Polo-like Kinase 1 Inhibition Research Articles

PLK1 protects intestinal barrier function during sepsis by targeting mitochondrial dynamics through TANK-NF-κB signalling

BackgroundIntestinal barrier integrity in the pathogenesis of sepsis is critical. Despite an abundance of evidence, the molecular mechanism of the intestinal barrier in sepsis pathology remains unclear. Here, we report a protective role of polo-like kinase 1 (PLK1) in intestinal barrier integrity during sepsis.MethodsMice with PLK1 overexpression (CAG-PLK1 mice) or PLK1 inhibition (BI2536-treated mice) underwent caecal ligation and puncture (CLP) to establish a sepsis model. The intestinal barrier function, apoptosis in the intestinal epithelium, mitochondrial function and NF-κB signalling activity were evaluated. To suppress the activation of NF-κB signalling, the NF-κB inhibitor PDTC, was administered. The Caco-2 cell line was chosen to establish an intestinal epithelial injury model in vitro.ResultsSepsis destroyed intestinal barrier function, induced excessive apoptosis in the intestinal epithelium, and disrupted the balance of mitochondrial dynamics in wild-type mice. PLK1 overexpression alleviated sepsis-induced damage to the intestinal epithelium by inhibiting the activation of NF-κB signalling. PLK1 colocalized and interacted with TANK in Caco-2 cells. Transfecting Caco-2 cells with TANK-SiRNA suppressed NF-κB signalling and ameliorated mitochondrial dysfunction, apoptosis and the high permeability of cells induced by lipopolysaccharide (LPS). Furthermore, TANK overexpression impaired the protective effect of PLK1 on LPS-induced injuries in Caco-2 cells.ConclusionOur findings reveal that the PLK1/TANK/NF-κB axis plays a crucial role in sepsis-induced intestinal barrier dysfunction by regulating mitochondrial dynamics and apoptosis in the intestinal epithelium and might be a potential therapeutic target in the clinic.

Design, synthesis, and biological evaluation of a potent PLK4 inhibitor WY29 with 1H-pyrazolo[3,4-d]pyrimidine scaffold.

Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Overexpression of PLK4 in somatic cells can lead to the excessive formation of centrioles, directly causing chromosome segregation errors and tumorigenesis. In this study, we described our efforts to develop a series of PLK4 inhibitors with 1H-pyrazolo[3,4-d]pyrimidine core, and further structure- and receptor-based design and optimization resulted in a potent inhibitor WY29 (IC50 = 0.027 μM), which exhibited good selectivity to other PLK family members (PLK1-3). At the cellular level, compound WY29 showed excellent antiproliferative activity against three breast cancer cell lines (MCF-7, BT474, and MDA-MB-231) while weak inhibitory activity was found on normal cell line HUVECs. In addition, the in vitro preliminary drug-like properties evaluation of compound WY29 showed outstanding stability in human plasma and liver microsomes, and weak inhibitory activity against the major subtypes of human cytochrome P450. Also, the drug-like properties prediction of compound WY29 displayed remarkable drug-like properties (drug-likeness mode score: 1.06). In conclusion, these results support the further development of compound WY29 as a lead compound for PLK4-targeted anticancer drug discovery.

Polyploidy Is Necessary for Apoptotic Cell Death in TP53-Mut AML in Response to Polo-like-Kinase 4 (PLK4) Inhibition and Results in Caspase 3 Cleavage

