Abstract 2642: BID expression levels determine cell fate upon AURKB inhibition
Authors: Bertrán-Alamillo Jordi , Travers John , Talbot Sara , Whiteley Rebecca , Giménez-Capitán Ana , Román Ruth , Rodríguez Sonia , Aldeguer Erika , Castellví Josep , Martínez-Bueno Alejandro , Martella Andrea , Saeh Jamal , Fabbri Giulia , Rosell Rafael , Polanska Urszula , Urosevic Jelena , Molina-Vila Miguel A. 
Published: June 2022

Abstract
Background: Aurora kinase B (AURKB) plays a key role in the regulation of mitosis, and AURKB inhibitors are in Phase II-III clinical trials. We reported previously that Chr22q11 amplification, leading to BID overexpression, was associated with apoptosis in upon AURKB inhibition (AURKBi) in NSCLC cell lines resistant to EGFR TKIs. However, it remains to be elucidated whether high levels of BID expression are associated with sensitivity to AURKBi beyond NSCLC and Chr22q11 amplification.
Methods: A panel of 11 solid tumor cell lines were used in the study. Also included were the EGFR-mut cell lines PC9 and 11-18, and 36 EGFR-TKI resistant clones derived from them. FISH and Q-PCR were used to determine copy number status; mRNA expression was studied by nCounter and RT-Q-PCR and protein expression by Western blotting. The effects of drugs were analyzed by MTT and flow cytometry. CRISPR-Cas9 technology was used for KO generation while ectopic expression was achieved by cloning the BID gene into a doxycycline-inducible pTRIPZ vector. Finally, in vivo experiments were performed by implanting tumor cells into athymic nude mice.
Results: First, we analyzed copy numbers of BID and other 22q11 genes, together with BID expression levels, in the 49 cell lines included in the study. Then, we determined the dose-response curves of the cell lines for AZD2811, a specific AURKB inhibitor. We found that tumor cells with high BID expression levels were consistently sensitive to AURKB inhibition, independently of 22q11 amplification, while cell lines with low BID expression were uniformly resistant (r=0.91, p&lt;0.0001). Also, the growth of xenografts derived from the BID-high expressor H1819 cell line was inhibited by AZD2811. Gene silencing consistently rendered BID-high expressing cell lines fully resistant to Aurora B inhibition. Conversely, ectopic BID expression in BID-low cell lines significantly increased sensitivity to AURKB inhibitors particularly in absence of KRAS or BRAF mutations. Experiments using doxycycline gradients established that cell growth inhibition after treatment with AZD2811 was dose-dependent on the BID expression levels (R2=0.97). Western blotting experiments in parental and BID-silenced cells demonstrated that AZD2811 induced apoptosis in sensitive cells by a mechanism mediated by CASP-2 activation, leading to CASP-3 cleavage. In addition, CRISPR KO of the PIDDosome components (CASP-2, PIDD1 and CRADD) mimicked the effects of BID silencing, rendering BID-high expressing cells resistant to AURKB inhibition. Finally, a prevalence study including 118 tumor samples of different solid malignancies revealed high BID mRNA levels in 6% of cases.
Conclusions: BID upregulation switches cell fate from survival to apoptosis in response to AURKB inhibition in a wide range of tumor cell lines from different origins. Our results pave the way to clinical exploration of AURKB inhibitors in BID overexpressing tumors.
Citation Format: Jordi Bertrán-Alamillo, John Travers, Sara Talbot, Rebecca Whiteley, Ana Giménez-Capitán, Ruth Román, Sonia Rodríguez, Erika Aldeguer, Josep Castellví, Alejandro Martínez-Bueno, Andrea Martella, Jamal Saeh, Giulia Fabbri, Rafael Rosell, Urszula Polanska, Jelena Urosevic, Miguel A. Molina-Vila. BID expression levels determine cell fate upon AURKB inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2642.