Abstract: Double heterozygosity is an extremely rare occurrence in hereditary breast and ovarian cancer syndrome (HBOC [MIM 604370; MIM 612555]) where two pathogenic variants, one in BRCA1 and one in BRCA2, are found in an individual. To date, only a few case reports and case series have been reported in the literature (1-3). Furthermore, little is known about the clinical characteristics, family history, and tumor histology in these patients. In this study, we utilized targeted gene testing with next-generation sequencing (NGS) technology in an early-onset metastatic breast cancer patient from France. We evaluated germline variants using Pathway Genomics' BRCATrueTM NGS test, which analyzes variants covering all exons and exon flanking regions in both the BRCA1 and BRCA2 genes. All variant calls were determined after alignment and mapping to the GRCh37/hg19 reference genome. Variant calls were confirmed by Sanger sequencing. In this patient, a c.1016dupA (p.V340GfsX6) frameshift variant was found in BRCA1 along with a c.6814delA (p.R2272EfsX8) frameshift variant in BRCA2. Both frameshift variants are predicted to truncate the BRCA proteins. The BRCA1 c.1016dupA variant is considered a Norwegian founder mutation but has also been observed in individuals who are of French-Canadian, French, Italian or Dutch ancestry (4-7). The BRCA2 c.6814delA (p.R2272Efs*8) pathogenic variant, also known as 7042delA, is predicted to truncate the BRCA2 protein and has been identified in individuals with a personal or family history of breast and/or ovarian cancer (8,9). To the best of our knowledge, the combination of these two pathogenic variants in an individual has not been previously reported. In a clinical diagnostic setting, the possibility of double heterozygosity of pathogenic variants in more than one susceptibility gene should be considered, especially in patients with early-onset metastatic cancers. Furthermore, genetic testing and genetic counseling should also be indicated for high-risk family members. 1. Heidemann, S. et al. (2012) Breast cancer research and treatment 134, 1229-1239 2. Lavie, O., et al. (2011) Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 22, 964-966 3. Nomizu, T., et al. (2012). Breast cancer 4. Andersen, T. I., Borresen, A. L., and Moller, P. (1996) American journal of human genetics 59, 486-487 5. Caputo, S., et al. (2012) Nucleic acids research 40, D992-1002 6. Dorum, A., et al. (1999). American journal of human genetics 65, 671-679 7. Simard, J., et al. (1994). Nature genetics 8, 392-398 8. Novakovic, S., et al. (2012) International journal of oncology 41, 1619-1627 9. Tea, M. K., et al. (2014) Maturitas 77, 68-72. Citation Format: Curtit E, Meynard G, Villanueva C, Mansi L, Chaix M, Vilalta A, Kuo JZ, Villa M, Neidich J, Tomar A, Arianpour A, Lebahar P, Pivot X. Double heterozygosity for BRCA1 and BRCA2 pathogenic variants in a French metastatic breast cancer patient. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-10.

Abstract: Kruppel-like factor 9 (KLF9) is a member of the KLF family of transcription factors. Previous studies report significant KLF member involvement in breast cancer estrogen response, notably KLF4 [1, 2] and KLF5 [3, 4] . KLF9 has been shown in uterine cancer cells to function as an estrogen-dependent regulator of the estrogen response pathway [5]. We aim to evaluate the role of KLF9 in breast cancer cell estrogen response. We have performed studies demonstrating that KLF9 exhibits an early-activated estrogen response. Five sites have been identified upstream of the KLF9 gene that interact with estrogen receptor alpha (ERα) [6-8] ; we observe ERα enrichment at three of these sites that is estradiol-dependent. Though KLF9 has been shown to act as an essential element in estrogen response in the uterus, KLF9 response in breast cancer cells has yet to be characterized. To study the role of KLF9 in estrogen-mediated responses in transcriptional and proliferative activity, we have manipulated the level of KLF9 expression in MCF-7 breast cancer cells. These cells have been shown to exhibit a clear transcriptional response to estradiol in luciferase reporter assays. We have optimized RNAi conditions to achieve significant knockdown of the KLF9 gene in MCF-7 cells. Additionally, we have produced and tested a KLF9 overexpression vector construct that significantly upregulates expression of KLF9 in this cell line. These tools will be used to more extensively characterize the role of KLF9 in MCF7 cell estrogen-stimulated transcription and proliferation. We are measuring transcriptional response to estrogen signaling with luciferase reporter assays and estrogen stimulated cell proliferation with BrdU proliferation assays. Elucidating KLF9 involvement in E2-mediated breast cancer cell signaling and response, therefore, is an important component of understanding of the regulatory mechanisms behind estrogen response in breast cancer. 1. Akaogi K et al. (2009). Oncogene 28(32): 2894. 2. Quintana AM et al. (2011). BMC Cancer 11: 30. 3. Guo P et al. (2010). International Journal of Cancer 126(1): 81. 4. Zhao KW et al. (2011). Biochemical Journal 437(2): 323. 5. Velarde MC et al. (2007). Molecular Endocrinology 21(12): 2988. 6. Carroll JS et al. (2006). Nature Genetics 38(11): 1289. 7. Hua S et al. (2008). Molecular Systems Biology 4:188. 8. Fullwood MJ et al. (2009). Nature 462(7269): 58. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 67. doi:1538-7445.AM2012-67

