Sequential Targeting of PLK1 and PARP1 Reverses the Resistance to PARP Inhibitors and Enhances Platin-Based Chemotherapy in BRCA-Deficient High-Grade Serous Ovarian Cancer with KRAS Amplification

Abstract

Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.

Keywords: BRCA2 deficiency; DNA damage; KRAS amplification; PARP inhibitor resistance; PLK1-based combinatorial therapy; high-grade serous ovarian cancer.

Figure 1

KRAS amplification mediates resistance to PARPi in HGSOC cells with BRCA2 deficiency. (A)—Treatment schedule: KURAMOCHI and OVSAHO cells were treated with increasing concentrations of the PARP inhibitor Olaparib, 2 µM to 20 µM. Cells were harvested after 24 h and 48 h, and further experiments were carried out. (B,C) Apoptosis was then assessed at the indicated time points by measuring Caspase 3/7 activity in the lysates of cells. The results are presented as mean ± SD. (n = 3). (D,E) Cell death was assessed by quantifying the sub-G1 phase. The results are presented as mean ± SD. (n = 3). (F,G) The cell cycle distribution of treated KURAMOCHI and OVSAHO was analyzed 24 h and 48 h post-Olaparib treatment using FACS. The resulting G2 fractions of both cell lines are displayed. The results are presented as mean ± SD. (n = 3). (H,I) OVSAHO and KURAMOCHI cell lysates treated as in (A) were prepared for Western blot analysis after 24 h and 48 h using the indicated antibodies. The results of the (B–G) are presented as mean ± SD. (n = 3, p ≤ 0.001, p ≤ 0.01, p ≤ 0.05).

Figure 2

Sequential PARPi and PLKi sensitize BRCA2-deficient HGSOC to Olaparib treatment independent of the KRAS status. (A) Treatment schedule: KURAMOCHI and OVSAHO cells were first incubated with a single treatment consisting of Olaparib (10 µM) or BI67272 (20 nM) and the sequential combinations Olaparib/BI6727 or BI6727/Olaparib at days 1 and 2. The cells were harvested 24 h and 48 h post-treatments, and further experiments were carried out. (B,C) Apoptosis was assessed by measuring Caspase 3/7 activity in cell lysates of cells incubated with the different single or combinatorial treatments. The results are presented as mean ± SD. (n = 3, p ≤ 0.001, p ≤ 0.01, p ≤ 0.05). (D,E) Cell death was then assessed after 24 h and 48 h using Annexin V/AAD. The results are presented as mean ± SD. The total apoptosis (early and late) was used for statistical analysis. (n = 3, p ≤ 0.001, p ≤ 0.01). (F,G) The cell lysates of KURAMOCHI and OVSAHO cells treated with the single and the combination treatments were prepared for Western Blot with the indicated antibodies.

Figure 3

Sequential PARPi and PLKi treatment reduced the 2D clonogenic potential of HR-deficient HGSOC. (A,C) OVSAHO and KURAMOCHI cells grown in colonies were subjected to Coomassie blue staining. (B,D) The number of colonies was counted and is represented as a bar graph. The results are presented as mean ± SD. (n = 3, p ≤ 0.001, p ≤ 0.01).

Figure 4

Combining PLK1i and PARPi enhances the DNA damage effect of MMS and reduces the viability of BRCA2-deficient HGSOC with KRAS amplification. (A)—Treatment schedule: KURAMOCHI cells were EdU labeled and pulse-treated with MMS (0.5 mM) for 1 h. The cells were released in EdU-containing medium for 4 h, then treated with the single agents or combined for 24 h in the presence of EdU. γ-H2AX foci were analyzed in EdU-negative G2 phase cells. (B) EdU-positive cells were identified as S phase cells, whereas EdU-negative cells were classified as G1 (small DNA content) or G2 (large DNA content). (C) IF images show KURAMOCHI cells with γ-H2AX foci and negative EdU staining at 24 h post-treatment. (D) Quantification of γ-H2AX foci 24 h post-treatment. The results are presented as mean ± SEM (n = 50 cells per treatment, p ≤ 0.001). (E) Cell death was assessed in treated KURAMOCHI cells as in (A) by quantifying the sub-G1 phase after 72 h. The results are presented as mean ± SD. (n = 3, p ≤ 0.001). (F) Treatment schedule: KURAMOCHI and OVSAHO cells were pulse-treated with MMS (0.5 mM) for 1 h on day 1. Following this, cells were sequentially treated with Olaparib (5 µM) on day 2 and BI6727 (20 nM) on day 3. Cells were harvested and counted 7 and 9 days upon the completion of the combination treatment. (G,H) Growth kinetics of KURAMOCHI and OVSAHO cells treated with the different single agents and in combinations over 7 and 9 days. The results are presented as mean ± SD. (n = 3, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05).

Figure 5

PLK1i and PARPi mediate sensitivity to HR-deficient HGSOC with KRAS amplification to Carboplatin. KURAMOCHI cells were treated with increasing concentrations of Carboplatin, 1 µM to 20 µM. Cells were harvested after 48 h. Cell lysates were prepared for Western blot using the indicated antibodies (A), and the cell cycle distribution of treated cells was analyzed using FACS (B). (C) Treatment schedule: KURAMOCHI cells were treated with Carboplatin (3 µM) on day 1. Following this, cells were sequentially treated with Olaparib (10 µM) on day 2 and BI6727 (20 nM) on day 3. Cells were harvested 24 h (day 4) and 48 h (day 5) after completion of the combinatorial treatments, and further experiments were carried out. (D,E) Apoptosis was first assessed by measuring Caspase 3/7 activity in cell lysates of cells incubated with the different single or combinatorial treatments and by measuring cell death after 24 h and 48 h using Annexin V/AAD. The results are presented as mean ± SD. (n = 3, p ≤ 0.05). (F) Cell lysates of KURAMOCHI cells treated with single agents or combinations, as in (C), were prepared for Western blot using the indicated antibodies.

Figure 6

PARPi and PLK1i combination strongly enhances apoptosis-dependent death of patients-derived tumor cells. (A–C) Primary tumor cells of patients were treated with increasing concentrations of Olaparib, BI6727, and Carboplatin as single agents or combinations (D–F). The cell viability was determined after 48 h, and the IC₅₀ of the different agents was calculated. (G,H) 3D cultures grown from tumor cells of patients were treated with 10 µM Olaparib, 20 nM BI6727, and 100 µM Carboplatin as single or combinatorial treatments for 8 days. Cells were stained, and the fraction of dead cells was quantified using immunofluorescence. The results are presented as mean ± SD and statistically analyzed (n = 5, p ≤ 0.001, p ≤ 0.01).