Plk1 Inhibitors Research Articles

Plk1 Inhibition Enhances the Efficacy of BET Epigenetic Reader Blockade in Castration-Resistant Prostate Cancer.

Polo-like kinase 1 (Plk1), a crucial regulator of cell-cycle progression, is overexpressed in multiple types of cancers and has been proven to be a potent and promising target for cancer treatment. In case of prostate cancer, we once showed that antineoplastic activity of Plk1 inhibitor is largely due to inhibition of androgen receptor (AR) signaling. However, we also discovered that Plk1 inhibition causes activation of the β-catenin pathway and increased expression of c-MYC, eventually resulting in resistance to Plk1 inhibition. JQ1, a selective small-molecule inhibitor targeting the amino-terminal bromodomains of BRD4, has been shown to dramatically inhibit c-MYC expression and AR signaling, exhibiting antiproliferative effects in a range of cancers. Because c-MYC and AR signaling are essential for prostate cancer initiation and progression, we aim to test whether targeting Plk1 and BRD4 at the same time is an effective approach to treat prostate cancer. Herein, we show that a combination of Plk1 inhibitor GSK461364A and BRD4 inhibitor JQ1 had a strong synergistic effect on castration-resistant prostate cancer (CRPC) cell lines, as well as in CRPC xenograft tumors. Mechanistically, the synergistic effect is likely due to two reasons: (i) Plk1 inhibition results in the accumulation of β-catenin in the nucleus, thus elevation of c-MYC expression, whereas JQ1 treatment directly suppresses c-MYC transcription; (ii) Plk1 and BRD4 dual inhibition acts synergistically in inhibition of AR signaling. Mol Cancer Ther; 17(7); 1554-65. ©2018 AACR.

Abstract 2845: The mechanisms of resistance to temozolomide in glioma cells

Abstract Glioblastoma is one of the most aggressive tumor in the central nervous system tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). One of the reasons of the worse prognosis of the tumor is the acquisition of resistance to TMZ in glioma cells. TMZ is a DNA-methylating agent. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair with thymine during the next DNA replication cycle, leading to futile DNA mismatch repair (MMR) by the MMR system, formation of double strand breaks (DSBs) and eventual cell death of glioma cells. Several mechanisms have been proposed about the acquisition of resistance to TMZ in glioblastomas, such as changes of expression of MGMT, MMR, nucleotide excision repair and homologous recombination (HR). To clarify the mechanisms of resistance to TMZ and to find the way to overcome the resistance to TMZ, several clones of U251 cells resistant to TMZ were obtained and analyzed. FACS analyses showed several patterns of cell cycle distributions of the cells treated with TMZ. #3 resistant clone showed G2 arrest at day3 after TMZ exposure and this arrest was abrogated sooner compared to the G2 arrest of parental U251. TMZ did not induce G2 arrest in #8 resistant clone. Consistent with the results, TMZ-induced DSBs in #3 resistant cells were rapidly disappeared compared to those in U251 cells, and the DSBs were least induced by TMZ in #8 resistant cells. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells, which mean MGMT was not associated with the resistance to TMZ in those cells. The protein levels of MSH6, which was associated with MMR, was reduced in #8 cells. The inhibitor of PLK1 or PARP reduced the cell growth independent of MMR. The ability of HR was increased in #3 resistant clone. By suppression of Rad51, which was major factor associated with HR, the resistant #3 clone was re-sensitized to TMZ, however #8 was not. These results suggested that inhibition of HR may be a viable means to resensitize some kinds of temozolomide-resistant gliomas to TMZ. Citation Format: Shigeo Ohba, Yuichi Hirose. The mechanisms of resistance to temozolomide in glioma cells [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 2845.

Abstract 4628: Tailoring personalized strategies for children with treatment-refractory liver cancer

