Abstract C49: Using single cell fluorescent cell cycle reporters to study mechanisms of action of selective inhibitors of nuclear export (SINE).
Authors: Marcus Joshua , Landesman Yosef , Shacham Sharon , Orth James 
Published: August 2014

SINE are novel small molecule drugs in phase I clinical trials for advanced cancers. SINE inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1), inducing forced nuclear retention of major tumor suppressor proteins including p53, p21, and pRb, resulting in selective cancer cell death. In contrast, non-transformed cells show only a transient reduction of S-phase and once drug is removed, they resume proliferation. In order to study SINE drug effects in individual cells we developed a panel of solid tumor-derived cell lines that stably express dynamic fluorescent ubiquitin cell cycle reporters, and followed individual cell responses and cell fate using continuous fluorescent and phase-contrast microscopy. We report that in fibrosarcoma-derived HT-1080 cells treated with ≥100nM SINE KPT-330 (EC50 100nM) for 48 hours, ≈30% of cells showed a penetrance of cell cycle arrest with &gt;80% in G1- or S-phase and ≤20% in G2-phase. These arrested cells were negative for the proliferation antigen Ki67. By 48 hours, ≈25% of dying cells were from interphase, where &gt;80% died from G1- or S-phase [[Unable to Display Character: &#8211;]] and not from G2-phase. Direct analysis showed that cell division (mitosis) occurred in &gt;30% of the treated population. Of these mitotic cells, a small number died while in mitosis, while the rest divided. The duration of cell division was &gt;2-fold longer than normal and we observed chromosome missegregations. Lineage tracking of the daughter cells from these abnormal divisions indicated that ≈50% remain in interphase, mostly in G1- or S-phase, while the remainder progress to cell death, also mainly from G1- or S-phase. In contrast, non-transformed retinal pigment epithelial cells showed very little death, and instead remained in prolonged interphase. At 48 hours, cancer cells lacking p53 showed no difference in cell death, however we observed more dividing cells compared to control. These data indicate that p53 does not affect immediate cell death, but it does have a role in SINE mediated cell cycle arrest. We conclude that SINE drugs are highly effective at arresting cell growth and inducing apoptosis in tumor cells, with most cell death occurring in G1- or S-phase. Importantly, cells that overcome cell cycle arrest and divide are so severally compromised that they either die while in mitosis or the daughter cells subsequently arrest or die. Taken together, KPT-SINE may exert anti-cancer effects by perturbing both interphase and cell division.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C49.

Citation Format: Joshua Marcus, Yosef Landesman, Sharon Shacham, James Orth. Using single cell fluorescent cell cycle reporters to study mechanisms of action of selective inhibitors of nuclear export (SINE). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C49.