(P 39) BUB1 AN OVEREXPRESSED KINASE IN SARCOMA: FINDING NEW TARGET THERAPY FOR OSTEOSARCOMA, LIPOSARCOMA, SYNOVIAL SARCOMA AND LEIOMYOSARCOMA

Objective: This study aimed to identify and validate common overexpressed kinases in osteosarcoma, liposarcoma, synovial sarcoma, and leiomyosarcoma, to establish novel therapeutic targets.

Methods: This work was developed in two steps:
First, we performed a bioinformatics workflow utilizing several data mining methods through the R programming language (4.1.1), the Bioconductor environment (3.13), and gene expression data sets. To establish the differentially expressed (DE) kinase genes in each type of sarcoma, we employed the DESeq2 package. Then, we developed a comparative analysis using the VennDiagram R package to establish common kinases with overexpression between the four sarcomas analyzed. The transcriptional regulation of BUB1 kinase was investigated, first determining its interaction with gene expression regulatory molecules such as transcription factors, mRNA-interacting molecules, and non-coding RNAs (microRNAs and long non-coding RNAs). Later, BUB1 interactions with transcription factors, RNA binding proteins, and long noncoding RNAs were tested using RNAinter.
The second step consisted of experimental validation. For that, the cell lines HTB-86 (U-2 OS), HTB-92 (SW-872), HTB-88 (SK-LMS-1), and HTB-93 (SW-982), derived from osteosarcoma, liposarcoma, leiomyosarcoma, and synovial sarcoma, respectively, were obtained from ATCC. Dental Pulp Stem Cells (DPSC) were used as non-tumoral mesenchymal cells. Total RNA and protein extracts were obtained from the cells to validate the BUB1 levels. To demonstrate functional inhibition of BUB1 kinase activity, the sarcoma cell lines were treated with the inhibitor 2OH-BNPP1. Phosphorylation levels of H2A and AKT (BUB1 targets previously reported) were evaluated. The biological implication of BUB1 activity was tested by colony formation and proliferation assays. Finally, an Overall Survival (OS) analysis was performed to explore the relation of BUB1 levels with the OS of sarcoma patients.

Results: In this work, we used gene expression databases and bioinformatic tools to identify the overexpressed kinases in the four most frequent types of sarcomas: osteosarcoma, synovial sarcoma, liposarcoma, and leiomyosarcoma. Then, over-expressed (OE) kinases shared in all analyzed sarcomas were determined. BUB1 kinase was identified as a key enzyme across all four types of sarcomas analyzed. The interaction network for BUB1 expression regulation was predicted and a gene set enrichment analysis was performed to determine biological implications. BUB1 interactions suggest a potential role in promoting cell proliferation and invasion. Signaling pathways that regulate malignancy and pro-gression of STS and BS were the most represented. Using sarcoma cell lines and non-tumoral mesenchymal cells, the gene expression and protein levels of BUB1 were measured. Pharmacological BUB1 inhibition assays showed an implication in cell pro-liferation of this kinase. Finally, a correlation between high levels of BUB1 and poor overall survival in patients of all analyzed sarcomas was determined.

Conclusion: This study highlights the overexpression of effector molecules such as kinases in the four most frequent types of sarcomas. Our analysis allowed us to identify a molecule that clearly could be a target of pharmacological inhibition in the treatment of both, bone and soft tissue sarcomas. This strengthens the idea of the use of target therapies focused on the inhibition of BUB1. As perspectives of this work remain the deep analysis of the molecular aspects that modulate the inhibition of this kinase and how it affects the biology of the assayed subtypes of sarcomas.