Background Approximately twenty percent of patients with newly diagnosed acute myeloid leukemia (AML) have TP53 mutations, associated with complex karyotype and the highest risk of relapse(Döhner et al., 2017; Giacomelli et al., 2018). Although combination therapies, such as Ara-C + anthracyclines and Venetoclax (VEN) + hypomethylating agents (HMA) result in higher remission rates in patients with TP53-mutant AML, the mutant clone is never eliminated and invariably leads to relapse(Short et al., 2021; Takahashi et al., 2016). Novel targets and mechanisms associated with TP53-mut AML need to be identified. Results: We performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations of TP53-mutant or TP53-wild-type (wt) high-risk AML patients before and after treatment. Amongst overexpressed genes, we identified Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis and therefore of polyploidy, as a potential target highly expressed in TP53-mutant AML using differential gene expression analysis of TP53-wt vs TP53-mutant samples collected (TP53-mutant=36, TP53-wt=31, PLK4 LOG2FC=~2, p<0.001). We validated this finding using an independent RNA-seq dataset from the Munich Leukemia Lab of 726 AML samples (TP53-mutant=72, TP53-wt=654, PLK4 LOG2FC=0.48, p<0.001) and investigated the impact of PLK4 expression levels on AML patient outcomes in the context of TP53-status. High levels of PLK4 expression in AML are associated with poor outcomes (Figure 1). Targeting PLK4 by CFI-400945 (25nM, Treadwell Therapeutics, Toronto, Canada) in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 for 72 hours resulted in increased polyploid cells in TP53-mut cells compared to TP53-wt AML (> 2 fold, p< 0.0001). PLK4 inhibition triggered higher levels of cleaved caspase 3 in polyploid TP53-mut polyploid MOLM-13 cells compared to TP53-wt MOLM-13 cells (Figure 2). Polyploidization in TP53-mut MOLM-13 cells 48 hours post treatment with 15nM of PLK4 inhibitor was not reversible and retained higher apoptogenic activity in TP53-mut MOLM-13 cells even after wash out for additional 48 hours. To translate our findings to in vivo models, we transplanted CRISPR-generated TP53-wt, TP53-ko, and TP53-248 mutant MOLM-13 cells (Boettcher et al., 2019) into NSG mice (n=58: untreated n= 28, treated n=30), confirmed successful engraftment, and treated the mice with CFI-400945 (oral gavage 7.5 mg/kg, 5 times/week) for 21 days. Our preliminary data indicates that all treated mice showed improved survival compared to untreated mice. The median survival for treated TP53-wt vs untreated TP53-wt was 17.5 vs 14.5 days (20.7% increase, n=20 (untreated n=10, treated n=10), p=0.00086). The median for treated TP53-R248Q vs untreated TP53-R248Q was 15.0 vs 12.0 days respectively (25% increase, n=19 (untreated n=9, treated n=10), p=0.00039). The median OS for treated TP53-ko vs untreated TP53-ko was 11 vs 8 days respectively (37.5% increase, n=19 (untreated n=9, treated n=10), p<0.0001). Next, we performed a Cox proportional hazard model to investigate the contribution of TP53 genotype and PLK4 inhibition on overall survival. The resulting model (Likelihood ratio test=83.09 on 3 degrees of freedom and p<2.2e-16) showed a higher hazard ratio in recipients with TP53-R248Q and TP53-ko (~4.3, and ~1.2 hazard coefficients respectively) in comparison to TP53-wt (p=2.67e-14, and 0.000708 respectively). Additionally, treated mice had statistically significant lower hazard ratios compared to untreated (hazard coefficients=-2.47277 and p=8.38e-10). The data suggest that PLK4 inhibition is a promising treatment approach for AML with TP53 mutation. We are presently testing different dosing schedules in other aggressive TP53-mut AML models to further ascertain the efficacy of PLK4 inhibition. Conclusion: We established an association between PLK4 overexpression and poor survival outcomes in patients with newly diagnosed AML. TP53-mutant AML express higher levels of PLK4 in comparison to TP53-wt AML and targeting PLK4 induces polyploidy and apoptotic cell death predominantly in TP53-mutant AML. We demonstrate that targeting of PLK4 improves overall survival in TP53-mut and TP53-ko AML cells in vivo. Data suggest that targeting PLK4 represents a potential treatment approach for TP53-mut AML. An early phase, multi-center clinical trial is ongoing with CFI-400945 in AML (NCT04730258, TWT-202). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Role for PLK1 in Leukemia Stem Cell Function in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. While most patients achieve initial remission following standard chemotherapy, the majority will relapse and ultimately die. Poor outcomes in AML are tied to the properties of leukemia stem cells (LSC), which drive therapy resistance and relapse. For successful therapeutic targeting, it is critical to understand the pathways that operate in LSC function and survival and how they are impacted by drug activity. We carried out an innovative stemness-focused high-throughput drug screen employing a hierarchical AML cell model and a Nanostring-based LSC signature assay to probe for genes and pathways that impact LSC function. All 4 small molecule inhibitors of Polo-like Kinase 1 (PLK1) included in the compound library were among the top hits. PLK1 inhibitor treatment selectively targeted the CD34⁺CD38⁻ fraction of OCI-AML-8227 cells, which is enriched for quiescent LSCs. PLK1 is an important cell cycle regulator; although there are reports for cell cycle-independent roles, they have not been systematically characterized. Importantly, the role of PLK1 has not been previously studied in the context of cancer stem cells. To investigate the role of PLK1 in LSCs, we performed single-cell (sc) multi-omics incorporating both scRNAseq and scATACseq on the CD34⁺CD38⁻ compartment of OCI-AML-8227 cells. Enrichment analysis of previously-identified chromatin accessibility signatures of primitive long-term hematopoietic stem cells (LT-HSC) and progenitors was used to identify cell clusters with distinct stemness and differentiation properties. Interestingly, PLK1 mRNA expression in OCI-AML-8227 was restricted to cells enriched for the LT-HSC epigenetic signature (Fig 1A). In healthy human bone marrow, PLK1 expression was restricted to committed progenitors and not detected in HSCs (Fig 1B). These data strongly suggest a previously unexplored role of PLK1 in regulating stemness that may be specific LSCs and not normal HSCs. To determine the effects of PLK1 against LSCs in vivo, we carried out PLK1 inhibitor treatment using the specific PLK1 inhibitor volasertib in a cohort of 9 primary AML samples in PDX models. Volasertib treatment significantly reduced leukemic burden in 7 of 9 samples, all of which had a high LSC17 score (associated with poor prognosis and standard chemotherapy response). To quantify effects on LSCs, we serially transplanted 2 samples at limiting doses into untreated secondary recipients and demonstrated a 5- to 25-fold reduction in LSC frequency following volasertib treatment, indicating that functional LSCs were targeted by PLK1 inhibition. Together, these data strongly suggest that PLK1 impacts LSC function and stemness, and that PLK1 inhibitors are acting through an uncharacterized cell-cycle independent mechanism to target quiescent LSCs. To discover novel PLK1 signaling pathways, we carried out proximity-dependent biotinylation (PDB) to characterize the PLK1 interactome in serum-starved cells. Preliminary analysis has revealed a number of previously uncharacterized PLK1 interactors, including components of autophagy and apoptosis machinery, that are currently being validated in the hematopoietic cell context. Our study highlights PLK1 as a specific vulnerability of LSCs, and furthers the understanding of malignant PLK1 signaling in non-cell cycle related processes. Further insights into the underlying mechanisms will enable the development of effective therapies to target these pathways and improve patient outcomes in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of Polo-like Kinase 4 Induced Both Cell Intrinsic and Non-Cell Intrinsic Anti-Leukemia Effects on TP53-Mutated Acute Myeloid Leukemia

