Abstract 5394: Uterine leiomyosarcoma with homologous recombination deficiency is highly sensitive to polo-like kinase 4 inhibitor
Authors: Chong Tsz Yan , Lee Horace HY , Yam Judy WP , Chow Kin Long , Wong Ho Shing , Tse Ka Yu , Bray Mark R. , Mak Tak Wah , Ip Philip PC 
Published: June 2022

INTRODUCTION: Uterine leiomyosarcoma (LMS) is rare and aggressive and without a well-established cancer-driver. Treatment is mainly surgical because most other therapies have limited benefits. Polo-like kinase 4 (PLK4) is a modulator of centriole duplication that is essential for proper cell division. Its inhibition by the PLK4 inhibitor, CFI-400945 resulted in centriole dysregulation, increased in double-strand DNA damage, and ultimately, mitotic catastrophe and cell death. Recent genomic studies have identified a subset of LMS with homologous recombination (HR) repair deficiency. We hypothesize that HR-deficient LMS is highly sensitive to CFI-400945.

METHODS: Gene knockouts (KO) were done by CRISPR-Cas9 system. Plasmids encoding the BRCA1- and BRCA2-targeting guide RNA, Cas9 protein, and selection markers were generated and were transfected into SK-UT-1 and SKN followed by selection with antibiotics. The KO and wild-type cell lines were treated with CFI-400945 in vitro. Percentage apoptosis was evaluated by Annexin V-PI staining and cell proliferation was examined by sulforhodamine B assay. To determine whether BRCA1- and BRCA2-KO had any effects on double-strand DNA damage, the γH2AX foci were studied by immunofluorescence staining.

RESULTS: CFI-400945 caused a greater degree of apoptosis and less cell viability in the KO than in the wild-type cell lines. In SK-UT-1, quantity of apoptotic cells for BRCA2-KO and BRCA1-KO were 77% and 34%, respectively, versus 27% in the wild-type cell line (p&lt;0.05). Less dramatic findings were observed in SKN with respective percentages of 20% and 25% versus 15% (p&lt;0.05). In SK-UT-1, the mean IC50 for BRCA2-KO and BRCA1-KO were 3.4 nM and 5.5 nM, respectively, versus 7.8 nM in the wild-type cell line (p&lt;0.05). Similar observations were noted in SKN, with respective mean IC50 of 6.5 nM and 7.1 nM versus 10.0 nM (p&lt;0.05). After CFI-400945 treatment, the γH2AX/Hoechst 33342 staining intensity ratio was 0.40 in SK-UT-1 BRAC2-KO and 0.44 in BRCA1-KO, where both were higher than in the corresponding wild-type cell line with 0.29 (p&lt;0.05). Similarly, the intensities in the two SKN KO cell lines were 0.29 and 0.31, respectively, and higher than in the wild-type cell line with 0.19 (p &lt;0.05).

CONCLUSIONS: PLK4 inhibitor induced DNA double-strand breaks in LMS and the repair of these breakages was dependent on HR repair. In vitro, LMS with BRCA1 or BRCA2 deficiencies were more sensitive to the effect of PLK4 inhibitor.

Citation Format: Tsz Yan Chong, Horace HY Lee, Judy WP Yam, Kin Long Chow, Ho Shing Wong, Ka Yu Tse, Mark R. Bray, Tak Wah Mak, Philip PC Ip. Uterine leiomyosarcoma with homologous recombination deficiency is highly sensitive to polo-like kinase 4 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5394.