Abstract 2312: Phospho-proteomics analysis to determine the signaling pathways affected by a novel CDC7-selective inhibitor TAK-931

Abstract

Abstract Background: Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. We developed the CDC7-selective small molecule inhibitor TAK-931. Treatment with TAK-931 results in delayed S phase progression, DNA replication stress (RS), centrosome disorganization, mitotic aberrations, and potent growth suppression in cancer cells. In this study, we conducted the time-dependent phospho-proteomics analysis to determine the signaling pathways affected by TAK-931. Materials and Methods: TAK-931 was synthesized by Takeda Pharmaceutical Company, Ltd. The SILAC-labeled COLO205 cells were treated with TAK-931 at 100 nM for 0, 4, 24 hours, applying for the phospho-proteomics analysis in the PhosphoScout® Service at Evotec AG (Munich, Germany). Results: The SILAC-labeled COLO205 cells with TAK-931 treatment were subjected to quantitative mass spectrometry, detecting 15,128 phosphorylation sites of the 4,426 phosphoproteins. At 4 h of treatment, only 2 phosphorylation sites were detected as significantly downregulated: MAX gene-associated protein (MGA) and alpha-enolase (ENO1). At 24 h of treatment, however, 51 phosphorylation sites were identified as being significantly altered, including 44 upregulation and 7 downregulation sites. The gene ontology (GO) enrichment analysis in the categories of biological process (BP) and cellular component (CC) revealed that 10 GO terms from BP and 2 GO terms from CC were significantly enriched in the treated cells after 24 h: cell cycle, cell cycle phase, cell cycle process, mitosis, nuclear division, organelle fusion, organelle organization, mitotic cell cycle, M phase of mitotic cell cycle, M phase, chromosomal part, and spindles. Network analysis on the changed phosphorylation prolife also revealed that significant network modules existed at 24 h of treatment, which included DNA replication/damage, mitotic progression/spindle assembly, nucleus assembly, GTPase activity, cell migration, cell adhesion, TP53, and protein translation. Conclusion: The time-dependent phospho-proteomics analysis revealed the dynamics of phospho-signal transduction affected by TAK-931 in the cancer cell, which may help to provide a deeper understanding of the molecular mechanisms of TAK-931. Citation Format: Kenichi Iwai, Masamitsu Gotou, Kazunori Yamanaka, Akihiro Ohashi. Phospho-proteomics analysis to determine the signaling pathways affected by a novel CDC7-selective inhibitor TAK-931 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2312.