Monopolar spindle 1 (MPS1) kinase inhibitor abrogated the spindle assembly checkpoint which resulted in insurmountable genome instability and cell death in uterine leiomyosarcoma

Abstract

Uterine leiomyosarcoma (LMS) is a rare and aggressive mesenchymal tumor. Management is problematic because its symptoms are similar to leiomyoma, and preoperative diagnostic tests are not accurate. Treatment is mainly surgical because other therapies are ineffective. According to The Cancer Genome Atlas, monopolar spindle 1 (MPS1) kinase is a key spindle assembly checkpoint gene that was overexpressed in uterine LMS. Its overexpression signified an impaired spindle assembly checkpoint and was also associated with poor overall survival. MPS1 is a crucial regulator of spindle assembly checkpoint which safeguards chromosome segregation through recruitment of downstream proteins to halt mitosis in the presence of unattached kinetochore. CFI-402257 is a potent MPS1 inhibitor that has been the subject of a few preclinical studies and occasional clinical trials. In this project, we investigated the in vitro and in vivo effects of this drug on uterine LMS cell lines. Since MPS1 was reported to influence DNA damage response in a few cancers, we also investigated whether CFI-402257 exerted similar effects on uterine LMS cell lines. To facilitate the future development of CFI-402257 as targeted therapy for uterine LMS, we explored the drug-induced mechanistic effects of this inhibitor on cellular pathways. The in vitro experiments were conducted on three LMS cell lines, SKUT1, SKN, and SKLMS1. The in vivo effects were studied by using a BALB/cAnN-nu (nude) mice model through subcutaneous injection of tumor cells. RNA sequencing was performed on pre- and post-drug treatment cells lines and pathfindR was used to perform pathway enrichment analysis. The sulforhodamine B assay showed that CFI-402557 was able to inhibit cell proliferation with IC50 values of 45 nM, 64 nM, and 193 nM in SKUT1, SKN and SKLMS1, respectively. Immunofluorescence imaging studies revealed chromosome missegregation and formation of micronuclei that characterizes mitotic catastrophe. CFI-402257 treatment induced aneuploidy in the propidium iodide assay, and an increase in apoptosis was observed through AnnexinV/Propidium iodide assay. A significantly reduced tumor size was observed in vivo. CFI-400257 caused double-stranded DNA damage as demonstrated by neutral comet assay, γH2AX foci formation, and elevated γH2AX protein levels. While the role of MPS1 in DNA damage repair remains controversial, we have demonstrated that MPS1 silencing did not affect CHK2-mediated DNA damage response in uterine LMS. RNA sequencing and pathway enrichment analysis indicated that several widely studied signaling pathways were upregulated through CFI-400257 treatment. Of these, drug-induced alterations were significantly enriched in the NF-κB signaling pathway. Most notably, IL-8 and IL-1β upregulation were striking. These findings provided us with new directions to further investigate the therapeutic potential of CFI-402257 as a single drug or use as a combination therapy with other agents. In conclusion, CFI-402257 demonstrated its ability as a potent inhibitor of uterine LMS cell lines in vitro and in vivo. CFI-402257 abrogated the spindle assembly checkpoint which resulted in insurmountable genome instability and cell death. The drug also induced double-stranded DNA breaks and upregulated NF-κB signaling pathway genes expression.