PLK1 inhibition enhances gemcitabine-induced apoptosis through PLK1-dependent ERK1/2-Bim and AKT1/Noxa signals in pancreatic cancer cells.

Background: BRCA1/2 mutant tumors cells are deficient for homologous recombination (HR)-mediated DNA repair and are particularly sensitive to PARP inhibitors (PARPi). PARPi have proved efficacy in breast, ovarian, prostate, and pancreatic cancers, particularly in HR-deficient tumors, while their activity is limited in HR-proficient tumors. However, PARPi resistance is inevitable and therapeutic resistance resulting from restoration of HR repair is a pressing clinical problem. Identifying combination treatments to sensitize tumors cells to PARPi and/or overcome PARPi resistance is critical to expand the benefit of these therapies. The Polo-like kinase 1 (PLK1), a serine threonine kinase, is a master regulator of mitosis, overexpressed in many cancers. PLK1 is also involved in the DNA damage response through the promotion of HR-mediated DNA repair and the recovery from the G2/M checkpoint. PLK1 roles in HR repair suggest that PLK1 inhibition may reverse PARPi resistance. Methods: To test the effect of PLK1 and PARP inhibitors combination, we used onvansertib, a highly selective, ATP-competitor PLK1 inhibitor currently in clinical development and the FDA-approved PARPi olaparib. The antitumor effect of the single and combined drug treatments was tested in 2 BRCA1 mutated high-grade serous ovarian cancer (HGSOC) patient derived (PDX) models resistant to olaparib. Orthotopically PDX transplanted mice were treated for 4 weeks and followed for survival. Results: The combination of onvansertib and olaparib was well tolerated and showed strong anti-tumor activity in both PDX models. The combination significantly increased mice survival in comparison to vehicle, olaparib and onvansertib, and showed that onvansertib can re-sensitize PARPi-resistant tumors to olaparib. Median survival increased by 2.7-fold and 8.1-fold respectively in the 2 PDX models in the combination group versus vehicle and the Kaplan Meyer survival curves of mice treated with the combination showed a statistically survival advantage versus control and single agent treated mice. Pharmacodynamic analyses showed an increase in mitotic, apoptotic and DNA damage markers in tumors treated with the combination versus vehicle. Conclusions: The combination of the PLK1 inhibitor onvansertib and the PARPi olaparib showed potent anti-tumor activity in olaparib-resistant BRAC1 mutant HGSOC PDX models. Additional studies are ongoing to further assess the potential of the combination in BRCA wild-type and mutant ovarian, prostate, pancreatic and breast cancer preclinical models. Citation Format: Michela Chiappa, Federica Guffanti, Alessandra Decio, Alessandro Aliverti, Francesca Ricci, Eugenio Scanziani, Federica Camin, Ilaria Craparotta, Maria Chiara Barbera, Marco Bolis, Maya Ridinger, Giovanna Damia. Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3237.

Objective: To clarify the potential therapeutic effects of thymoquinone (TQ) on pancreatic cancer and its gemcitabine (GEM) sensitivity.Methods: The expression levels of hypoxia inducible factor-1α (HIF-1α), collagens (COL1A1, COL3A1, and COL5A1), and transforming growth factor-β1 (TGFβ1) in pancreatic cancer and para-carcinoma tissues were compared using immunohistochemical methods, and their relationships with TNM staging were analyzed. The effects of TQ on apoptosis, migration, invasion, and GEM sensitivity of pancreatic cancer cells were assessed using in vitro and in vivo experiments. Western blot and immunohistochemistry were used to detect the expression levels of HIF-1α, extracellular matrix (ECM) production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins.Results: The expression levels of HIF-1α, COL1A1, COL3A1, COL5A1, and TGFβ1 in pancreatic cancer tissues were significantly higher than those in para-carcinoma tissues and correlated with TNM staging (p < 0.05). TQ and GEM administration inhibited the migration and invasion of the human pancreatic cancer cell line PANC-1 and promoted the apoptosis of PANC-1 cells. The combination of TQ and GEM was more effective than GEM alone. Western blot analysis showed that the expression levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins were significantly decreased when TQ was used to treat PANC-1 cells (p < 0.05), and the expression levels of these proteins in the TQ + GEM group were significantly more decreased than those in the GEM group. Overexpression or knockdown of HIF-1α in PANC-1 cells showed the same effects as those induced by TQ administration. In vivo experiments showed that in PANC-1 tumor-bearing mice, tumor volume and tumor weight in mice treated with GEM and TQ were significantly lower than those in control or GEM-treated mice, whereas cell apoptosis was significantly increased (p < 0.05). Western blot and immunohistochemistry results showed that the levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins in the GEM + TQ treatment group were further decreased compared to the control group or the GEM treatment group (p < 0.05).Conclusion: In pancreatic cancer cells, TQ can promote apoptosis, inhibit migration, invasion, and metastasis, and enhance the sensitivity to GEM. The underlying mechanism may involve the regulation of ECM production through the TGFβ/Smad pathway, in which HIF-1α plays a key role.