Abstract Tumour heterogeneity is probably the reason for different sensitivity to the same treatment in patients. Therefore, we urgently need to develop tools to help assign the right treatment to the right patient. This is particularly challenging for paediatric cancers. Hepatoblastoma (HB) is the most frequent malignant embryonal tumor of the liver in childhood. About 25% of HB patients will develop metastatic disease which is associated with a poor prognosis. Because of the small number of patients eligible for enrollment, clinical trials in most of cases could not provide clinicians with statistically significant results. In oncology, preclinical data are generally poorly predictive of treatment efficacy in patients, probably due to oversimplification of tumor biology and underrepresentation of the genetic tumor heterogeneity observed in patients. To face this issue, we have launched a program to generate patient-derived xenografts, the gold standard in preclinical oncology, from surgical tissue from HB patients after chemotherapy, at relapse and from local and distant metastases. Therefore, the models developed are representative of relapsing, metastatic HB. So far, a panel of 24 HB PDXs models has been established. From these models 10 cell lines could be established so far to be used for large-scale in vitro screening of anticancer compound libraries. The aim of this project is to combine in vitro and in vivo drug screening to provide strong preclinical rationale to the development of novel therapeutic options for children with HB, and to identify candidate biomarkers predictive of tumor response. In vitro screening of a HB PDX-derived cells with a library of 60 compounds corresponding to treatments already available in the clinic lead us to identify 4 compounds with very strong cytotoxic activity on HB cells. One of them, Volasertib, a PLK1 inhibitor, was selected to be tested in the HB PDX the cells were derived from. The in vivo study compared the effect of volasertib as single agent or in combination with irinotecan to irinotecan alone or in combination with temozolomide. The results showed that high dose volasertib induce tumor regression with efficacy comparable to irinotecan/temozolomide combination. Following these encouraging results, Volasertib was tested in the complete panel of HB PDX-derived cells, and the majority of them showed strong sensitivity to the drug. This sensitivity was associated with specific molecular alterations that might be used as biomarkers to predict tumor sensitivity. Overall, these results suggest that volasertib could be an effective second line treatment for children with recurrent HB, and that this platform could be a very important preclinical tool to foster translational research in HB. Citation Format: Stefano Cairo, Bénédicte Noblet, Samuel Rasmussen, Matthew N. Svalina, Katell Mevel, Enora Le Ven, Delphine Nicolle, Olivier Déas, Charlotte Mussini, Sophie Branchereau, Jean-Gabriel Judde, Charles Keller. Tailoring personalized strategies for children with treatment-refractory liver cancer [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 4628.

Abstract 3174: PLK4 is a rational target for very high-risk medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite aggressive therapy (maximal safe surgical resection, craniospinal irradiation and/or chemotherapy) only 65% of patients survive, and survivors are left with severe neuro-cognitive sequelae. MB is comprised of at least four distinct molecular subgroups (SHH, Wnt, Group 3 and Group 4). Within the Sonic hedgehog (SHH) subgroup, a subset harboring inactivating mutations within TP53 represent a very high-risk group with almost universally fatal outcome. TP53 mutated SHH tumors have a high degree of genomic instability with frequent chromothripsis and are resistant to the current generation of SHH pathway inhibitors. The protein PLK4 has a key role in the maintenance of chromosome numeral integrity and as such dysregulation of PLK4 expression causes genomic instability. The PLK4 inhibitor (CFI-400945, produced by the Campbell Family Institute for Breast Cancer Research) was shown to increase genomic instability in aneuploid cells from adult solid tumors and induce these cells to die through mitotic catastrophy. Here we leverage a sporadic transposon based metastatic mouse model (Ptch+/-/Math1-SB11/T2Onc) that faithfully mimics very high-risk MB in children. In order to mimic treatment in children, tumors are microsurgically subtotally resected, followed with the oral administration of CFI-400945 and cranial MRI to monitor response. Blood brain barrier penetration of CFI-400945 was confirmed using mass spectroscopy, specifically pharmacokinetics study revealed an expected availability of CFI-400945 in the circulating blood and concentrated compound in the tumor compared to the normal brain. The administration of CFI-400945 confers an advantage of survival to the mice in this model, median of 18.5 days for surgery group and 49 days for surgery plus CFI-400945 group (p=0.005). In human tumor datasets PLK4 is highly expressed across a variety of malignancies, including MB. Specifically, across two independent, non-overlapping datasets higher PLK4 levels correlate with a trend toward worse outcome, however, when restricting this analysis only to SHH tumors, there is a significant survival difference between high and low PLK4 expression (p=0.012). In paired primary-metastatic MB PLK4 expression is retained at the time of recurrence, specifically in metastatic recurrences, suggesting PLK4 inhibition is a suitable treatment option at the time of recurrence. Taken together, the data reveals PLK4 inhibition as a novel and rational therapeutic target for a subset of very high risk MB and a broad range of other pediatric cancers. Citation Format: Carolina Nor, Mark Bray, Tak Mak, Michael Taylor. PLK4 is a rational target for very high-risk medulloblastoma [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 3174.

