Improvement of drug efficacy of polo-like kinase 4 inhibitor in the treatment of uterine leiomyosarcoma

Abstract

Introduction: Uterine leiomyosarcoma (Ut-LMS) is aggressive. Recurrence usually develops in 50 - 70% of patients and effective chemotherapy regime are limited. Polo-like kinase 4 (PLK4) is vital modulator of centriole duplication and cell mitotic progression. Inhibition of PLK4 disrupts mitosis, which leads to polyploidy, further genomic instability, mitotic catastrophe, and cell death. Recently, CFI-400945, a PLK4 inhibitor, has been found effective in preclinical studies in the treatment of several malignancies but its effectiveness on Ut-LMS has not been investigated. In phase I studies, CFI-400945 was well tolerated although toxicity, primarily reversible neutropenia, was observed at higher doses. Ataxia-telangiectasia mutated (ATM) is a key kinase in the DNA damage response pathway. AZD0156, an ATM kinase inhibitor, has recently been proposed to be used with other DNA damaging agents in cancer treatments. We explored the effectiveness of a combined approach with CFI-400945 and AZD0156 in Ut-LMS.

Methods: Ut-LMS cell lines SK-UT-1, SKN and SK-LMS-1 cultured in-vitro were treated with CFI-400945 and AZD0156. PLK4 gene expression in these cell lines was also determined by RT-qPCR assays. Cell proliferation was examined using the sulforhodamine B (SRB) assay while apoptosis was examined by Annexin V-PI and caspase 3/7 assays. Immunofluorescence confocal centrosome quantification and flow cytometry PI cell cycle analysis were used to detect centrosome overduplication and generation of aneuploidy cells. The effect of gene expression in normal uterine smooth muscle cells and Ut-LMS cell lines treated with CFI-400945 was performed by RNA-Seq. DNA damage was detected using neutral comet assay.

Results: Ut-LMS cell lines expressed high levels of PLK4 and were highly sensitive (IC50 in nM range) to CFI-400945. Centrosome overduplication, aneuploidy cells, and cell growth inhibition were observed in a dose-dependent manner, and prominent apoptosis was observed at 48 hours after drug treatment. Gene set enrichment analysis on RNA-Seq data indicated that cells were under genotoxic stress after CFI-400945 treatment while comet assay verified DNA damage. Cell arrest and apoptosis with CFI-400945 was further enhanced by the addition of AZD0156. Combined treatment of CFI-400945 and AZD0156 produced synergistic antineoplastic effects in all three Ut-LMS cell lines, with combination indices <1. DNA damage was also enhanced in the combined treatment group versus the single treatment group.

Conclusion: The PLK4 inhibitor CFI-400945 was a potent treatment for Ut-LMS in vitro. This effect was further augmented when combined with the DNA damage response pathway inhibitor AZD0156. The synergistic effect of these agents is promising, and should be investigated further in the treatment of Ut-LMS