Diffuse Midline Gliomas (DMGs) are devastating and incurable pediatric brain tumours. DMGs are characterised by epigenetic aberrations in the form of histone modifications, coupled with mitotic abnormalities, which together drive a stem-like transcriptional cell state that is critically dependent on the cell cycle. Polo-like kinase 1 (PLK1) is a pivotal regulator of mitosis and cell cycle progression. To determine an effective therapeutic strategy against DMG, we characterised the anti-tumour potential of PLK1 inhibition, either alone or in combination with radiotherapy. We show that targeting PLK1 with small molecule inhibitors BI2536 and BI6727 (volasertib) reduced DMG colony formation and cell proliferation, and induced G2/M checkpoint arrest and apoptosis in vitro at physiologically achievable, clinically relevant concentrations. When combined with radiotherapy, PLK1 inhibitors acted as potent radiosensitisers with synergistic suppression of DMG survival. We further show that PLK1 inhibitors exert potent anti-tumour effects in highly aggressive orthotopic DMG models, effectively extending the median survival of tumour harbouring mice both as a single agent and enhanced in combination with radiotherapy. We further demonstrate a potential novel role for PLK1 in defining an oncogenic transcriptional state in DMG by acting as a conduit between cell cycle progression and the DMG epigenome through modulation of polycomb repressive complex 2 (PRC2) activity and its resultant histone methyltransferase function. Together, these data indicate that targeting PLK1 is a highly promising therapeutic strategy to arrest DMG tumorgenicity. This data has formed the basis for a planned international Phase 1/2 trial of the PLK1 inhibitor volasertib in combination with radiotherapy in paediatric patients with DMG. Citation Format: Elisha Hayden, Laura Franshaw, Dannielle Upton, Jie Liu, Hieu Nguyen, Swapna Joshi, Maria Tsoli, Emmy Dolman, Benjamin Rayner, David Ziegler. Preclinical examination of PLK1 inhibitors for the treatment of diffuse midline gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1561.

BackgroundMedulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and regulates critical steps in mitotic progression. Recent studies suggest that targeting PLK1 with small molecule inhibitors is a promising approach to tumor therapy.MethodsWe examined the expression of PLK1 mRNA in medulloblastoma tumor samples using microarray analysis. The impact of PLK1 on cell proliferation was evaluated by depleting expression with RNA interference (RNAi) or by inhibiting function with the small molecule inhibitor BI 2536. Colony formation studies were performed to examine the impact of BI 2536 on medulloblastoma cell radiosensitivity. In addition, the impact of depleting PLK1 mRNA on tumor-initiating cells was evaluated using tumor sphere assays.ResultsAnalysis of gene expression in two independent cohorts revealed that PLK1 mRNA is overexpressed in some, but not all, medulloblastoma patient samples when compared to normal cerebellum. Inhibition of PLK1 by RNAi significantly decreased medulloblastoma cell proliferation and clonogenic potential and increased cell apoptosis. Similarly, a low nanomolar concentration of BI 2536, a small molecule inhibitor of PLK1, potently inhibited cell growth, strongly suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, BI 2536 pretreatment sensitized medulloblastoma cells to ionizing radiation. Inhibition of PLK1 impaired tumor sphere formation of medulloblastoma cells and decreased the expression of SRY (sex determining region Y)-box 2 (SOX2) mRNA in tumor spheres indicating a possible role in targeting tumor inititiating cells.ConclusionsOur data suggest that targeting PLK1 with small molecule inhibitors, in combination with radiation therapy, is a novel strategy in the treatment of medulloblastoma that warrants further investigation.