Abstract 4130: TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma

Abstract Centrosomes are the major microtubule-organizing centers in animal cells. Centrosomes catalyze microtubule assembly to accelerate formation of the bipolar mitotic spindle that segregates chromosomes during cell division. To analyze the effect of centrosome removal in normal and cancer cells, we developed centrinone, a potent, specific and cellularly-active inhibitor of the protein kinase polo-like kinase 4 (Plk4), which is essential for centrosome duplication (Wong et al., 2015, Science 348:1155-60). Centrosome-less cells take longer to execute mitosis, and trigger a mitotic stress signaling pathway that results in p53 stabilization and apoptosis and/or senescence of the resulting daughter cells. Failure to form a spindle can also lead to apoptosis during mitosis or exit from mitosis without chromosome segregation (mitotic slippage). In a genome-wide CRISPR/Cas9 loss-of-function screen for resistance to centrinone, we had previously shown that loss of the ubiquitin ligase TRIM37 makes spindle assembly more robust to centrosome removal (Meitinger et al., 2016, J Cell Biol. 214:155-66), raising the possibility that high levels of TRIM37 might increase sensitivity to centrosome removal. Here, we describe the use of single-cell live imaging assays to identify determinants of vulnerability of cancer cells to centrosome removal. Among a panel of 37 tested cell lines, neuroblastoma-derived cell lines were the most sensitive to centrosome removal, exhibiting high levels of apoptosis and rapid loss of viability following treatment with centrinone. We found that sensitivity to centrosome removal correlated with TRIM37 expression levels. Upon centrosome depletion, cells with high TRIM37 levels, particularly neuroblastoma cells, exhibited greatly increased mitotic duration and frequency of mitotic failure. p53 positive neuroblastoma cell lines exhibited apoptosis resulting from mitotic stress-mediated p53 activation and from mitotic failure. Surprisingly, p53 negative neuroblastoma cell lines, which are unable to sense mitotic stress, were also highly sensitive to centrosome removal, with cell death in this case resulting from high rates of mitotic failure. TRIM37 deletion restored the ability of cells to proliferate in centrinone. Thus, centrosome removal via Plk4 inhibition appears to be a promising strategy for therapeutic treatment of neuroblastomas and potentially other cancers with high levels of TRIM37 expression. Citation Format: Franz Meitinger, Robert L. Davis, Ruth Kabeche, John V. Anzola, Yao Liang Wong, Andrew K. Shiau, Arshad Desai, Karen Oegema. TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma [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 4130.

Abstract 4032: Pharmacogenomic analysis of acquired resistance can reveal effective drug combination in glioblastoma

Abstract Targeted cancer therapies have produced substantial clinical responses, but glioblastoma multiforme (GBM) has poor response due to developing resistance. The goal is thus to design strategies to overcome resistance to targeted drugs. To address this, we have embarked on a new type of screening for resistant targets, by developing acquired resistant glioma models and using pharmacogenomic platform to identify the resistant targets. We have established several patient-derived in vivo glioma stem cells (GSC) models using a PI3K specific inhibitor BKM-120 by a selective pressure after a chronic drug treatment. The resistant cells were subjected to RNA-seq analysis as well as pharmacologic analysis utilizing high-throughput kinase drug library screening. Analysis of integrated RNA-seq and kinase drug library screening of resistant cells identified multiple effective drug combination. Activation of Aurora kinase A (Aurora A)/Polo-like kinase 1 (Plk1)/cyclin-dependent kinase (CDK) signaling pathway was predominant in resistant GSCs. Concomitant targeting of PI3K together with Aurora A, Plk1 or CDK1 had a synergistic growth inhibitory effect. In addition, the combined inhibition of Plk1 and PI3K resulted in tumor growth suppression and significant delay of acquired resistance in vivo. These observations suggest that cell cycle pathway serves as a major survival signal upon PI3K inhibition and provides a rationale for the combined use of PI3K and cell cycle inhibition to prevent acquired resistance in human GBM. Citation Format: Xiaolong Li, Juan Martinez-Ledesma, Chen Zhang, Feng Gao, W.K. Alfred Yung, Dimpy Koul. Pharmacogenomic analysis of acquired resistance can reveal effective drug combination in glioblastoma [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 4032.

Abstract 3459: Dual targeting of BTK and PLK1 causes lethal synergistic effects in mantle cell lymphoma

Abstract 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.

