Precision Phosphoproteomic Analysis In Chr22q11.2 Amplified Nsclc Cells Reveals Distinct Signaling Corruption And Response To Aurora Kinase B Inhibition.

Abstract Background: NSCLC cells carrying EGFR mutations can gain resistance to cognate TKIs through amplification of Chr22q11.2 (Chr22amp), a chromosome segment containing CRKL. This also specifically associates with exquisite sensitivity to inhibitors of Aurora Kinase B (AZD2811), potentially mediated by other Chr22 genes. Furthermore, a phenotypic rewiring occurs in the response to AZD2811, from a senescent polyploidy in wildtype (WT) cells to apoptosis in Chr22amp cells. Here, we aimed to elucidate the underlying signaling alterations in this background by phosphoproteomic pathway analysis. Methods: The EGFR mutant cell line PC9 and 8 TKI resistant derivatives were profiled (4 Chr22amp and 4 WT). Kinetics of response to AZD2811 (100nM) and osimertinib (160 nM) were identified by flow cytometry. Samples (n=3) were prepared for phosphoproteomics, after 6, 24, and 48 h AZD2811 and 1 h osimertinib, with time matched controls. Cells were washed and lysed in urea, then digested with trypsin. Phosphorylated peptides were enriched with TiO2 and analyzed by Orbitrap LC-MS/MS. Computational analyses quantified peptides across samples. KScanTM bioinformatics identified differential phosphopeptides between Chr22amp and WT to determine kinase substrate profiles by KSEA, putative downstream targets (PDT) and differential compound target activity markers (CTAM). Results: Single cell time-course analysis of phenotypic response to AZD2811 in Chr22amp cells showed that >60% of cells become Annexin V+ by 48 h post-treatment. We took earlier timepoints of 6, 24 and 48 h post treatment. We focused the phosphoproteomic analysis on three comparisons of Chr22amp amplified cells to: 1) the basal signaling state compared to WT; 2) the signaling response to osimertinib in parental PC9; and 3) the altered kinetics of signaling in response to AZD2811 compared to WT. At the basal level, Chr22amp had CK1e, CDK2, p38a substrates differentially enriched, and MTOR inhibitor and Aurora B inhibitor modulated sites (p<10-3). The response to osimertinib was largely differential in the maintenance of ERK1/2 signaling to P90RSK1 but not MEK1 in Chr22amp cells. In cells treated with AZD2811, alterations in signaling were associated with Aurora B in all cells as expected. However in amplified cells, we observed key differences at 24h such in cell death and metabolic processes in specific hierarchical clusters of temporally modulated sites, underpinned by relative down regulation of multiple signaling nodes such as ARAF (z = 4.87, p<10-2), ERN1 (z = 4.56, p<10-2), and CDK2 (z = 4.30, p<10-2). Conclusions: Here, we identified significant pathway deregulation in Chr22amp cells that subverted EGFR inhibition and enhanced sensitivity to AZD2811. Intriguingly, we detected enhanced Aurora B activity in Chr22amp cells at basal levels, and surprising impact of AZD2811 on the EGFR pathway. Citation Format: Arran Dokal, Jordi Bertran-Alamillo, Edmund Wilkes, Hilary Lewis, Ana Gimenez-Capitan, Calum Greenhalgh, Ruth Osuntola, Maruan Higazi-Vega, Shona Ellison, Vinothini Rajeeve, Giulia Fabbri, Urszula Polanska, J. Elizabeth Pease, Pedro Rodriguez-Cutillas, Jelena Urosevic, Miguel Angel Molina-Vila, David Britton, Jon Travers. Precision phosphoproteomic analysis in Chr22q11.2 amplified NSCLC cells reveals distinct signaling corruption and response to Aurora kinase B inhibition [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 1107.