Inhibition of Polo-like Kinase 4 Induced Both Cell Intrinsic and Non-Cell Intrinsic Anti-Leukemia Effects on TP53-Mutated Acute Myeloid Leukemia

Single-cell transcriptomics reveals the role of Macrophage-Naïve CD4 + T cell interaction in the immunosuppressive microenvironment of primary liver carcinoma

BackgroundLiver carcinoma generally presents as an immunosuppressive microenvironment that promotes tumor evasion. The intercellular crosstalk of immune cells significantly influences the construction of an immunosuppressive microenvironment. This study aimed to investigate the important interactions between immune cells and their targeting drugs in liver carcinoma, by using single-cell and bulk transcriptomic data.MethodsSingle-cell and bulk transcriptomic data were retrieved from Gene Expression Omnibus (GSE159977, GSE136103, and GSE125449) and The Cancer Genome Atlas (TGCA-LIHC), respectively. Quality control, dimension reduction, clustering, and annotation were performed according to the Scanpy workflow based on Python. Cell–cell interactions were explored using the CellPhone database and CellChat. Trajectory analysis was executed using a partition-based graph abstraction method. The transcriptomic factors (TFs) were predicted using single-cell regulatory network inference and clustering (SCENIC). The target genes from TFs were used to establish a related score based on the TCGA cohort; this score was subsequently validated by survival, gene set enrichment, and immune cell infiltration analyses. Drug prediction was performed based on the Cancer Therapeutics Response Portal and PRISM Repurposing datasets.ResultsThirty-one patients at four different states, including health, hepatitis, cirrhosis, and cancer, were enrolled in this study. After dimension reduction and clustering, twenty-two clusters were identified. Cell–cell interaction analyses indicated that macrophage-naive CD4 + T cell interaction significantly affect cancerous state. In brief, macrophages interact with naive CD4 + T cells via different pathways in different states. The results of SCENIC indicated that macrophages present in cancer cells were similar to those present during cirrhosis. A macrophage-naive CD4 + T cell (MNT) score was generated by the SCENIC-derived target genes. Based on the MNT score, five relevant drugs (inhibitor of polo-like kinase 1, inhibitor of kinesin family member 11, dabrafenib, ispinesib, and epothilone-b) were predicted.ConclusionsThis study reveals the crucial role of macrophage-naive CD4 + T cell interaction in the immunosuppressive microenvironment of liver carcinoma. Tumor-associated macrophages may be derived from cirrhosis and can initiate liver carcinoma. Predictive drugs that target the macrophage-naive CD4 + T cell interaction may help to improve the immunosuppressive microenvironment and prevent immune evasion. The relevant mechanisms need to be further validated in experiments and cohort studies.Graphical