Abstract 2882: Elucidation of the mechanism of action of the multi-kinase inhibitor midostaurin in NSCLC

Abstract Background: Non-small cell lung cancer (NSCLC) constitutes a strong unmet medical need in spite of recent successes with targeted drugs and immunotherapy in some subsets. We had observed the recently FDA-approved drug midostaurin to exhibit potent anticancer activity in a subset of NSCLC cell lines that was unrelated to its known targets FLT3 or PKC. We here explored a new repurposing opportunity and investigated the mechanism of action (MoA) of midostaurin in NSCLC utilizing a multilayered functional proteomics approach. Methods: Chemical proteomics identified kinase targets and global and tyrosine phosphoproteomics generated a network view of intracellular signaling upon midostaurin treatment. Functional validation was performed using RNA interference and selective small-molecule probes. Cell viability assays were carried out using CellTiterGlo and colony formation assays. Induction of apoptosis was determined by immunoblotting and fluorescent microscopy. Flow cytometry assessed cell cycle stages. Signaling changes were determined by immunoblotting. Results: Viability screening confirmed anticancer activity in several midostaurin-sensitive NSCLC cell lines at clinically relevant concentrations independent of its canonical targets. Using chemical proteomics, we identified multiple midostaurin kinase targets. Network-based integration of the chemical proteomics and quantitative tyrosine and global phosphoproteomics data using STRING for protein-protein interactions and NetworKIN for substrate-kinase predictions revealed several nodes to be relevant for the MoA of midostaurin. Subsequent functional validation showed that simultaneous inhibition of TBK1, PDK1 and AURKA was required to mimic midostaurin's cellular effects in these cells. Immunoblotting for downstream targets of these kinases revealed PLK1 as a critical signaling effector. Rational combination of midostaurin with the more potent PLK1 inhibitor BI2536 caused strong synergy. Conclusion: Midostaurin showed activity in a subset of NSCLC cells. Combination of complementary functional proteomics approaches and subsequent network-based integration revealed novel insight into the MoA of midostaurin and NSCLC vulnerabilities. We utilized this mechanistic knowledge for the rational combination of midostaurin with the clinical PLK1 inhibitor BI2536, which is highly synergistic and has strong potential for clinical translation. Citation Format: Claudia Ctortecka, Brent Kuenzi, Bin Fang, Natalia Sumi, Victoria Izumi, Fumi Kinose, Eric Haura, John Koomen, Uwe Rix. Elucidation of the mechanism of action of the multi-kinase inhibitor midostaurin in NSCLC [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 2882.

Abstract 2810: Computationally predicted sensitivity of clinical cohorts identifies drug relationships and biomarkers associated with response to PCM-075, a PLK1 selective inhibitor

Abstract Polo-like kinase 1 (PLK1) is a serine-threonine kinase which regulates various cellular processes, including mitosis, DNA replication, and the stress response, and is over-expressed in many malignancies. PCM-075, a PLK1 selective inhibitor, is currently in a phase1b/2 clinical trial in AML in combination therapy with low dose cytarabine (LDAC) (NCT03303339). To identify potential drug relationships and biomarkers associated with response to PCM-075, a two-step computational approach was conducted. Using a modification of the ‘imputed drug-wide association study' (IDWAS) method(1), we first performed ridge regression model training on IC50 values and gene expression signatures for PCM-075 and 138 other drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) database . In the second step, we applied these models to clinical patient datasets (The Cancer Genome Atlas (TCGA) populations containing 32 Cancer types and 9,968 total patients), generating a predicted sensitivity value to all drugs for each patient. Hierarchical clustering of imputed sensitivities across all patients showed that the PCM-075 activity profile is most closely related to cytarabine, etoposide and cisplatin, all of which are compounds that impact DNA replication and lead to apoptosis. Correlation of predicted response values was significant both within and between cancer types, with the acute myeloid leukemia (AML) cohort showing highest increased sensitivity to PCM-075, cytarabine and etoposide relative to the other 31 cancer types examined. Interestingly, other PLK1 inhibitors present in the GDSC (BI-2536 and GW843682X) do not closely cluster with PCM-075. Association of imputed TCGA sensitivity values with somatic variant data shows a high degree of concordance between predicted biomarkers, including TP53 mutation status, further suggesting a functional relationship. Given that this computational analysis suggests that similar pathways are affected by both cytarabine and PCM-075, we tested this experimentally by assessing the level of interaction between these two compounds within an AML cell line (HL-60). The combination resulted in statistically significant synergy (BRAID, k~2.7(1.31-4.25)) for anti-proliferative activity. The mechanistic cause of synergy may be related to DNA damage caused by cytarabine which is known to inhibit and degrade the PLK1 protein. In conclusion, we have identified a pathway signature that clusters PCM-075 with a subset of current oncology therapeutics. Synergy between these compounds in combination suggests the potential for use in treating AML and potentially other cancer types. 1.Genome Res. 27: 1743-1751 (2017). Citation Format: Penn Whitley, Peter J. Croucher, Barbara Valsasina, Dario Ballinari, Italo Beria, Jeffrey N. Miner, Mark G. Erlander. Computationally predicted sensitivity of clinical cohorts identifies drug relationships and biomarkers associated with response to PCM-075, a PLK1 selective inhibitor [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 2810.