Targeting Polo-like Kinase in Acute Myeloid Leukemia

Integrated analysis of CRLF2 signaling in acute lymphoblastic leukemia identifies Polo-like kinase 1 as a potential therapeutic target

Mantle cell lymphoma (MCL) is an incurable, aggressive B cell malignancy. Ibrutinib, an oral irreversible Bruton's tyrosine (BTK) inhibitor, was FDA-approved in 2013 for patients with relapsed/refractory MCL. In further support of BTK inhibition for the treatment of MCL, acalabrutinib, an irreversible second-generation BTK inhibitor, was recently approved by the FDA for previously treated MCL. Although BTK inhibition has resulted in remarkable MCL patient outcomes, primary resistance and acquired resistance to ibrutinib remains a challenging hurdle to the treatment of MCL patients. In this study, we aimed to discover alternative therapies to overcome resistance to ibrutinib for MCL patients. To identify therapies that will overcome resistance to BTK inhibition, we performed a combinational screen for ibrutinib with FDA-approved drugs and novel agents and found that combination treatment of ibrutinib with the polo-like kinase 1 (PLK1) inhibitors volasertib or GSK461364 achieved synergistic growth inhibition in both ibrutinib-sensitive and -resistant MCL cells. PLK1, a master regulator of entry into mitosis, is overexpressed in many types of cancer, is correlated with poor clinical outcome, and plays an important role in the development of drug resistance. Volasertib, a second-generation PLK1 inhibitor, has been intensively studied, and there is an ongoing phase III trial to further determine the efficacy and safety of this agent in a large AML cohort. Phase I study of GSK461364 has been completed, and promising antitumor activity, especially in non-Hodgkin lymphoma and AML, has been determined. We found that PLK1 is highly expressed in MCL cells, and treatment with the PLK1 inhibitors volasertib and GSK461364 as single agents significantly arrested the cell cycle in the G2/M phase, potently reduced cell viability with IC50 values ranging from 6-28 nM and 8-33 nM, respectively, via induction of apoptosis in both ibrutinib-sensitive (n=3) and -resistant (n=6) MCL cells. Interestingly, treatment with the BTK inhibitor alone induced PLK1 expression, suggesting a compensatory mechanism in MCL cells to combat BTK inhibition. Dual BTK and PLK1 inhibition caused cell cycle arrest in the G2/M phase at a similar level to that of the PLK inhibitors alone; however, synergistic effects on cell viability and cleavage of PARP and caspase 3 were observed. The detailed mechanisms underlying these synergistic effects are currently under investigation. We are also validating the in vitro findings using patient-derived xenograft models in vivo. These data suggest that targeting PLK1 in MCL is a promising therapy and that dual targeting of BTK and PLK1 is a promising therapeutic strategy to improve MCL patient outcomes and to overcome ibrutinib resistance. Citation Format: Changying Jiang, Shengjiang Huang, Yang Liu, Krystle Nomie, Leo Zhang, Michael Wang. Dual targeting of BTK and PLK1 causes lethal synergistic effects in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3459.

Targeting Polo-like Kinase 4 for Therapeutic Benefit in the Patients with Diffuse Large B-Cell Lymphoma

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase and plays a critical role in mitosis. PLK1 has also been regarded as a valuable target for cancer treatment, and several PLK1 inhibitors are currently undergoing clinical investigations. In this study, our data show that the expression level of PLK1 is upregulated in human pancreatic cancer cells. Molecular modeling studies indicate that DMTC inhibits PLK1 activity through competitive displacement of ATP from its binding pocket. Our data further show that DMTC suppresses the proliferation of pancreatic cancer cells and induces the formation of multinucleated cells, ultimately resulting in apoptosis. In addition, combination index analysis demonstrates that DMTC acts synergistically with the chemotherapeutic drug gemcitabine in inhibiting the proliferation of pancreatic cancer cells. These results thus suggest a potential of using PLK1 inhibitors for the treatment of pancreatic cancer.

