Abstract 4724: DNA demethylation and double-strand breaks affect levels of the BRCA2 Δ3 alternate splicing isoform

Introduction:BRCA2 is one of two principal tumor suppressors responsible for hereditary breast/ovarian cancer syndrome (HBOC). Patients with strong family histories of breast/ovarian cancer may carry BRCA2 DNA sequence variants of unknown clinical significance (VUSs). To evaluate which of these VUSs may contribute to cancer risk by altering splicing patterns, naturally occurring BRCA2 mRNA alternate splicing events have been extensively characterized, but it is not yet known which if any of these alternate splicing events plays a role in BRCA2 function. One variant lacking exon 3 (Δ3), which maintains the full length translational reading frame, lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of Δ3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. It is therefore of interest to characterize the factors that influence the frequency of naturally occurring BRCA2 exon 3-skipping, including cell types and therapies. Methods: 1) To determine whether therapeutic DNA damaging agents can alter the levels of Δ3 alternate splicing isoforms, the breast cancer cell line MCF7 was treated with either doxorubicin or bleomycin, and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 2) To determine whether DNA demethylating agents known to promote expression of some tumor suppressors can alter the levels of Δ3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 29-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and again isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. 3) To determine whether the BRCA2 Δ3 isoform was equally accessible to translational machinery in all cell types, RNA was prepared from separated nuclear and cytoplasmic fractions of MCF7 and MCF 10A and isoform-specific RT-PCR was used to estimate levels of cytoplasmic Δ3 available to translational machinery. Results and conclusions: 1) While bleomycin decreases the relative levels of Δ3 compared with untreated controls, doxorubicin increases relative levels of Δ3 in MCF7. This suggests that some DNA damaging agents may cause changes in splicing patterns that may affect therapeutic effectiveness. 2) While both 5-AzaC and 5-AzadC reduce relative levels of Δ3 in MCF7, 5-azadC does not reduce relative levels of Δ3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. 3) MCF 10A limits the level of Δ3 that can accumulate in the cytoplasm, but this regulatory mechanism is absent from MCF7, indicating there is a cell type- specific mechanism that limits the amount of alternately spliced mRNA variants that are available to translational machinery. Citation Format: Talia Ishfaq, Zaain Ahmad, Nourhan Mohamed, Milica Janosevic, Ziyad Abdelrahim, Jessica Georgopulos, James Fackenthal. DNA demethylation and double-strand breaks affect levels of the BRCA2 Δ3 alternate splicing isoform [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 4724.