Abstract PS8-32: Effects of DNA-damaging and demethylating agents on alternate mRNA splicing in BRCA2

Abstract

Abstract Introduction DNA sequence variants of unknown clinical significance (VUSs) are routinely identified during genetic testing of BRCA2 and other tumor suppressor genes associated with hereditary breast/ovarian cancer syndrome (HBOC). To determine which VUSs could be classified as pathogenic mutations, sequence variants near intron-exon boundaries are tested for effects on normal splicing patterns. However, these investigations can be complicated by potential genome-wide disruptions of normal splicing patterns caused by therapeutic agents. To test the potential contribution of systemic therapies to alternative splicing events in BRCA2, we have tested the effects of two DNA damaging agents and two demethylating agents on the relative levels of the naturally occurring alternative splicing variant BRCA2Δ3. While the protein product of this alternative splicing event has not been characterized, the BRCA2Δ3 mRNA maintains the full-length translational reading frame, and 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 BRCA2Δ3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. Thus, alternative splicing events that alter relative levels of the BRCA2Δ3 mRNA variant may serve both as an indicator of the effects of some systemic therapies on genome-wide splicing defects and also BRCA2 gene function per se. 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 2’-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: James D. Fackenthal, Talia Ishfaq, Zaain Ahmad, Nourhan Mohamed, Milica Janosevic, Ziyad Abdelrahim, Jessica Georgopulos. Effects of DNA-damaging and demethylating agents on alternate mRNA splicing in BRCA2 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-32.