Drug combination therapies in the cancer setting often succeed where mono-therapies fail, facilitating durable and robust responses that may curtail metastases and even be accompanied by milder side-effects. Here we incorporate transcriptional splicing dynamics with a gene level expression analysis to assess the transcriptional landscape of MCF-7 (ER+) cell-lines across mono or combination therapies in a time course short-read RNA-seq experiment. We leverage the most comprehensive known splicing database of MCF7 to assess the sensitivity and specificity our short-read splicing inference by interrogating the long-read PacBio isoform sequencing (IsoSeq) MCF7 database. We infer robust isoforms which are strongly associated with phenotypic synergy and demonstrate their novel functional role by computing gene-gene coexpression networks which are sensitive to their expression.
Briefly, we use RNA-seq to study the transcriptional response over time (0, 3, 6, 9, 12, and 24 h) for three drugs (A, B and C) and their combinations (AB, AC and BC) in MCF-7 (ER+) breast cancer cells lines. Cell viability measurements show that one of the combinations (AB) is strongly synergistic, whereas the other two (AC and BC) are merely additive. We show that in addition to a novel transcriptional signature driven by differential expression, the combination AB transcriptional landscape is characterized by persistent alternative splicing signatures mostly comprised of genes which are not differentially expressed with respect to A or B, but whose functional role has been dramatically changed by the addition (deletion) of a key regulatory protein domain encoded by the extra(missing) exon. We integrate this synergistic splicing signal into a gene regulatory network (GRN) via adaptive network refinement to show that it strongly contributes to the emergence of extensive transcriptional cascades by creating and removing key gene-gene correlations and altering the modular structure of the network. Using this approach we show that a key (heretofor poorly understood) isoform of Protein beta-arrestin-1 (ARBB1), which participates in the desensitization of G-protein coupled receptors through intracellular response to Estrogen, becomes enriched in the calcium ion-dependent exocytosis coexpression module for synergistic therapies only, despite being absent in the baseline MCF-7 long-read PacBio IsoSeq data.
Our results directly show that alternative splicing is crucial in any molecular characterization of phenotypic drug synergy in drug-tumor interactions.
Citation Format: Xintong Chen, Ali Bashir, Gustavo Stolovitzky, Bojan Losic. Dynamic alternative splicing correlates with drug synergy and induces novel gene regulatory networks in MCF7. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-286.