The kinase PLK1 promotes the development of Kras/Tp53-mutant lung adenocarcinoma through transcriptional activation of the receptor RET.

Increased abundance of polo-like kinase 1 (PLK1) is observed in various tumor types, particularly in lung adenocarcinoma (LUAD). Here, we found that PLK1 accelerated the progression of LUAD through a mechanism that was independent of its role in mediating mitotic cell division. Analysis of human tumor databases revealed that increased PLK1 abundance in LUAD correlated with mutations in KRAS and p53, with tumor stage, and with reduced survival in patients. In a mouse model of KRASG12D-driven, p53-deficient LUAD, PLK1 overexpression increased tumor burden, decreased tumor cell differentiation, and reduced animal survival. PLK1 overexpression in cultured cells and mice indirectly increased the expression of the gene encoding the receptor tyrosine kinase RET by phosphorylating the transcription factor TTF-1. Signaling by RET and mutant KRAS in these tumors converged to activate the mitogen-activated protein kinase (MAPK) pathway. Pharmacological inhibition of the MAPK pathway kinase MEK combined with inhibition of either RET or PLK1 markedly suppressed tumor growth. Our findings show that PLK1 can amplify MAPK signaling and reveal a potential target for stemming progression in lung cancers with high PLK1 abundance.

100 (PB090) - Synergy of the novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) BAL0891 with paclitaxel and carboplatin in mouse models of human cancer

Background: BAL0891 is a first-in-class dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). Both kinases collaborate in activating the spindle assembly checkpoint (SAC) to regulate chromosome alignment and segregation before the cell can exit mitosis. In vitro treatment of tumor cells with BAL0891 leads to rapid SAC disruption and accumulation of cells with aberrant chromosome numbers. The resultant genetic instability suggests a potential for combination effects with approved cytotoxic therapies, such as paclitaxel and carboplatin.

87 (PB077) - Optimal dosing of a novel PLK4 inhibitor nested in a phase II study of CFI-400945 in patients with Advanced/Metastatic Breast Cancer (MBC): Canadian Cancer Trials Group (CCTG) IND.237

