Abstract 1020: Amplification of 22q11 is associated with hypersensitivity to AURKB inhibition in cell line models of resistance to EGFR TKIs
Authors: Bertran-Alamillo Jordi , Travers Jon , Giménez-Capitán Ana , Floch Nicolas , Román Ruth , Swaih Aisha , Rodríguez Sonia , Martin Matthew , Aldeguer Erika , Miragaia Ricardo , Castellví Josep , Smith Paul , Martínez-Bueno Alejandro , Pease Elizabeth , Ditzel Henrik , Fabbri Giulia , Rosell Rafael , Polanska Urszula , Molina-Vila Miguel A , Urosevic Jelena 
Published: July 2021

Background: Non-small cell lung cancer (NSCLC) tumors with mutations in the EGF receptor (EGFR) ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (TKIs). Cell line models of resistance to EGFR TKIs have been described to be sensitive to AURKB inhibitors, such as AZD2811, but the mechanisms and markers of hypersensitivity remain to be elucidated.

Methods: A panel of 36 clones with acquired in vitro resistance to EGFR TKIs was used in the study. Some of them had been previously demonstrated to be exquisitely sensitive to AZD2811 and to show a phenotypic response switch from polyploidy and senescence to apoptosis. Whole exome sequencing were employed for genotyping, while FISH and Q-PCR were used to determine copy number gains in particular genes. mRNA expression was studied by whole transcriptome sequencing, nCounter and RT-Q-PCR; and protein expression by proteomics and Western blotting. The effects of several drugs were analyzed by MTT, flow cytometry and beta-galactosidase staining. Finally, CRISPR was used to silence specifics genes. In vivo experiments were performed by implanting heterogenous pools of resistant cells in athymic nude mice. Selection against Chr22q11 amplification was assessed by FISH copy number analysis and polysomy assessment.

Results: Transcriptomics, proteomics, RT-Q-PCR and Western blotting of the EGFR TKI resistant clones revealed that hypersensitivity to AZD2811 was consistently associated with up-regulation of &gt; 40 genes in the q11.2 segment of chromosome 22; including MAPK1, CRKL, BID or BCL2L13. FISH using probes against HIRA, CRKL and MAPK1 demonstrated 7-10 fold 22q11 amplification in all AZD2811 hypersensitive clones, compared to lack of amplification in parental and resistant clones. Amplification of 22q11, confirmed by WES, was mutually exclusive with known mechanisms of resistance to EGFR-TKIs. Cell cycle and beta-galactosidase staining revealed G2/M arrest upon AZD2811 treatment followed by apoptosis in 22q11-amplified cells and senescence in non-amplified clones. Silencing of one 22q11 gene - BID - specifically abolished sensitivity to AZD2811 in 22q11 cells with little or no effect on the IC50s to other drugs. Silencing of CRKL only partially restored sensitivity to osimertinib in 22q11 amplified cells. Furthermore, clonal selection analysis in vivo demonstrated that a nanoparticle formulation of AZD2811(NP) in combination with osimertinib selectively eliminated 22q11 amplified cells and rendered the remaining non-amplified cells polyploid.

Conclusions: Amplification of 22q11, leading to BID overexpression, predicts sensitivity to AURKB inhibition in cell line models of resistance to osimertinib and other EGFR TKIs. These data are consistent with overdrive in 22q11 amplified cells of a recently reported mitotic checkpoint mediated by AURKB phosphorylation of CASP2, and suppression of BID cleavage.

Citation Format: Jordi Bertran-Alamillo, Jon Travers, Ana Giménez-Capitán, Nicolas Floch, Ruth Román, Aisha Swaih, Sonia Rodríguez, Matthew Martin, Erika Aldeguer, Ricardo Miragaia, Josep Castellví, Paul Smith, Alejandro Martínez-Bueno, Elizabeth Pease, Henrik Ditzel, Giulia Fabbri, Rafael Rosell, Urszula Polanska, Miguel A Molina-Vila, Jelena Urosevic. Amplification of 22q11 is associated with hypersensitivity to AURKB inhibition in cell line models of resistance to EGFR TKIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1020.