Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer

Abstract

Objective: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer.

Methods: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model.

Results: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability.

Conclusion: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Keywords: Entinostat; Olaparib HR-proficient ovarian cancer.

Figure 1.. Olaparib combined with entinostat decreases viability and clonogenicity in HR-proficient ovarian cancer cells.

(A) Cell proliferation and combination index: Cells (SKOV-3, OVCAR-3, patient derived primary cells) were pre-treated with 0.25 μM entinostat or media for 24 h followed by 0.1, 0.25, 0.5, 1 or 2 μM Ent and/or 2.5, 5, 10, 20 or 40 μM Ola (alone or combination) for 72 h followed by SRB assay to measure cell proliferation. All cell lines and primary cells showed enhanced inhibition of cell viability in Ola+Ent treated groups compared to Ola or Ent alone. Combination index (CI) showed synergy (CI value <1) between Ent and Ola at all concentrations, in all cell lines tested, except two earlier concentrations for OVCAR-3. Strong synergy was seen at all concentrations in SKOV-3 cells and several combinations in other cells (CI value <0.3). CI was calculated by Chou-Talalay method. (B) Clonogenicity: 500 SKOV-3 cells/well were plated overnight on 6 well plates. Cells were pre-treated with 0.25 μM Ent for 24 h and subsequently treated with 0.5 μM Ent or 10 μM Ola for 24 h. Drugs were replaced by regular media, cells allowed to grow until colonies formed and stained with crystal violet using standard procedure. Quantification of clonogenic data was carried out using ImageJ and graph plotted for each treatment as percent of control. Difference between control and each treatment was significant, with larger difference between Con and Ola+Ent. **p < 0.001; ***p<0.0001; ****p<0.00001; determined by one-way ANOVA, Sidak’s multiple comparisons. [Con – Control; Ola – Olaparib; Ent – Entinostat].

Figure 2.. Olaparib combined with entinostat decreases peritoneal tumor load in HR-proficient SKOV-3-IP-luc xenograft and increases survival in CCNE1 amplified HGSOC HR-proficient PDX mice.

(A) SKOV-3 xenograft treatment timeline. (B) SKOV-3-xenograft in NOD-SCID mouse model: Bioluminescent imaging showing peritoneal tumor spread in four treatment groups (Control, 100 mg/kg olaparib, 15 mg/kg entinostat, combination of olaparib+entinostat) on day 29. Total flux value (indicative of peritoneal tumor spread) was plotted for each treatment group at weekly intervals and water fall plot for day 29 shows slower tumor progression in combination treatment group. Statistical significance determined using 2-way ANOVA multiple comparisons test (*p < 0.0170). (C) PDX induction and treatment timeline. (D) Large amount of ascites (red asterisk) and tumor nodules around liver yellow arrows) and omentum (white arrow) developed in NOD-SCID mice injected intraperitoneally with patient derived tumors. (E) H&E staining of tumor section and immunohistochemistry showing positive expression of p53, PAX8 and WT1, indicative of high-grade serous carcinoma (20X, 100micron scale bar). (F) Copy caller assay indicating the tumor was CCNE1 amplified. (G) PDX mouse model pre-treated with 15 mg/kg entinostat or vehicle for one week, followed by two-week treatment with vehicle, 100 mg/kg olaparib or 15 mg/kg entinostat or olaparib+entinostat showed that treatment with combination significantly improved the median survival (*p<0.0325, Log-rank (Mantel-Cox) test). [Con – Control; Ola – Olaparib; Ent – Entinostat].

Figure 3.. Olaparib combined with entinostat causes significant DNA damage in HR-proficient ovarian cancer cells.

(A) γ-H2AX foci localization in SKOV-3 cells showed high number of foci in Ola treated cells, whereas merged foci were seen in Ola+Ent treated cells, indicative of extensive DNA damage. Statistical analysis showed enhanced foci in Ola+Ent (60X, 50-micron scale bar; *p<0.01; **p< 0.001; ***p,0.001; ****p<0.0001; one-way ANOVA; Sidak’s multiple comparison tests). (B) HR-proficient ovarian cancer cells pre-treated with 0.25 μM Ent for 24 h followed by 0.5 μM Ent or 10 μM Ola alone or Ola+Ent for 24 h were processed for comet assay to determine DNA damage. Both SKOV-3 and OVCAR-3 showed significant DNA damage with Ola and even greater extent of DNA damage with Ola+Ent (*p<0.01; **p< 0.001; ***p,0.001; ****p<0.0001; one-way ANOVA; Sidak’s multiple comparison tests). Fluorescent images show the classic comet tails indicative of DNA damage and the graphs show the relative length of comet tails for individual cells. [Con – Control; Ola – Olaparib; Ent – Entinostat].

Figure 4.. Olaparib combined with entinostat decreases proliferation, alters HR machinery and increases apoptosis in HR-proficient SKOV-3 cells.

(A). Immunofluorescence showing that Ola+Ent significantly reduces BRCA1 and Ki-67 expression levels in SKOV-3 cells. DAPI was used as nuclear stain (40X 100 micron scale bar). Quantification of BRCA1 and Ki-67 indicated significant downregulation in cells treated with Ola+Ent (*p < 0.05, **p<0.005 Students T-test). (B) BRCA1 transcript levels showed significant downregulation in all treatment groups, with enhanced downregulation in Ola+Ent (*p < 0.012; **p<0.0036; ***p<0.0005, one-way ANOVA; Sidak’s multiple comparison test) indicating that BRCA1 expression is affected markedly at the mRNA levels compared to protein levels. (C) Western blot analysis of SKOV-3 cells treated with Ola+Ent as described for SRB assay showing increased cleaved PARP expression in olaparib and Ola+Ent treated cells. Quantification showed significant upregulation of cleaved PARP, a marker of cell death (*p< 0.05; **p<0.005, Students T-test). The antibody used was specific for fragments of PARP1. [Con – Control; Ola – Olaparib; Ent – Entinostat].

Figure 5.. Entinostat slows replication fork progression in HR-proficient SKOV-3 cells.

A) Experimental scheme for DNA fiber assays and representative images for each condition. B) Dot plot and median values (purple line) of total tract lengths (IdU+CldU) upon mock treatment or treatment with Ola (10 μM), Ent(0.5 μM), or combination of Ola+Ent. Ent and Ola+Ent combination treatment lead to decreased fork progression relative to Con and Ola treated SKOV-3 cells (n=4; ****P<0.0001; Kruskal-Wallis test followed by Dunn’s multiple comparison test). [Con – Control; Ola – Olaparib; Ent – Entinostat].

Figure 6.. Graphical summary indicating the mechanism for entinostat-induced olaparib sensitivity in HR-proficient cells.

As described, entinostat causes breakdown of HR-DNA repair pathway, whereas olaparib inactivates PARP, crippling the cell’s potential to repair SSBs. This in turn leads to accumulation of DSBs, which the cell is unable to repair due to defective HR repair pathway, accumulating unrepaired DNA ultimately resulting in cell death.