Background: CFI-400945 is a selective oral inhibitor of Polo-like Kinase 4 (PLK4), a controller of centriole duplication and mitotic progression. CFI-400945 was identified by functional screening of genomically unstable breast cancer (BC). The phase I study escalated doses from 3 to 96 mg/d in a 3 + 3 design; dose-dependent neutropenia (ANC) was noted, and the recommended phase 2 dose (RP2D) declared at 64 mg. IND.237 (NCT01954316) is a phase 2 study in MBC with 3 cohorts, 2 enriched for PTEN status. Enrollment started in 2018 at 64 mg. The initial patients had higher than expected grade 3/4 ANC. Materials and Methods: The primary endpoint of the safety cohort was to confirm the RP2D for CFI-400945. Patients with MBC who met inclusion criteria for CCTG IND.237 were enrolled. A reverse 3 + 3 design was used, and patients enrolled at 64 mg, 48 mg, 40 mg and 32 mg dose levels (DL). In cycle (C)1, patients were treated 7 days on 7 days off for 28 d; for c2 onwards, CFI-400945 was administered continuously. Results: The most common severe adverse event (AE) was dose dependent ANC (table 1). After 80% of the first 5 patients developed grade 3 or 4 ANC, accrual was held while the protocol was amended to add a dose exploration cohort with pharmacokinetics in a reverse dose-finding approach. Based on the frequency of ANC and dose intensity, 32 mg was declared the new RP2D for CFI-400945. There were no non-hematologic severe AEs. PK analysis confirmed similar AUC for the 32 mg DL compared to the PK from the phase I study RP2D.Table 1Frequency of severe neutropenia by DL during C1 and C2 of safety cohort of IND.237DL# PatientsGrade 3 ANC(n(%))Grade 4 ANC(n(%))64 mg51 (20%)3(60%)48 mg65 (50%)2(33%)40 mg82 (25%)2(25%)32 mg60 (0%)0(0%) Open table in a new tab Refinement of the manufacturing process used for IND.237 and subsequent studies incorporated a more stable polymorph of CFI-400945 drug substance had the unexpected property of higher exposure at a given dose and was hypothesized to be due to slightly faster dissolution of the new tablets in the acidic environment of the stomach. Conclusions: The RP2D for CFI-400945 in patients with advanced breast cancer is 32 mg/day. This is 50% lower than the RP2D previously established in the phase I study due to an unexpected increase in exposure resulting from tablets containing a more stable polymorph of CFI-400945 drug substance. Acknowledgements: Sponsored by CCTG. Research supported by a Stand Up to Cancer Canada (SU2C), Canadian Breast Cancer Foundation Breast Cancer Dream Team Research Funding, with supplemental support of the Ontario Institute for Cancer Research through funds provided by the Government of Ontario (Funding Award Number: SU2C-AACR-DT-18-15). Research funding is administered by the American Association for Cancer Research International - Canada, the scientific partner of SU2C Canada. Conflict of interest: Ownership: none Advisory Board: Anthony Lott - Novartis, Eisai, Astra-Zeneca, Ipsen Terry Ng - Novartis, Knight Therapeutics, Boehringer-Ingelheim Arif Awan - AstraZeneca, Eli Lily, Exact Sciences, Exactis, Novartis, Pfizer, Roche Honoraria: Apotex, Roche Rossanna Pezo - Merck, Novartis, Pfizer, Astra Zeneca, Lilly, BMS, Exact Sciences, Myriad Genomics, Mylan Philippe Bedard - Seattle Genetics, Lilly, Amgen, Merck, Gilead Sciences, BMS, Pfizer John Hilton - Bristol-Myers-Squibb (BMS), AstraZeneca (AZ), Pfizer, Novartis, Eli Lilly, Puma, and Genomic Health Jacques Antoun Raphael - Lilly, Merck, AstraZeneca and Novartis. Moira Rushton - Gilead, Pfizer, Viatris David Cescon - Pfizer, AstraZeneca, Novartis, GlaxoSmithKline, Merck, Roche/Genentech, Exact Sciences, Gilead Sciences, Eisai Board of Directors: none Corporate-sponsored Research: Terry Ng - Takeda Oncology Rossanna Pezo - Merck and Novartis Philippe Bedard - Bristol-Myers Squibb, Sanofi, AstraZeneca, Genentech/ Roche, GlaxoSmithKline, Novartis, Nektar, Merck, Seattle Genetics, Immunomedics, Lilly, Amgen, Bicara, Zymeworks David Cescon - Merck, Roche/Genentech, GlaxoSmithKline, Pfizer, Inivata, AstraZeneca, Gilead Sciences Other Substantive Relationships: Terry Ng - Pharma-sponsored education session - ARIAD and Takeda Oncology Arif Awan - Travel: Roche Mark Bray - Full time employee of Treadwell Therapeutics Lesley Seymour & Pierre-Olivier Gaudreau - Funding to CCTG from UHN to partially support trial conduct Moira Rushton - Medical Montior: Pharmamatrix, Edmonton, Alberta David Cescon - Patent (US62/675,228) for methods of treating cancers characterized by a high expression level of spindle and kinetochore associated complex subunit 3 (ska3) gene. note CFI-402257 // CFI-400945 were developed at UHN

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phaseI/II/III clinical trials. Erythrocytes do not have anucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin‑V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Design and Synthesis of Aminopyrimidinyl Pyrazole Analogs as PLK1 Inhibitors Using Hybrid 3D-QSAR and Molecular Docking.