A novel L-shaped ortho-quinone analog as PLK1 inhibitor blocks prostate cancer cells in G2 phase

BackgroundAdrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer.MethodsWe used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis.ResultsKnocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53.ConclusionsThese preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

Objective To investigate the effect of gemcitabine on proliferation,apoptosis and polo-like kinase-1 (PLK-1) expression of human pancreatic cancer cell line AsPC-1 cells.Methods AsPC-1 cells were treated with various concentrations of gemcitabine for different time.The effect of gemcitabine on survival of AsPC-1 cells was determined by methyl thiazol tetrazolium (MTT) assay.The effect of gemcitabine on apoptosis of AsPC-1 cells was mearsured by flow cytometry with various concentrations of 2 and 5 μmol/L,and at different time points (12,24,48,and 72 h).The expression levels of PLK-1 mRNA and protein were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting,respectively.Results Gemcitabine decreased the proliferation of pancreatic cancer cells (P ＜ 0.05),and the apoptosis rate was significantly increased (P ＜ 0.05).After treatment with gemcitabine,the expression levels of PLK-1 mRNA (control:0.70 ±0.15,0.65 ±0.27,0.72 ±0.13,0.68 ±0.30;2 μmol/L:0.88 ±0.58,0.95 ±0.53,2.36 ±0.57,3.53 ±0.89;5 μmol/L:0.89 ± 0.60,1.02 ± 0.32,3.95 ± 1.84,4.52 ± 2.25) and protein (control:0.82 ± 0.41,0.78 ± 0.46,1.01 ±0.05,0.88 ±0.25 ;2 μmol/L:0.89 ±0.28,1.61 ±0.88,1.66 ±0.18,3.28 ± 1.33 ;5 μmol/L:0.85 ± 0.36,1.89 ± 0.18,2.73 ± 0.78,4.32 ± 2.13) were also increased significantly in pancreatic cancer cells (P ＜ 0.05).Conclusion Gemcitabine can influence the proliferation and apoptosis of AsPC-1 cells probably by the expression changes of PLK-1. Key words: Pancreatic carcinoma; Gemcitabine ; Apoptosis ; Polo-like kinase-1

ABSTRACTMechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1′s and PLK1′s functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive and malignant pediatric brain tumor. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and essential for mitosis. Overexpression of PLK1 promotes chromosome instability and aneuploidy by overriding the G2-M DNA damage and spindle checkpoints. Recent studies suggest that targeting PLK1 by small molecule inhibitors is a promising approach to tumor therapy. We investigated the effect of PLK1 inhibition in ATRT. Gene expression analysis showed that PLK1 was overexpressed in ATRT patient samples and tumor cell lines. Genetic inhibition of PLK1 with shRNA potently suppressed ATRT cell growth in vitro. Treatment with the PLK1 inhibitor BI 6727 (Volasertib) significantly decreased cell growth, inhibited clonogenic potential, and induced apoptosis. BI6727 treatment led to G2-M phase arrest, consistent with PLK1’s role as a critical regulator of mitosis. Moreover, inhibition of PLK1 by BI6727 suppressed the tumor-sphere formation of ATRT cells. Treatment also significantly decreased levels of the DNA damage proteins Ku80 and RAD51 and increased γ-H2AX expression, indicating that BI 6727 can induce DNA damage. Importantly, BI6727 significantly enhanced radiation sensitivity of ATRT cells. In vivo, BI6727 slowed growth of ATRT tumors and prolonged survival in a xenograft model. PLK1 inhibition is a compelling new therapeutic approach for treating ATRT, and the use of BI6727 should be evaluated in clinical studies.