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds.

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.

Hsa-let-7b inhibits cell proliferation by targeting PLK1 in HCC

Previous studies have shown that high levels of PLK1 are expressed in HCC, and PLK1 inhibitors are being tested in clinical trials. However, the mechanisms, which regulate PLK1 expression in HCC, have not been clarified. Here, we show that induction of let-7b over-expression inhibits the PLK1-regulated luciferase activity in HEK-293T cells, and decreases the levels of PLK1 expression in HCC cells. Furthermore, the levels of let-7b expression were negatively correlated with PLK1 expression in HCC tissues. Let-7b over-expression inhibited the proliferation of HCC cells and promoted their apoptosis, which were partially rescued by increased PLK1 expression. Let-7b over-expression decreased the levels of PLK1, CDC25C and Survivin phosphorylation and CDC2, β-catenin, TCF-4 expression, which were mitigated by increased PLK1 expression in MHCC-97H cells. Let-7b over-expression inhibited the development and growth of implanted HCC tumors in mice by decreasing PLK1 and Survivin expression in the tumors. Together, our data indicated that let-7b targeted PLK1 to inhibit HCC growth and induce their apoptosis by attenuating the PLK1-mediated Survivin phosphorylation. Our findings may provide new insights into the pathogenesis of HCC.

Inhibition of Prostate Smooth Muscle Contraction by Inhibitors of Polo-Like Kinases.

Background: Prostate smooth muscle contraction plays an important role for pathophysiology and treatment of male lower urinary tract symptoms (LUTS) but is incompletely understood. Because the efficacy of available medication is limited, novel options and improved understanding of prostate smooth muscle contraction are of high demand. Recently, a possible role of polo-like kinase 1 (PLK1) has been suggested for smooth muscle contraction outside the lower urinary tract. Here, we examined effects of PLK inhibitors on contraction of human prostate tissue.Methods: Prostate tissues were obtained from radical prostatectomy. RT-PCR, Western blot and immunofluorescence were performed to detect PLK expression and phosphorylated PLK. Smooth muscle contractions were induced by electric field stimulation (EFS), α1-agonists, endothelin-1, or the thromboxane A2 analog U46619 in organ bath.Results: RT-PCR, Western blot, and immunofluorescence suggested expression of PLK1 in the human prostate, which may be located and active in smooth muscle cells. EFS-induced contractions of prostate strips were reduced by SBE 13 (1 μM), cyclapolin 9 (3 μM), TAK 960 (100 nM), and Ro 3280 (100 nM). SBE 13 and cyclapolin 9 inhibited contractions by the α1-agonists methoxamine, phenylephrine, and noradrenaline. In contrast, no effects of SBE 13 or cyclapolin 9 on endothelin-1- or U46619-induced contractions were observed.Conclusion: Alpha1-adrenergic smooth muscle contraction in the human prostate can be inhibited by PLK inhibitors. PLK-dependent signaling may be a new pathway, which promotes α1-adrenergic contraction of prostate smooth muscle cells. As contractions by endothelin and U46619 are not susceptible to PLK inhibition, this reflects divergent regulation of adrenergic and non-adrenergic prostate smooth muscle contraction.

Synthetic lethality in CCNE1-amplified high grade serous ovarian cancer through combined inhibition of Polo-like kinase 1 and microtubule dynamics.

The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.