Cancer continues to be one of the world’s most severe public health issues. Polo-like kinase 1 (PLK1) is one of the most studied members of the polo-like kinase subfamily of serine/threonine protein kinases. PLK1 is a key mitotic regulator responsible for cell cycle processes, such as mitosis initiation, bipolar mitotic spindle formation, centrosome maturation, the metaphase to anaphase transition, and mitotic exit, whose overexpression is often associated with oncogenesis. Moreover, it is also involved in DNA damage response, autophagy, cytokine signaling, and apoptosis. Due to its fundamental role in cell cycle regulation, PLK1 has been linked to various types of cancer onset and progression, such as lung, colon, prostate, ovary, breast cancer, melanoma, and AML. Hence, PLK1 is recognized as a critical therapeutic target in the treatment of various proliferative diseases. PLK1 inhibitors developed in recent years have been researched and studied through clinical trials; however, most of them have failed because of their toxicity and poor therapeutic response. To design more potent and selective PLK1 inhibitors, we performed a receptor-based hybrid 3D-QSAR study of two datasets, possessing similar common scaffolds. The developed hybrid CoMFA (q2 = 0.628, r2 = 0.905) and CoMSIA (q2 = 0.580, r2 = 0.895) models showed admissible statistical results. Comprehensive, molecular docking of one of the most active compounds from the dataset and hybrid 3D-QSAR studies revealed important active site residues of PLK1 and requisite structural characteristics of ligand to design potent PLK1 inhibitors. Based on this information, we have proposed approximately 38 PLK1 inhibitors. The newly designed PLK1 inhibitors showed higher activity (predicted pIC50) than the most active compounds of all the derivatives selected for this study. We selected and synthesized two compounds, which were ultimately found to possess good IC50 values. Our design strategy provides insight into development of potent and selective PLK1 inhibitors.

Structure-based discovery of 1-(3-fluoro-5-(5-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)-3-(pyrimidin-5-yl)urea as a potent and selective nanomolar type-II PLK4 inhibitor

Polo-like kinase 4 (PLK4) is a serine/threonine protein kinase involved in regulating cell mitosis and centriole duplication, and has emerged as a therapeutic target for treating multiple cancers. At first, the design and in vitro validation of PLK4 inhibitors (12a-12e, 17a-17f, 22a-22e) bearing 1H-pyrazolo[3,4-b]pyridine scaffold was described and lead compound 22a (IC50 = 0.106 μM) was identified. Then, selectivity- and activity-guided development of a series of potent and selective type-II PLK4 inhibitors using a homology model approach was carried out. Further structure-based optimization resulted in a potent type-II PLK4 inhibitor 29u (IC50 = 0.026 μM), which exhibited outstanding selectivity in a panel of 47 kinases at a single concentration of 1.0 μM. Furthermore, compound 29u significantly inhibited the proliferation of breast cancer cell line MCF-7 with an IC50 value of 1.52 μM, while it exhibited no inhibitory effect on normal cell lines (L02 and HUVECs). Meanwhile, the clone formation, senescence and migration abilities of compound 29u were evaluated using MCF-7 cells. The detailed biological evaluation revealed that compound 29u could arrest cell division in S/G2 phase by inhibiting PLK4, and then affect the expression of downstream signalling pathway proteins regulated by PLK4. Moreover, the in vitro preliminary evaluation of the drug-like properties of compound 29u exhibited outstanding plasma stability, moderate liver microsomal stability, and low risk of drug-drug interactions (DDIs). The current discovery will support the further development of compound 29u as a lead compound for PLK4-targeted anticancer drug discovery and as a useful chemical probe for the further biological research of PLK4.

A high-throughput drug screen reveals means to differentiate triple-negative breast cancer

Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.

The mitotic regulator polo-like kinase 1 as a potential therapeutic target for c-Myc-overexpressing canine osteosarcomas.

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo-like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c-Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self-renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c-Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c-Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c-Myc and PLK1-overexpressing cOSA cell line (D17). PLK1 and c-Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c-Myc overexpression was associated with a significantly reduced overall survival (p= .003). Western Blot and RT-qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5-15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G2 /M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c-Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c-Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c-Myc.

