Uterine leiomyosarcoma with homologous recombination deficiency is highly sensitive to polo-like kinase 4 inhibitor

Abstract

Uterine leiomyosarcoma (LMS) is a rare and aggressive smooth muscle neoplasm with poor prognosis. Most current treatment methods are ineffective. Recent genomic profiling studies have identified frequent loss-of-function alterations in homologous recombination (HR) genes, in particular, BRCA1 and BRCA2 which are well-known tumor suppressor genes responsible for the repair of DNA double-strand breaks (DSBs). Thus, tumors with this BRCAness phenotype are more vulnerable to DNA damaging agents. Polo-like kinase 4 (PLK4) precisely modulates mitosis and its inhibition could lead to chromosome missegregation and increase in DNA damage. We have previously demonstrated that PLK4 was overexpressed in LMS and affirmed that it is a potential therapeutic target. PLK4 inhibitor, CFI-400945, is an oral drug and its use has been shown to be successful in the treatment of several malignancies in preclinical studies and a few clinical trials. We hypothesized that CFI-400945 was also effective in the treatment of LMS and those with HR deficiency are even more sensitive to this drug. Even though a significant proportion of clinical LMS cases are HR-deficient, the commercially available LMS cell lines are all BRCA1/2-proficient. In order to study the effect of CFI-400945 in HR-deficient LMS, we generated BRCA1 and BRCA2 gene knockouts from the wild-type cell lines SKUT1 and SKN by CRISPR gene editing system. Successful knockouts were confirmed by Sanger sequencing and validated by Western blot. Both the wild-type cell lines and the BRCA1/2-KO models were treated with either CFI-400945 or DMSO as control, and the in-vitro effects were compared. Cell proliferation was evaluated by sulforhodamine B assay, and apoptosis and cell death were analyzed by Annexin V and propidium iodide flow cytometry. Drug-induced DNA damage was assessed and quantified by γH2AX immunofluorescence staining, and DNA repair by an I-SceI-based DSB repair reporter assay. In the wild-type cell lines, CFI-400945 effectively suppressed cell proliferation, induced apoptotic cell death, generated DNA DSBs, and activation of HR repair. In the BRCA1- and BRCA2-deficient cell lines, there was more significant proliferation suppression, greater degree of DNA damage, and more cell death. The increased in drug-sensitivity was explained by results of the DSB repair reporter assay. In the wild-type cell lines, DNA damage induced by CFI-400945 was repaired by HR as well as non-homologous end joining (NHEJ) mechanisms. In the BRCA1- and BRCA2-deficient cell lines, both HR and NHEJ repair was significantly suppressed. The impairment of DNA repair by two major mechanisms resulted in accumulation of DNA damage to an intolerable level, leading to a greater degree of cell death. In conclusion, BRCA1- and BRCA2-deficient LMS were more susceptible to the anti-tumor effects of CFI-400945. In a newly diagnosed LMS, genomic analysis of BRCA1/2 status would facilitate the selection of most suitable patients for treatment with CFI-400945.