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.

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.

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1 amplification to paclitaxel in vitro by targeting APC/C and the pro-survival protein MCL-1.

Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells.

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells.

Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.

Mitotic arrest and slippage induced by pharmacological inhibition of Polo-like kinase 1.

Exposure to drugs that interfere with microtubule dynamics block cell cycle progression at mitosis by prolonged activation of the spindle assembly checkpoint (SAC). Cells can evade mitotic arrest and proceed to interphase without chromosome segregation by a process termed mitotic slippage that involves Cyclin B1 degradation without checkpoint inactivation. Here, we explored the cellular response to small-molecule inhibitors of Polo-like kinase 1 (Plk1), an important regulator of cell division. We found that the clinical Plk1 inhibitors BI 2536 and BI 6727, both unexpectedly, induced a dose-dependent cellular drug response: While mitotic arrest was induced in cancer cell lines and primary non-transformed cells across the entire range of concentrations tested, only high concentrations seemed to promote mitotic slippage. Since this observation contrasts with the effects expected from studies reporting RNAi-mediated Plk1 depletion in cancer cells, we wondered whether both ATP-competitive inhibitors target unknown kinases that are involved in signaling from the spindle assembly checkpoint (SAC) and might contribute to the mitotic slippage. A chemical proteomics approach used to profile the selectivity of both inhibitors revealed that SAC kinases are not targeted directly. Still, the activities of Cdk1/Cyclin B1 and Aurora B, which plays important roles in the error correction of false microtubule-kinetochore attachments and in checkpoint signaling, were shown to be downregulated at high inhibitor concentrations. Our data suggest that the inhibition of Plk1 activity below a certain threshold influences Aurora B activity via reduced phosphorylation of Fox M1 and Survivin leading to diminished levels of Aurora B protein and alteration of its subcellular localization. Within the spectrum of SAC proteins that are degraded during mitotic slippage, the degradation of Cyclin B1 and the downregulation of Aurora B activity by Plk1 inhibition seem to be critical promoters of mitotic slippage. The results indicate that careful dose-finding studies in cancer trials are necessary to limit or even prevent mitotic slippage, which could be associated with improved cancer cell survival.

Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells.

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.

Downregulation of Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles.

Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.

Down-regulation of Polo-like kinase 1 elevates drug sensitivity of breast cancer cells in vitro and in vivo.

Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.

Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1.

The cyclin-dependent kinase 1 (Cdc2)/cyclin B1 complex performs cardinal roles for eukaryotic mitotic progression. Phosphorylation of four serine residues within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at the G(2)/M transition. Still, the role of individual phosphorylation sites and their corresponding kinases remain to be elucidated. Polo-like kinase 1 (Plk1) shows a spatial and temporal distribution which makes it a candidate kinase for the phosphorylation of cyclin B1. We could demonstrate the interaction of both proteins in mammalian cells. Plk1 phosphorylated wild-type cyclin B1 expressed in bacteria and in mammalian cells. Ser-133 within the cytoplasmic retention signal (CRS) of cyclin B1, which regulates the nuclear entry of the heterodimeric complex during prophase, is a target of Plk1. In contrast, MAPK (Erk2) and MPF phosphorylate Ser-126 and Ser-128 within the CRS. Phosphorylation of CRS by MAPK (Erk2) prior to Plk1 treatment induced enhanced phosphorylation of cyclin B1 by Plk 1 suggesting a synergistic action of both enzymes towards cyclin B1. In addition, pretreatment of cyclin B1 by MAPK (Erk2) altered the phosphorylation pattern of Plk 1. Mutation of Ser-133 to Ala decreased the phosphorylation of cyclin B1 in vivo. An immunofluorescence study revealed that a mutation of Ser-133 reduced the nuclear import rate of cyclin B1. Still, multiple serine mutations are required to prevent nuclear translocation completely indicating that orchestrated phosphorylation within the CRS triggers rapid import of cyclin B1.