Effects of the PLK4 inhibitor Centrinone on the biological behaviors of acute myeloid leukemia cell lines.

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, is frequently overexpressed in cancer cells. However, roles and the mechanism of PLK4 in the leukemiagenesis of acute myeloid leukemia (AML) remain unclear. In this study, the PLK4 inhibitor Centrinone and the shRNA knockdown were used to investigate roles and the mechanism of PLK4 in the leukemiagenesis of AML. Our results indicated that Centrinone inhibited the proliferation of AML cells in a dose- and time-dependent manner via reduced the expression of PLK4 both in the protein and mRNA levels. Moreover, colony formation assay revealed that Centrinone reduced the number and the size of the AML colonies. Centrinone induced AML cell apoptosis by increasing the activation of Caspase-3/poly ADP-ribose polymerase (PARP). Notably, Centrinone caused the G2/M phase cell cycle arrest by decreasing the expression of cell cycle-related proteins such as Cyclin A2, Cyclin B1, and Cyclin-dependent kinase 1 (CDK1). Consistent with above results, knockdown the expression of PLK4 also inhibited cell proliferation and colony formation, induced cell apoptosis, and caused G2/M phase cell cycle arrest without affecting cell differentiation. All in all, this study suggested that PLK4 inhibited the progression of AML in vitro, and these results herein may provide clues in roles of PLK4 in the leukemiagenesis of AML.

Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.

In chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (PLK1), two serine-threonine kinases involved in the maintenance of genomic stability by preserving a functional G2/M checkpoint, have been implicated in BCR::ABL1-independent resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate and in leukemic stem cell (LSC) persistence. It can be speculated that the observed deregulated activity of Aurora A and Plk1 enhances DNA damage, promoting the occurrence of additional genomic alterations contributing to TKI resistance and ultimately driving progression from chronic phase to blast crisis (BC). In this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or PLK1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Danusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors from BC patients. Our results may have implications for the development of innovative therapeutic approaches aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.

Polo-like Kinase 1 Inhibitors in Human Cancer Therapy: Development and Therapeutic Potential.

Polo-like kinase 1 (PLK1) plays an important role in a variety of cellular functions, including the regulation of mitosis, DNA replication, autophagy, and the epithelial-mesenchymal transition (EMT). PLK1 overexpression is often associated with cell proliferation and poor prognosis in cancer patients, making it a promising antitumor target. To date, at least 10 PLK1 inhibitors (PLK1i) have been entered into clinical trials, among which the typical kinase domain (KD) inhibitor BI 6727 (volasertib) was granted "breakthrough therapy designation" by the FDA in 2013. Unfortunately, many other KD inhibitors showed poor specificity, resulting in dose-limiting toxicity, which has greatly impeded their development. Researchers recently discovered many PLK1i with higher selectivity, stronger potency, and better absorption, distribution, metabolism, and elimination (ADME) characteristics. In this review, we emphasize the structure-activity relationships (SARs) of PLK1i, providing insights into new drugs targeting PLK1 for antitumor clinical practice.

Investigation of the potential antitumor activity of PLK1 inhibitor SBE13 in colon cancer cell line HT29

Background: High levels of Polo-like kinase 1 (PLK1), which have been found to be abnormally expressed in many tumor types, are known to contribute to tumorigenesis and poor prognosis. Therefore, specific targeting of PLK1 is an important strategy for cancer therapy. In this study, it was aimed to investigate the cytotoxic effect of SBE13, one of the PLK1 inhibitors, against HT29 colon adenocarcinoma cells and its apoptotic potential.&#x0D; Methods: The cytotoxic effect of SBE13 on HT29 was determined by XTT colorimetric assay. Flow cytometry was also used to determine apoptosis. &#x0D; Results: SBE13 showed a dose-dependent cytotoxic effect in HT29 cells and its IC50 value was calculated as 11.79 µM for 48 h. Moreover, the Annexin V binding assay revealed that SBE13 treatment significantly increased the apoptosis in HT29 cells.&#x0D; Conclusion: Generally, SBE13 exerts a cytotoxic effect promoted by apoptosis in colon cancer cells HT29. Although the anticancer efficacy of SBE13 in colon cancer is promising, this potential effect should be reinforced by further studies.

