The Poly (ADP-ribose) polymerase inhibitor olaparib and pan-ErbB inhibitor neratinib are highly synergistic in HER2 overexpressing epithelial ovarian carcinoma in vitro and in vivo

Abstract

Introduction: Ovarian cancer (OC) is associated with the highest gynecologic cancer mortality. The development of novel, effective combinations of targeted therapeutics remains an unmet medical need. We evaluated the preclinical efficacy of the Poly (ADP-ribose) polymerase (PARP) inhibitor (olaparib) and the pan-ErbB inhibitor (neratinib) as single agents and in combination in ovarian cancer cell lines and xenografts with variable HER2 expression.

Methods: In vitro cell viability with olaparib, neratinib, and their combination was assessed using flow-cytometry based assays against a panel of OC primary cell lines with variable HER2 expression. Immunoblotting experiments were performed to elucidate the mechanism of activity and synergism. The in vivo antitumor activity of the olaparib/neratinib combination versus single agents was tested in HER2 positive xenograft OC models.

Results: HER2 + OC cell lines demonstrated higher sensitivity to olaparib and neratinib when compared to HER2 negative tumors (i.e., IC₅₀: 2.06 ± 0.33 μM vs. 39.28 ± 30.51 μM, p = 0.0035 for olaparib and 19.42 ± 2.63 nM vs. 235.0 ± 165.0 nM, p = 0.0035 for neratinib). The combination of olaparib with neratinib was more potent when compared to single-agent olaparib or neratinib both in vitro and in vivo, and demonstrated synergy in all primary HER2 + OC models. Western blot experiments showed neratinib decreased pHER2/neu while increased Poly(ADP-ribose) (PAR) enzymatic activity; olaparib increased pHER2/Neu expression and blocked PAR activatio. Olaparib/neratinib in combination decreased both pHER2/Neu as well as PAR activation.

Conclusion: The combination of olaparib and neratinib is synergistic and endowed with remarkable preclinical activity against HER2+ ovarian cancers. This combination may represent a novel therapeutic option for ovarian cancer patients with HER2+, homologous recombination-proficient tumors resistant to chemotherapy.

Keywords: HER2; Neratinib; Olaparib; Ovarian cancer; PARP inhibitors.

Figure 1.

Cell viability assay of the four epithelial ovarian cancer cell lines with high HER2 expression. Four cell lines with high HER2 expression were treated with olaparib, neratinib, and the combination of both at the indicated concentration for 72 hours. Cell viability was analyzed by flow cytometry and was normalized to the mean of the control group receiving no drug. One-way ANOVA was used to determine the statistical significance of the effects of the combination treatment on ovarian cancer cell lines in vitro when compared to the two single-agent treatment. The one-way ANOVA was corrected with the Bonferroni’s statistic for comparing each group with each other. (*p < 0.05, **p < 0.01, and ***p < 0.005)

Figure 2.

Western blotting analyses after the treatment with olaparib, neratinib, or the combination of olaparib and neratinib. A representative cell line was treated with olaparib (0.6 μM), neratinib (6nM), and the combination of olaparib (0.6 μM) and neratinib (6nM) after 48–72 hours.

Figure 3.

In vivo antitumor activity of the combination of olaparib and neratinib compared to single-agent olaparib and neratinib. Mice were treated with olaparib (50mg/kg), neratinib (20mg/kg) or the combination of olaparib and neratinib for 60days. The mice treated with combination showed a statistically significant difference in tumor growth inhibition compared to the mice treated with single-agent olaparib or neratinib (A). A statistically significant difference in tumor growth inhibition of the combination (olaparib + neratinib) compared to olaparib single-agent treatment was detected on day 22 and beyond. (p = 0.0350 on day 22). A statistically significant difference in tumor growth inhibition of the combination compared to neratinib began on day 36 and continued as in vivo experiment continued until day 47 (p = 0.0463 on day 36 and p = 0.0001 on day 47). The mice treated with the combination of olaparib and neratinib had a significantly longer overall survival when compared to control mice (p = 0.0005), mice treated with olaparib (p = 0.0005), mice treated with neratinib (p = 0.0013) (B). The mice treated with the combination treatment tolerated the treatment well (C).