Combination treatment using DDX3 and PARP inhibitors induces synthetic lethality in BRCA1-proficient breast cancer

Abstract

Triple-negative breast cancers have unfavorable outcomes due to their inherent aggressive behavior and lack of targeted therapies. Breast cancers occurring in BRCA1 mutation carriers are mostly triple-negative and harbor homologous recombination deficiency, sensitizing them to inhibition of a second DNA damage repair pathway by, e.g., PARP inhibitors. Unfortunately, resistance against PARP inhibitors in BRCA1-deficient cancers is common and sensitivity is limited in BRCA1-proficient breast cancers. RK-33, an inhibitor of the RNA helicase DDX3, was previously demonstrated to impede non-homologous end-joining repair of DNA breaks. Consequently, we evaluated DDX3 as a therapeutic target in BRCA pro- and deficient breast cancers and assessed whether DDX3 inhibition could sensitize cells to PARP inhibition. High DDX3 expression was identified by immunohistochemistry in breast cancer samples of 24% of BRCA1 (p = 0.337) and 21% of BRCA2 mutation carriers (p = 0.624), as compared to 30% of sporadic breast cancer samples. The sensitivity to the DDX3 inhibitor RK-33 was similar in BRCA1 pro- and deficient breast cancer cell lines, with IC50 values in the low micromolar range (2.8-6.6 μM). A synergistic interaction was observed for combination treatment with RK-33 and the PARP inhibitor olaparib in BRCA1-proficient breast cancer, with the mean combination index ranging from 0.59 to 0.62. Overall, we conclude that BRCA pro- and deficient breast cancers have a similar dependency upon DDX3. DDX3 inhibition by RK-33 synergizes with PARP inhibitor treatment, especially in breast cancers with a BRCA1-proficient background.

Keywords: BRCA; Breast cancer; DDX3; DEAD box RNA helicase; DNA damage repair; PARP inhibitor.

Fig. 1

DDX3 expression in BRCA1-related breast cancer. Example of low (a) and high (b) immunohistochemical DDX3 expression in breast cancer occurring in patients with a germline BRCA1 mutation. Scale bar is 20 μm

Fig. 2

Sensitivity of BRCA1 pro- and deficient cell lines to the DDX3 inhibitor RK-33. a Immunoblot showing DDX3 and β-actin expression in BRCA1-proficient cell lines (BRCA1+) and cell lines with mutated BRCA1 (BRCA1−). b MTS assay showing RK-33 cytotoxicity in BRCA1-proficient cell lines and cell lines with mutated BRCA1. Graphs represent mean of independent experiments ± SD

Fig. 3

Synergy between RK-33 and the PARP inhibitor olaparib. a Bar graphs representing the surviving fraction in a colony formation assay after RK-33, olaparib or combination treatment in BRCA1-proficient (MCF7 and MDA-MB-468) and BRCA1-deficient breast cancer cell lines (HCC1937 and SUM149-PT). b CI-Fa plots showing the combination index (CI) and 95% confidence intervals of RK-33 olaparib combination therapy for different fractions affected (Fa) in BRCA1 pro- and deficient cell lines. Lines represent modeled CI curves. Points represent CI values calculated from measured data points. Dashed red lines represent 95% confidence intervals. Blue lines represent additivity reference line. Graphs represent mean of independent experiments ± SD