Polo-like kinase 4 is associated with advanced TNM stages and reduced survival and its inhibition improves chemosensitivity in colorectal cancer.

High expression of polo-like kinase 4 (PLK4) promotes tumorigenesis and is correlated with poor prognosis in several kinds of cancer. However, the prognostic value of PLK4 in colorectal cancer (CRC) has not been elucidated. The aim of the present study was to investigate the association between PLK4 and the prognosis and effect of PLK4 inhibition on chemosensitivity in CRC. A total of 142 patients with CRC were enrolled, and 142 pairs of CRC and para-carcinoma tissues were used to measure PLK4 protein expression using immunohistochemistry (IHC). Among them, 69 pairs were used to detect PLK4 mRNA expression using reverse transcription-quantitative PCR. In addition, PLK4-small interfering RNA (siRNA) was transfected into CRC cells, followed by 5-fluorouracil (5-FU) treatment for it was a fundamental chemotherapy for CRC. In addition, western blotting was used to detect PLK4 protein expression among human colonic epithelial cell and human CRC cell lines, including HCT-116, LoVo, SW480 and HT-29, as well as nuclear translocation of β-catenin. The IHC score and mRNA expression of PLK4 were higher in CRC tissues compared with para-carcinoma tissues (both P<0.001). Furthermore, the IHC score of tumor PLK4 was not correlated with pathological grade (P=0.585), T stage (P=0.357), N stage (P=0.107225) or tumor-node-metastasis (TNM) stage (P=0.093). The mRNA expression of tumor PLK4 was positively correlated with N stage (P=0.019) and TNM stage (P=0.004), but not with pathological grade (P=0.498) or T stage (P=0.112). Of note, the high protein expression of tumor PLK4 was an independent factor for poor overall survival (OS; P=0.048). In addition, PLK4 was elevated in CRC cell lines; PLK4-siRNA reduced the 50% inhibitory concentration value of 5-FU in HCT-116 (4.4±0.1 µM vs. 7.6±1.4 µM) and LoVo cells (5.5±0.6 µM vs. 9.9±1.8 µM) (both P<0.05). Besides, PLK4-siRNA decreased nuclear translocation of β-catenin. In conclusion, the high expression of tumor PLK4 was associated with advanced TNM stage and shorter OS in patients with CRC. In addition, targeting PLK4 improved chemosensitivity in CRC cells.

Antitumor activity of NMS-P937, specific small-molecule polo-like kinase 1 inhibitor, in PC3 human prostate cancer, HeLa cervical cancer, and SKOV-3 ovarian cancer cell lines

Objectives: We aimed to investigate the antitumor activity of NMS-P937, a specific small-molecule polo-like kinase 1 (PLK1) inhibitor, in PC3 human prostate cancer, HeLa cervical cancer, and SKOV-3 ovarian cancer cell lines.&#x0D; Methods: PC3, HeLa, and SKOV-3 cells were treated with NMS-P937 for 48 h. The viability was analyzed by XTT colorimetric assay, and since PC3 was found to be the most sensitive cell line, total oxidant status (TOS) values were evaluated in NMS-P937-treated and non-treated PC3 cells via TOS assay.&#x0D; Results: The proliferation of cancer cell lines was moderately inhibited by NMS-P937 in conjunction with the increase in concentration. The IC50 values of NMS-P937 in PC3, HeLa, and SKOV-3 cells were recorded as 27.3, 69.7, and 79.3 μM respectively, for 48 h. TOS was measured in control and NMS-P937-treated PC3 cells and calculated as 3.15±0.36 and 4.49±0.64, respectively, indicating the increased oxidative stress under the influence of the study compound (p=0.035).&#x0D; Conclusions: The PLK1 inhibitor NMS-P937 reduces the activity of cancer cell lines consisting of PC3 human prostate cancer, HeLa cervical cancer, and SKOV-3 ovarian cancer in a dose-dependent manner. This compound increases oxidative stress, and this may play a pivotal role in the cytotoxic activity of the compound in PC3 cells. However, there is still a need to carry out both in vitro and in vivo studies, including different cancer cell lines and tumor models, and to reveal the adverse effects that may develop.