression is coordinately regulated in a spatial and temporal manner to produce quite different patterns of expression, even from the same promoter. The analysis of the murine and human syntenic region and its control has important implications for the regulation of human and murine BRCA1/NBR1 expression and the interpretation of animal models of disease.

Transcriptional regulation of the murine brca2 gene by CREB/ATF transcription factors

The brca2 gene encodes a nuclear protein which is mainly involved in DNA repair and, when mutated, is responsible for some of the hereditary breast cancers. However, brca2 expression is also deregulated in sporadic breast tumors. In the mouse brca2 gene we had earlier identified a region of 148 bp upstream of the transcription start site sufficient to activate its expression. In the present report, we show that the −92 to −40 bp region is essential for the transcription of brca2 in murine mammary cells and that this nucleotide sequence contains one putative CREB/ATF consensus site (cAMP responsive element: CRE). We demonstrated that the mutation of this binding site led to a highly significant reduction of the mouse brca2 transcription, and that CREB, CREM, and/or ATF-1 functionally bound to and regulated this promoter. Therefore, the regulation of the promoter of the mouse brca2 gene is driven by this family of transcription factors.

Tumor formation in mice with conditional inactivation of Brca1 in epithelial tissues.

The BRCA1 tumor-suppressor protein has been implicated in the regulation of transcription, DNA repair, proliferation, and apoptosis. BRCA1 is expressed in many proliferative tissues and this is at least in part due to E2F-dependent transcriptional control. In this study, inactivation of a conditional murine Brca1 allele was achieved in a variety of epithelial tissues via expression of the Cre recombinase under the control of a keratin 5 (K5) promoter. The K5 Cre:Brca1 conditional knockout mice exhibited modest epidermal hyperproliferation, increased apoptosis, and were predisposed to developing tumors in the skin, the inner ear canal, and the oral epithelium after 1 year of age. Overexpression of the E2F1 transcription factor in K5 Cre:Brca1 conditional knockout mice dramatically accelerated tumor development. In addition, Brca1 heterozygous female mice that had elevated E2F1 expression developed tumors of the reproductive tract at high incidence. These findings demonstrate that in mice Brca1 functions as a tumor suppressor in other epithelial tissues in addition to the mammary gland. Moreover, inactivation of Brca1 is shown to cooperate with deregulation of the Rb-E2F1 pathway to promote tumorigenesis.

An adenoviral vector containing an arg-gly-asp (RGD) motif in the fiber knob enhances protein product levels from transgenes refractory to expression.

Genetic manipulation of the adenovirus type 5 represents one strategy to modify viral transduction properties in vitro and in vivo. In the majority of studies to date, reporter gene activity has been monitored to assess transduction efficiency. BRCA1 is a gene whose protein product is clinically important, biologically toxic, difficult to overexpress, and difficult to detect as an untagged protein species. Thus, it represents an attractive candidate from which to evaluate the efficacy of a gene delivery system. In the present study, transgene expression was assessed employing otherwise isogenic viruses, which differed only in the presence or absence of an RGD integrin-binding motif in the HI loop of the Ad fiber knob. We utilized a combination of BRCA1 expression level comparisons among several human BRCA1/mutant BRCA1/murine Brca1 constructs and reporter gene activity following transduction of a panel of human breast and ovarian tumor cell lines representative of both sporadic and hereditary cases. A general overall concordance in efficiency was observed, whether the biological readout measured was reporter gene activity or steady-state level of ectopic BRCA1 protein produced. Importantly, the expression of full-length wild-type BRCA1 protein, clinically relevant mutant BRCA1 proteins or murine Brca1 was superior when the gene was delivered via the RGD-modified Ad. The ectopic BRCA1 stabilized endogenous BARD1 and this functional effect was evident at lower input viral doses when BRCA1 was delivered via the RGD-modified Ad. Quantitative, noninvasive, real-time image analysis of reporter gene function in nude mice harboring human ovarian tumor xenographs demonstrated a similar enhancement of expression in vivo by the RGD fiber modification, with low levels of transduction of normal mouse mesothelium. These results provide additional evidence supporting the concept that rational modification of viral vectors can result in the delivery of functionally active therapeutic proteins such as BRCA1 that present with technical difficulties with regard to their expression.

Defective Brca2 influences topoisomerase I activity in mammalian cells.

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.

Defective Brca2 influences topoisomerase I activity in mammalian cells.

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.

Brca1 regulates in vitro differentiation of mammary epithelial cells.

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.