Cell Cycle Specific Effects And Associated Dna Damage Of Selective Inhibitors Of Nuclear Export (Sine)

Nuclear export of proteins is fundamental for cell growth and function. Selinexor is a SINE compound that is in clinical development for the treatment of different cancers. Selinexor forms a slowly reversible covalent bond to Exportin-1 (XPO1), preventing its association with protein cargos, thereby resulting in their nuclear retention. XPO1 cargos include the majority of tumor suppressor proteins (TSP) and cell cycle regulators such as p53, p21 and p27 that have key roles in cancer progression and drug response. It is unclear how selinexor affects cell cycle progression in individual cells and the subsequent stress and fate of those cells. To elucidate selinexor action, we developed cell lines that stably express fluorescent ubiquitin cell cycle reporters (FUCCI), and followed individual cells longitudinally using continuous time-lapse microscopy for 72 hours. We report that in fibrosarcoma-derived HT-1080 cells that express wildtype p53 and p21, 18% of the initial cell population became arrested with u003e90% in G1- or S-phase and 40% died with 64% from G1- or early S-phase after a cell cycle delay or arrest. We also found that 42% of cells divided, but the progeny died or arrested in G1- or S-phase of the next cell cycle - often after cell cycle arrest or slowed cell cycle progression. Using FUCCI, we tracked the response of cells treated acutely in specific cell cycle stages. Cells treated in G1-phase most often arrested or died in G1- or S-phase, whereas cells treated in G2-phase usually progressed to cell division. As FUCCI revealed S-phase progression defects and associated cell death, we further characterized this phenotype. Using nucleotide incorporation with fluorescent detection, we observed that as early as 2 hours after selinexor treatment only a subset of cells is undergoing DNA replication. Cells that were able to replicate their DNA are doing that inefficiently as both the rate and maximal levels of nucleotide incorporation are significantly reduced. S-phase arrest and progression defects may manifest as DNA double-strand breaks. We find a strong association between S-phase status and DNA damage. In some cells, the damage occurs within hours of selinexor treatment and appears as a striking cluster of foci. At 8 hours, nearly 35-45% of cells contain DNA damage clusters. Importantly, the damage clusters sometimes repair as determined by fixed cell time-course analysis and live-cell microscopy. We are exploring the nature of these DNA damage structures and the mechanisms of their formation and repair. In summary, selinexor is fast acting, shows cell cycle selectivity, results in DNA damage and is highly effective at arresting cell growth and inducing apoptosis in tumor cells. These data suggest that selinexor may exert anti-cancer effects even on slow growing tumors where the bulk of the cell mass presents a G1-like state and that it likely combines well with other cell cycle targeted therapeutics. Citation Format: Russell T. Burke, Joshua Marcus, John DeSisto, Yosef Landesman, James D. Orth. Cell cycle specific effects and associated DNA damage of selective inhibitors of nuclear export (SINE). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4645.