Abstract: Estrogen stimulation triggers ERα binding to distant estrogen response elements (DEREs) through intra- or inter-chromatin interactions (Hsu et al. 2013; Hsu et al. 2015). Prolonged or repetitive stimulation causes alterations of histone marks and DNA methylation patterns in neighboring regions of DEREs, establishing three-dimensional structures for concordant transcription of estrogen-responsive genes. DEREs from multiple chromosome regions can be mobilized to form transcription depots for synchronized transcription of neighboring target genes. Heterochromatin protein 1 (HP1) acts as an anchor in regulating chromatin movement of DERE-mediated transcription assemblages in the nucleus (Hsu et al. 2015). Although HP1 is a fundamental unit of heterochromatin and acts a transcriptional repressor, we found that this protein may serve as a lamina buttress to support free movement of flexible chromatin in assembling transcription factories (Beisel and Paro 2011; Hsu et al. 2015). Neutralizing HP1 function deregulates the formation of DERE depots for concordant transcription of target genes. Furthermore, deletion of DEREs using the CRISPR/Cas9 genomic editing system alters transcription profiles of proliferation-associated networks, resulting in reduced cellular growth of luminal breast cancer cells. The epigenetic control of DERE-related chromatin movement has profoundly geographic influences on local genomic structures of gene clusters. Frequent chromatin interactions increase DNA breaks at or nearby looping sites, contributing to genomic instability in cancer cells (Kaiser and Semple 2018). Inappropriate DNA repair leads to genomic fusion and duplication of DEREs and target promoters. Amplified DEREs potentially cooperate with epigenetic machineries for long-range transcription regulation, leading to intensification of gene activation and repression for advanced development of luminal breast cancer. Specifically, we have identified DEREs on chromosome 20q13 frequently amplified and translocated to other chromosomes in this cancer subtype (Hsu et al. 2015). Genome-wide survey of The Cancer Genomic Atlas (TCGA) breast cancer cohort identified 17 contiguous chromosome regions that contain gene clusters (n=16 to 33 genes) with high correlation coefficients between their amplification and expression levels. Five of these identified regions were from chromosome 17q, and, moreover, upregulation of their gene expression was linked to luminal breast cancer with adverse clinical outcomes. Specifically, we found that six genes BCAS3, BRIP1, INTS2, MED13, METTL2A and TLK2 displayed concordantly high expression are located on the active or open topologically associating domains of 17q23 enriched with permissive chromatin marks H3K36me3 and H3K4me3. Complex translocation patterns between 17q23 and 20q13 were detected in breast tumors. The expression of these genes was increased in breast cancer cells after estrogen stimulation. However, this synchronized regulatory function was abolished in breast cancer cells harboring CRISPR/Cas9-knockout of 20q13 DEREs. These findings further establish that the 17q23 region can serve as an estrogen-driven transcriptional hub to promote ERα-mediated gene regulation and also extend previously published results showing that 17q23 genes are indeed DERE-bound ERα target genes. One of these DERE-regulated loci is Tousled Like Kinase 2 (TLK2), known to participate in checkpoint regulation and DNA damage response (Bruinsma et al. 2016), is an actionable target. Amplification-associated upregulation of TLK2 can be suppressed in breast cancer cells treated with phenothiazine antipsychotics, thioridazine, perphenazine and trifluoperazine (Sudeshna and Parimal 2010). Antiproliferative effects of phenothiazines were prominent especially in high TLK2-expressing cells, compared to those low TLK2-expressing cells that were relatively insensitive to phenothiazines. To further extend the potential use of TLK2 inhibition, we determined the efficacy of phenothiazine treatment on ex vivo cultures of patient-derived circulating tumor cells (CTCs). Enriched populations of CTCs were obtained from blood samples of breast cancer patients with metastatic disease. CTCs derived from patients with ERα-positive breast cancer appeared to be more sensitive to TLK2 inhibition compared to those of ERα-negative patients. Therefore, these antipsychotics can potentially be used as an adjunct for treating breast cancer tumors carrying DERE-mediated 17q23 amplification. Citations: Beisel C, Paro R. 2011. Silencing chromatin: comparing modes and mechanisms. Nature reviews Genetics 12: 123-135. Bruinsma W, van den Berg J, Aprelia M, Medema RH. 2016. Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest. EMBO reports doi:10.15252/embr.201540767: e201540767. Hsu P-Y, Hsu H-K, Lan X, Juan L, Yan Pearlly S, Labanowska J, Heerema N, Hsiao T-H, Chiu Y-C, Chen Y et al. 2013. Amplification of Distant Estrogen Response Elements Deregulates Target Genes Associated with Tamoxifen Resistance in Breast Cancer. Cancer Cell 24: 197-212. Hsu PY, Hsu HK, Hsiao TH, Ye Z, Wang E, Profit AL, Jatoi I, Chen Y, Kirma NB, Jin VX et al. 2015. Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells. Oncogene 35: 2379. Kaiser VB, Semple CA. 2018. Chromatin loop anchors are associated with genome instability in cancer and recombination hotspots in the germline. Genome biology 19: 101-101. Sudeshna G, Parimal K. 2010. Multiple non-psychiatric effects of phenothiazines: A review. European Journal of Pharmacology 648: 6-14. Citation Format: Chun-Lin Lin, Tim Hui-Ming Huang. Long-range epigenetic control of estrogen-responsive transcription [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 SY43-03.