Abstract 1468: MYC-C interacts with Aurora kinase A in Ewing sarcoma cells

Abstract

Abstract EWSR1-ETS fusion genes result in upregulation and high expression of the oncogene MYC-C in Ewing sarcoma (ES). MYC-C is therefore an attractive therapeutic target in these aggressive cancers with limited treatment options. Unsuccessful efforts to target MYC-C proteins directly have resulted in the development of inhibitors to associated stabilizing proteins, such as Aurora kinase A (AURKA), to induce proteasomal degradation1. In this study we test the hypothesis that MYC-C and AURKA interact in ES. Peptide pull-down assays were performed using Strep-Tactin Sepharose beads and N-biotinyl MYC-C peptides. After incubation with AURKA, eluted proteins were analyzed by SDS-PAGE. Intracellular colocalization of proteins was examined by immunofluorescence of TC-32 and SK-N-MC cell lines using confocal microscopy (Zeiss LSM880) and high content imaging (CD7). Cells were stained with anti-MYC-C and anti-AURKA antibodies, and DAPI used to visualize nuclei. DuoLink® proximity ligation assays (PLA; Sigma) were performed on TC-32 and SK-N-MC cell lines. Images were captured on the confocal microscope and the number of nuclear foci quantified.MYC-C binds AURKA through its transcriptional activation domain (residues 1-155). Residues 10-34 of MYC-C are sufficient for binding with AURKA (residues 122-403) in cell-free peptide assays. Colocalization of MYC-C and AURKA was visualized in mitotic SK-N-MC cells, but not in TC-32 cells. The proteins colocalize at the spindle poles. PLA confirmed an interaction between MYC-C and AURKA in TC-32 and SK-N-MC ES cells. The number of foci per nucleus was significantly higher in the SK-N-MC p53-null cells compared to the wild-type p53 TC-32 cells (p<0.0001). In conclusion, MYC-C and AURKA colocalize and interact in the nucleus of ES cells. The frequency of interactions is greater in p53-null cells compared to cells with wild-type p53. Peptide pull-down assays identified residues 10-34 of MYC-C as the minimal AURKA interacting region. Disruption of this interaction by AURKA inhibitors may provide a mechanism for MYC-C degradation in ES, which could be exploited to develop novel therapeutic combinations to improve outcomes. 1Richards MW, Burgess SG, Poon E, Carstensen A, Eilers M, Chesler L, Bayliss R. Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc Natl Acad Sci U S A. 2016 ;113(48):13726-13731. Citation Format: Molly McNae, Selena G. Burgess, Richard Bayliss, Susan A. Burchill. MYC-C interacts with Aurora kinase A in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1468.