DECIPHERING THE ROLE OF AURORA KINASE A IN HUMAN RETINOBLASTOMA AND ITS THERAPEUTIC TARGETING IN-OVO IN A CHORIOALLANTOIC MEMBRANE XENOGRAFT MODEL

Abstract

Purpose : Chemotherapy is a primary treatment approach for the pediatric intraocular neoplasm retinoblastoma (RB). The immediate and long-term side effects of current chemotherapeutic drugs necessitate the development of new treatment strategies. Aurora Kinases have emerged as promising therapeutic targets in cancers and their inhibitors are currently in clinical development. However, their role in RB remains elusive. Here, we investigated the role of Aurora Kinase A (AURKA) in RB tumorigenesis and progression using in-vitro and in-ovo models.

Methods : The expression status of AURKA was determined in 60 archived patient tissue specimens using immunohistochemistry. AURKA was targeted in RB cells (Y79, WERI-RB1, patient derived LRB1, LRB2) with small molecule inhibitors (MLN8237, MK8745, VX689) and shRNA-mediated lentiviral knockdown. Cell viability, cell cycle and apoptosis analyses were performed with the inhibitor-treated and knockdown cells. AURKA protein levels in RB cells with MYCN knockdown were estimated using immunoblotting. The binding of MYCN on the AURKA promoter was validated with Chromatin Immunoprecipitation (ChIP)-qPCR. A xenograft model of RB was created by implanting cells on the chorioallantoic membrane (CAM) of the developing chick embryo and AURKA inhibition was studied.

Results : In the immunohistochemistry study, 43 out of 60 (71.6%) patient specimens showed expression of AURKA. Inhibition of AURKA with small molecule inhibitors and shRNA-mediated knockdown led to – decreased cell viability, increased apoptosis, cell cycle arrest at the G2/M phase and subsequent induction of polyploidy. Decreased levels of AURKA were detected in RB cells with MYCN knockdown. ChIP-qPCR confirmed the enrichment of MYCN binding sites at -510 and -718 bases upstream of the AURKA transcription start-site. In the in-ovo study, treatment with AURKA inhibitors led to a significant reduction in tumor weights. This was corroborated by an increase in p53 expression, indicating induction of apoptosis.

Conclusions : AURKA is overexpressed in RB, could be specifically targeted and is regulated by MYCN. In the in-ovo study, AURKA inhibition hampered tumor growth while simultaneously inducing apoptosis. Overall, our data suggest that AURKA is a potential therapeutic target for limiting RB tumor growth and further validations could lead to innovative treatment avenues.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.