Abstract P5-17-11: Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing
Authors: Manzanares Miguel A , Dhingra Priyanka , Anderson Kendall , Frederick Vanessa , Geier Adam , Casill Alyssa , Akerman Martin , Arun Gayatri 
Published: March 2022

Abstract
Aberrant splicing is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. The oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors have been demonstrated in multiple studies. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for the treatment of insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore technology is an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed &gt;2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with the potential to translate into therapeutic candidates for TNBC. Interestingly, one of our leading AS targets is an exon skipping isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival, without showing differences in gene expression between all the breast cancer subtypes and healthy patients studied. In addition, SpliceCore was used to predict and design a set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. The skipping isoform plays a critical role in tumor progression via a TGFβ-dependent mechanism as demonstrated by detailed isoform switching studies using SSO-0205. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation modulated TGFβ pathway related protein levels and cellular localization and reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. In summary, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Our results provide experimental proof of concept that demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent therapeutic development. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Moreover, SpliceCore can be applied to multiple other indications opening a new avenue for therapeutic development in cancer.
Citation Format: Miguel A Manzanares, Priyanka Dhingra, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-11.