Abstract LB-171: Assessing the impact of PIK3CA mutation on the three-dimensional organization of chromatin

Abstract

Abstract Background: Three-dimensional organization of chromatin within the nucleus is an important and dynamic regulator of cellular transcription. Changes in chromatin topology are implicated in processes like cell differentiation and cell fate decisions, but have been largely unstudied with regards to mutations and cancer cell transformation. Our goal is to assess the changes in chromatin topology that occur in response to a single oncogenic mutation and how these changes may influence cancer cell transformation. Methods: Using AAV-mediated homologous recombination, a E545K “hotspot” PIK3CA mutation was incorporated into MCF10A breast epithelial cells and conversely, corrected back to wild-type in MCF7 breast cancer cells that natively harbor a E545K PIK3CA mutation. To interrogate the chromatin topology of these cell lines and the unmodified versions we will use a technique called tethered chromosome conformation capture (TCC). TCC maps chromatin topology by combining the proximity ligation of interacting DNA segments with next generation sequencing to create a matrix of interacting DNA elements. We can analyze and overlay these matrices to look for significant differences in topologically associating domains (TADs). Results: Preliminary results comparing MCF7 parental cells and MCF7 cells with the PIK3CA exon 9 mutation corrected suggest rearrangement of TADs at many key cancer-associated genes. One of these rearrangements is the formation of a de novo TAD on chromosome 10 near PTEN, a tumor suppressor gene that acts antagonistically to PIK3CA. Rearrangement of chromatin topology at this locus could lead to downregulation of a negative actor on this pathway further stimulating downstream elements of the PIK3CA pathway. Conclusions: Oncogenic mutations can cause changes that can have a drastic impact on transcription. Rearrangement of chromatin topology is a key mechanism by which the transcriptional program of a cell can be modified. Our experiments reveal key sites of chromatin rearrangement caused by a single oncogenic mutation that may promote a cancer phenotype in our cells. We plan to investigate the degree to which the expression of PTEN is altered by chromosomal rearrangements as well as perform CRISPR-KO of the interacting loci to determine the transcriptional effect of removing the loop. These assays will also be conducted for other sites of chromatin rearrangement. Results from these experiments will reveal the influence oncogenic mutations may have on chromatin topology and expression. Citation Format: Adam X. Miranda, Ben H. Park, Emily Hodges. Assessing the impact of PIK3CA mutation on the three-dimensional organization of chromatin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-171.