Roß, Christian

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Abstract The post-genomic era ushered in a cadre of techniques to study epigenetic mechanisms involved in disease progression and cancer. One understudied mechanism of gene expression/transcriptional control is 3-dimensional architecture of the genome. Several high-throughput technologies have been adapted to examine 3D genome architecture; in particular those derived from chromatin conformation capture (3C) sequencing technologies. Of 3C-derived techniques, HiC-seq, is the most extensive, and examines all chromatin-chromatin contact sites within a genome, elucidating the entire 3D genome architecture. Although, HiC-seq technique was published 2009, it has been under-utilized in research laboratories due to its technical complexity, expense, and requirement for large volumes of starting material (50 million cells/500 μg of DNA). Other barriers to use of HiC-seq include the requirement for specialized equipment and laborious extraction techniques. However, the benefits of HiC-seq are high for cancer researchers because a genome-wide "snap-shot" of the entire 3D genome of the cancer is displayed. This allows for analysis of higher-order epigenetic control of gene expression. Furthermore, understanding the chromatin conformation of the genome provides new insights into the cellular disease state and potentially novel therapeutic approaches to drug-resistant cancers. Our study presents significant technical innovations, permitting HiC-seq technique to be adopted in laboratories without extensive experience in 3C-seq technologies, and with the use of standard equipment and reagents, as well as smaller volumes of starting materials. Our modified technique utilizes up to 100 fold lower amounts of starting material (500,000 cells/5-10 μg of DNA) per reaction. This adaptation of HiC-seq preparation offers the potential for simultaneous extraction of total RNA and Hi-C processed chromatin for sequencing. To optimize our technique we utilized the estrogen-responsive breast cancer cell line, MCF7. This cell line is typically utilized to study potential drug therapies in the context of estrogen-sensitive cancers. We stream lined HiC-seq by optimizing nuclei extraction and allowing for collection of a cytoplasmic fraction for mRNA extraction and sequencing analysis. We also optimized enzymatic digestion, ligation and DNA extraction methods by utilizing newer enzymatic chemistries, commercially available silica column extraction methods and reagents in place of multiple phenol-based DNA extraction methods. Our quality control results suggest that sample processing for reduced amounts of material is similar if not superior to conventional HiC-seq. We leverage this powerful genomic high-throughput technique in order to simultaneously examine the 3D architecture and transcriptional profiles of MCF7 breast cancer cells. Our computational workflow includes further quality controls using HiC-User Pipeline software from the Bahraham Institute and the R Bioconductor package HiTC package. Overall our optimizations in MCF7 breast cancer cells, and post-acquisition work-flow, make HiC-seq an accessible technology. These innovations permit simultaneous analysis of architectural and transcriptional profiles in cancer cells. Citation Format: Jennifer D Davis, Christian Ross, Hu Li, Amy Brock. Examining 3-dimensional genome architecture and transcriptional profiles using an optimized HiC-seq protocol in breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-16.

Background— Genetic studies of cardiomyopathy and heart failure have limited throughput in mammalian models. Adult zebrafish have been recently pursued as a vertebrate model with higher throughput, but genetic conservation must be tested. Methods and Results— We conducted transcriptome analysis of zebrafish heart and searched for fish homologues of 51 known human dilated cardiomyopathy–associated genes. We also identified genes with high cardiac expression and genes with differential expression between embryonic and adult stages. Among tested genes, 30 had a single zebrafish orthologue, 14 had 2 homologues, and 5 had ≥3 homologues. By analyzing the expression data on the basis of cardiac abundance and enrichment hypotheses, we identified a single zebrafish gene for 14 of 19 multiple-homologue genes and 2 zebrafish homologues of high priority for ACTC1 . Of note, our data suggested vmhc and vmhcl as functional zebrafish orthologues for human genes MYH6 and MYH7 , respectively, which are established molecular markers for cardiac remodeling. Conclusions— Most known genes for human dilated cardiomyopathy have a corresponding zebrafish orthologue, which supports the use of zebrafish as a conserved vertebrate model. Definition of the cardiac transcriptome and fetal gene program will facilitate systems biology studies of dilated cardiomyopathy in zebrafish.