PLK1 as a potential therapeutic target of lupus

Abstract System lupus erythematosus (SLE) is a complex autoimmune disease with high mortality and morbidity. A targeted therapy is desirable for personalized medication of SLE. Recently we used a novel Activity-based Kinome Screening and identified several novel signaling molecules which are up- or downregulated in murine lupus. Totally we screened 196 kinases in spleens from two lupus mouse models, B6.Sle1.Sle3 and MRL.lpr. Compared to the B6 control, PLK1 and its upstream regulator Aurora A exhibited significantly increased activities. We further found the activation of both Aurora-A and PLK1 were significantly upregulated in splenic B220+ B cells of lupus mice MRL.lpr. We found a PLK1 specific inhibitor NMS-P064 ameliorated splenomegaly (P < 0.05), anti-dsDNA antibody production (P < 0.001), proteinuria and BUN, and renal pathology GN score in MRL.lpr. In addition, we found IL-6, IL-17 and kidney injury molecule 1 (KIM-1) were significantly decreased in the serum of MRL.lpr mice after PLK1 inhibition. In vitro, after 24 h stimulation of splenocytes with CD40 ligand, IL-6 secretion was significantly reduced upon PLK1 blockade (P < 0.05). Whereas IL-10 secretion was significantly increased in anti-IgM Fab’ fragment stimulated splenocytes upon PLK1 blockade. Given the phosphorylation of PLK1 was significantly increased on Thr 210 in murine lupus, it is possible that this could be the consequence of the direct phosphorylation of PLK1 by Aurora A. Taken together, PLK1 may contribute to the pathogenesis of lupus via Aurora-A/PLK1 signaling cascade and might be a potential therapeutic target of lupus.

Targeting Polo-like Kinase 1 by a Novel Pyrrole-Imidazole Polyamide–Hoechst Conjugate Suppresses Tumor Growth In Vivo

The serine/threonine kinase Polo-like kinase 1 (Plk1) plays a pivotal role in cell proliferation and has been validated as a promising anticancer drug target. However, very limited success has been achieved in clinical applications using existing Plk1 inhibitors, due to lack of sufficient specificity toward Plk1. To develop a novel Plk1 inhibitor with high selectivity and efficacy, we designed and synthesized a pyrrole-imidazole polyamide-Hoechst conjugate, PIP3, targeted to specific DNA sequence in the PLK1 promoter. PIP3 could specifically inhibit the cell cycle-regulated Plk1 expression and consequently retard tumor cell growth. Cancer cells treated with PIP3 exhibited severe mitotic defects and increased apoptosis, whereas normal cells were not affected by PIP3 treatment. Furthermore, subcutaneous injection of PIP3 into mice bearing human cancer xenografts induced significant tumor growth suppression with low host toxicity. Therefore, PIP3 exhibits the potential as an effective agent for targeted cancer therapy. Mol Cancer Ther; 17(5); 988-1002. ©2018 AACR.

1103 Functional genomic evaluation of targetable pathways in three metastatic cutaneous squamous cell carcinomas

Cutaneous squamous cell carcinoma (cSCC) is increasing, paralleled by increases in deadly invasive and metastatic primary skin tumors. To date there are neither reliable predictive biomarkers of disease progression nor medical treatments as standard of care. To address these issues, we exposed primary cultured cells from invasive or metastatic cSCC tissues to 118 small molecule inhibitors as single agents and in combination with an EGFR inhibitor, gefitinib. In-house bioinformatics tools were used to cross analyze drug responses and DNA mutations in tumors detected by whole exome sequencing with patient-matched whole blood DNA standards. According to a paradigm that (in)activating mutations may predict sensitivity to targeted inhibitors, we associated drug sensitivities with DNA mutations predicted to be “damaging” by SIFT. Two cases sensitive to AKT inhibitor in combination with gefitinib had mutations in AKT3. One case, the only one with FLT or CSFR mutations, was sensitive to a single agent inhibitor of FLT1 and CSFR1R. Two cases with sensitivity to a PLK1 inhibitor in combination with gefitinib lacked mutations in PLK1. The most sensitive of these cases harbored mutations in p53BP1, which participates in a cell cycle pathway regulated by TBK1 and PLK1 phosphorylation. All three cases had damaging mutations in TP53, two cases had HRAS mutations (G13D and G13R), and two cases had mutations in the DNA damage repair genes FANCD2 and XRCC3. These studies form a plausible foundation for biomarkers of progression to high mortality/morbidity cSCC applicable in dermatopathology and for patient-specific therapeutic options for aggressive cSCC.

Essential Role of Polo-like Kinase 1 (Plk1) Oncogene in Tumor Growth and Metastasis of Tamoxifen-Resistant Breast Cancer.

The most common therapy for estrogen receptor-positive breast cancer is antihormone therapy, such as tamoxifen. However, acquisition of resistance to tamoxifen in one third of patients presents a serious clinical problem. Polo-like kinase 1 (Plk1) is a key oncogenic regulator of completion of G2-M phase of the cell cycle. We assessed Plk1 expression in five chemoresistant cancer cell types and found that Plk1 and its downstream phosphatase Cdc25c were selectively overexpressed in tamoxifen-resistant MCF-7 (TAMR-MCF-7) breast cancer cells. Real-time monitoring of cell proliferation also showed that TAMR-MCF-7 cells were more sensitive to inhibition of cell proliferation by the ATP-competitive Plk1 inhibitor BI2536 than were the parent MCF-7 cells. Moreover, BI2536 suppressed expression of epithelial-mesenchymal transition marker proteins and 3D spheroid formation in TAMR-MCF-7 cells. Using TAMR-MCF-7 cell-implanted xenograft and spleen-liver metastasis models, we showed that BI2536 inhibited tumor growth and metastasis in vivo Our results suggest that Plk1 could be a novel target for the treatment of tamoxifen-resistant breast cancer. Mol Cancer Ther; 17(4); 825-37. ©2018 AACR.

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 ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ)

ABSTRACTPolo-like kinase 1 (Plk1) is an instrumental kinase that modulates many aspects of the cell cycle. Previous investigations have indicated that Plk1 is a target of the DNA damage response, and Plk1 inhibition is dependent on ATM/ATR and Chk1. But the exact mechanism remains elusive. In a proteomic screen to identify Chk1-interacting proteins, we found that myosin phosphatase targeting protein 1 (MYPT1) was present in the immunocomplex. MYPT1 is phosphorylated by CDK1, thus recruiting protein phosphatase 1β (PP1cβ) to dephosphorylate and inactivate Plk1. Here we identified that Chk1 directly interacts with MYPT1 and preferentially phosphorylates MYPT1 at Ser20, which is essential for MYPT1-PP1cβ interaction and subsequent Plk1 dephosphorylation. Phosphorylation of Ser20 is abolished during mitotic damage when Chk1 is inhibited. The degradation of MYPT1 is also regulated by Chk1 phosphorylation. Our results thus unveil the underlying machinery that attenuates Plk1 activity during mitotic damage through Chk1-induced phosphorylation of MYPT1.

Antitumor activity of the polo-like kinase inhibitor, TAK-960, against preclinical models of colorectal cancer

BackgroundPolo-like kinase 1 (Plk1) is a serine/threonine kinase that is a key regulator of multiple stages of mitotic progression. Plk1 is upregulated in many tumor types including colorectal cancer (CRC) and portends a poor prognosis. TAK-960 is an ATP-competitive Plk1 inhibitor that has demonstrated efficacy across a broad range of cancer cell lines, including CRC. In this study, we investigated the activity of TAK-960 against a large collection of CRC models including 55 cell lines and 18 patient-derived xenografts.MethodsFifty-five CRC cell lines and 18 PDX models were exposed to TAK-960 and evaluated for proliferation (IC50) and Tumor Growth Inhibition Index, respectively. Additionally, 2 KRAS wild type and 2 KRAS mutant PDX models were treated with TAK-960 as single agent or in combination with cetuximab or irinotecan. TAK-960 mechanism of action was elucidated through immunoblotting and cell cycle analysis.ResultsCRC cell lines demonstrated a variable anti-proliferative response to TAK-960 with IC50 values ranging from 0.001 to > 0.75 μmol/L. Anti-proliferative effects were sustained after removal of drug. Following TAK-960 treatment a highly variable accumulation of mitotic (indicating cell cycle arrest) and apoptotic markers was observed. Cell cycle analysis demonstrated that TAK-960 treatment induced G2/M arrest and polyploidy. Six out of the eighteen PDX models responded to single agent TAK-960 therapy (TGII< 20). The addition of TAK-960 to standard of care chemotherapy resulted in largely additive antitumor effects.ConclusionTAK-960 is an active anti-proliferative agent against CRC cell lines and PDX models. Collectively, these data suggest that TAK-960 may be of therapeutic benefit alone or in combination with other agents, although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations.