Key Mitotic Regulator Research Articles

Aurora Kinases: Their Role in Cancer and Cellular Processes

Aurora kinases, belonging to a highly conserved family of serine/threonine kinases with critical roles in the regulation of the cell cycle, comprise three members: Aurora kinase A, B, and C, which serve as key mitotic regulators essential for maintaining chromosome stability. Aurora kinases play crucial roles in multiple events in mitotic such as the coordination of chromosomal and cytoskeletal events, regulation of the spindle assembly checkpoint pathway and cytokinesis to ensure the smooth progression of the cell cycle. Besides their mitotic functions, Aurora kinases are also involved in the regulation of meiosis. Gene amplification/mutation and overexpression of Aurora kinases have been detected in various solid and haematological cancers. In human tumours, Aurora kinases exhibit oncogenic roles associated with their mitotic roles, which drive the cancer cell proliferation and survival. Deregulation of Aurora kinase activity causes failure in centrosome function, spindle assembly, chromosomal alignment, and cytokinesis, eventually resulting in the mitotic abnormalities and genetic instability. These findings emphasize the crucial functions of Aurora kinases in cancer, prompting their recognition as valuable targets for cancer therapy. This review provides an overview of the structures and functions of Aurora kinases and sheds light on their oncogenic roles in cancer.

Discovery of novel, potent and orally available benzoazipinone derivatives that elicit MKLP2-inhibitory phenotypes

Mitotic kinesin-like protein 2 (MKLP2/KIF20A) is a key mitotic regulator frequently overexpressed in human malignancies and its abundance is positively correlated with poor outcomes of the disease. Despite extensive research on MKLP2 as a potential target for oncology, the development of small-molecule inhibitors specific to MKLP2 remains limited. We have previously identified a benzoazipinone compound, HJ81 as a potent disruptor of Aurora kinase B (AURKB) localization during late mitosis. This study reveals that such disruption results from a failure of AURKB relocation at the onset of anaphase and this phenomenon can be specifically attributed to the disablement of MKLP2, a recognized facilitator of the relocation process. Further optimization of HJ81 leads to identifying compounds such as 12a as promising lead inhibitors of MKLP2-mediated processes, with improved pharmacokinetic properties. 12a inhibits the microtubule-stimulated ATPase activity of the recombinant MKLP2 in vitro. Significant suppression of tumor growth was observed in mice bearing the Calu-6 lung cancer cell line when treated with 12a at a well-tolerated dose. Overall, our findings suggest that benzoazipinone derivatives represent a novel chemical scaffold with the potential to be developed to mimic MKLP2 inhibition for cancer treatment.

Aurora B facilitates cholangiocarcinoma progression by stabilizing c-Myc.

Cholangiocarcinoma (CCA), a malignancy that arises from biliary epithelial cells, has a dismal prognosis, and few targeted therapies are available. Aurora B, a key mitotic regulator, has been reported to be involved in the progression of various tumors, yet its role in CCA is still unclarified. Human CCA tissues and murine spontaneous CCA models were used to assess Aurora B expression in CCA. A loss-of-function model was constructed in CCA cells to determine the role of Aurora B in CCA progression. Subcutaneous and liver orthotopic xenograft models were used to assess the therapeutic potential of Aurora B inhibitors in CCA. In murine spontaneous CCA models, Aurora B was significantly upregulated. Elevated Aurora B expression was also observed in 62.3% of human specimens in our validation cohort (143 CCA specimens), and high Aurora B expression was positively correlated with pathological parameters of tumors and poor survival. Knockdown of Aurora B by siRNA and heteroduplex oligonucleotide (HDO) or an Aurora B kinase inhibitor (AZD1152) significantly suppressed CCA progression via G2/M arrest induction. An interaction between Aurora B and c-Myc was found in CCA cells. Targeting Aurora B significantly reduced this interaction and accelerated the proteasomal degradation of c-Myc, suggesting that Aurora B promoted the malignant properties of CCA by stabilizing c-Myc. Furthermore, sequential application of AZD1152 or Aurora B HDO drastically improved the efficacy of gemcitabine in CCA. Aurora B plays an essential role in CCA progression by modulating c-Myc stability and represents a new target for treatment and chemosensitization in CCA.

Loss of the mitochondrial protein SPD-3 elevates PLK-1 levels and dysregulates mitotic events.

In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early Caenorhabditis elegans embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the spd-3(oj35) mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

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.

Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.

Abstract 2299: Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability

Abstract Ewing sarcoma is the second most common bone cancer that develops in adolescents and young adults. Majority of Ewing sarcoma patients (~85%) carry an EWSR1-FLI1 fusion gene derived from t(11;22)(q24;q12) chromosomal translocation. The formation of EWSR1-FLI1 leads to the loss of one wild type EWSR1 allele, however, how loss of the gene contributes to the pathogenesis of Ewing sarcoma remains unclear. Our previous study revealed that the EWSR1 is required for a faithful mitosis. We demonstrated that the mutant ewsa (a homologue of human EWSR1) zebrafish induces mitotic defects, aneuploidy, and promotes tumorigenesis (with tp53 mutation). Consistently, Ewing sarcoma patients display high incidence of aneuploidy (e.g. approximately 47% of the patients display trisomy 8). For these reasons, we aim to elucidate the molecular function of EWSR1 in faithful chromosome segregation and in maintenance of chromosome stability. To accomplish our goal, we employed the Auxin-Inducible Degron (AID) system because it allows us to conditionally degrade EWSR1 proteins in a cell line. Both EWSR1 alleles were tagged with mini-AID using CRISPR/Cas9 system in a stable DLD-1 cell that expresses a plant E3 ligase TIR1. The western blot verified the efficient degradation of EWSR1 when the cells were treated with Auxin (AUX+) for 24hrs. To determine whether EWSR1 maintains chromosomal stability, the cells were synchronized to pro-metaphase using thymidine/nocodazole protocol, and were treated with AUX for 24hrs (underwent one cell cycle) concurrently. The EWSR1 knockdown (AUX+) cells displayed significantly higher incidence of lagging chromosomes compared to the control (AUX-) cells. Furthermore, immunocytochemistry using the antibody against the key mitotic regulator, Aurora B, revealed that the EWSR1 knockdown (AUX+) cells displayed significantly less incidence of Aurora B localization at inner centromere, but higher incidence of localization on kinetochore compared to the control (AUX-) cells. Consistently, when the cells that were treated with AUX for 48hrs (underwent two cell cycles), it displayed significantly higher incidence of aberrant numbers of chromosome compared to the control (AUX-) cells. In general, impaired chromosome segregation leads to mitotic delay, accompanied by the activation of mitotic checkpoint. However, the EWSR1 knockdown (AUX+) cells did not undergo any arrest in mitosis. Therefore, our data suggest that the EWSR1 knockdown (AUX+) cells not only induces aneuploidy by inducing lagging chromosome, but also it may override mitotic checkpoint. Currently, we are in a process of elucidating the molecular mechanism of EWSR1 knockdown dependent mitigation of mitotic arrest. Successful completion of the study may shed light on how/whether haploinsufficiency of EWSR1 contributes to the molecular pathogenesis of Ewing sarcoma. Citation Format: Haeyoung Kim, Mizuki Azuma. Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability [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 2299.

Crosstalk between Plk1, p53, cell cycle, and G2/M DNA damage checkpoint regulation in cancer: computational modeling and analysis

Different cancer cell lines can have varying responses to the same perturbations or stressful conditions. Cancer cells that have DNA damage checkpoint-related mutations are often more sensitive to gene perturbations including altered Plk1 and p53 activities than cancer cells without these mutations. The perturbations often induce a cell cycle arrest in the former cancer, whereas they only delay the cell cycle progression in the latter cancer. To study crosstalk between Plk1, p53, and G2/M DNA damage checkpoint leading to differential cell cycle regulations, we developed a computational model by extending our recently developed model of mitotic cell cycle and including these key interactions. We have used the model to analyze the cancer cell cycle progression under various gene perturbations including Plk1-depletion conditions. We also analyzed mutations and perturbations in approximately 1800 different cell lines available in the Cancer Dependency Map and grouped lines by genes that are represented in our model. Our model successfully explained phenotypes of various cancer cell lines under different gene perturbations. Several sensitivity analysis approaches were used to identify the range of key parameter values that lead to the cell cycle arrest in cancer cells. Our resulting model can be used to predict the effect of potential treatments targeting key mitotic and DNA damage checkpoint regulators on cell cycle progression of different types of cancer cells.

The RAD51 recombinase protects mitotic chromatin in human cells

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

Modelling the Functions of Polo-Like Kinases in Mice and Their Applications as Cancer Targets with a Special Focus on Ovarian Cancer.

Polo-like kinases (PLKs) belong to a five-membered family of highly conserved serine/threonine kinases (PLK1-5) that play differentiated and essential roles as key mitotic kinases and cell cycle regulators and with this in proliferation and cellular growth. Besides, evidence is accumulating for complex and vital non-mitotic functions of PLKs. Dysregulation of PLKs is widely associated with tumorigenesis and by this, PLKs have gained increasing significance as attractive targets in cancer with diagnostic, prognostic and therapeutic potential. PLK1 has proved to have strong clinical relevance as it was found to be over-expressed in different cancer types and linked to poor patient prognosis. Targeting the diverse functions of PLKs (tumor suppressor, oncogenic) are currently at the center of numerous investigations in particular with the inhibition of PLK1 and PLK4, respectively in multiple cancer trials. Functions of PLKs and the effects of their inhibition have been extensively studied in cancer cell culture models but information is rare on how these drugs affect benign tissues and organs. As a step further towards clinical application as cancer targets, mouse models therefore play a central role. Modelling PLK function in animal models, e.g., by gene disruption or by treatment with small molecule PLK inhibitors offers promising possibilities to unveil the biological significance of PLKs in cancer maintenance and progression and give important information on PLKs’ applicability as cancer targets. In this review we aim at summarizing the approaches of modelling PLK function in mice so far with a special glimpse on the significance of PLKs in ovarian cancer and of orthotopic cancer models used in this fatal malignancy.

Phase I study of the combination of alisertib (MLN8237) and gemcitabine in advanced solid tumors.

3589 Background: Aurora Kinase A (AKA) is a key mitotic regulator overexpressed in multiple solid tumors. This open-label dose escalation and expansion phase I study evaluated the safety and tolerability of alisertib (MLN8237), an oral AKA inhibitor, in combination with gemcitabine. Methods: In dose escalation, patients (pts) > 18y with refractory solid tumors received 28-day cycles of gemcitabine on days 1, 8, 15 and alisertib twice daily on days 1-3, 8-10, and 15-17. Gemcitabine was given at 1000mg/m2. Four dose levels (DL) of alisertib (20-50mg) were given per 3+3 design to investigate dose limiting toxicities (DLT) in cycle 1, to determine maximum tolerated dose (MTD) and recommended phase II dose (RP2D). In dose expansion, advanced pancreatic adenocarcinoma pts received the MTD dose twice daily on a modified dosing schedule to allow for pharmacokinetic (PK) evaluation. Anti-tumor activity was assessed by response rate (RECIST 1.1) and progression-free survival (PFS). PK evaluation of plasma gemcitabine and alisertib was performed on all pts enrolled in the dose expansion. PK sampling was performed before treatment, immediately after gemcitabine infusion, and at other pre-specified post-infusion timepoints. Results: Twenty-six pts were treated in total: 21 pts in dose escalation and 5 pts in dose expansion. Overall, median age was 57y [42-82]; 50% male; 62% PS 1 (16 pts); 2 [0-7] median prior therapies. In the dose escalation phase, 9 tumor types were included and NSCLC was most common (7 pts). Maximum administered dose (DL4) achieved 900 mg alisertib per cycle and was tolerated (1 DLT in 6 pts). The dose expansion phase enrolled 5 pts with advanced pancreatic adenocarcinoma; median age 63y [48-82]; 60% male; 60% PS 1 (3 pts); 2 [1-2] median prior therapies. Grade ≥3 TRAEs were observed in 73% of all pts and were predominantly hematologic, including neutropenia (54%), leukopenia (50%), and lymphopenia (31%). Similar TRAEs were seen at DL4; all 14 pts experienced neutropenia with 64% experiencing grade ≥3 neutropenia. Fourteen of 23 evaluable pts (61%) had stable disease and 2 pts (9%) had partial response (PR) as best overall response. Median PFS was 2.9 months (95% CI 2.0-4.2). Analysis of PK data is ongoing and will be reported. Conclusions: Alisertib can be safely administered with gemcitabine. RP2D for alisertib is 50 mg PO BID in combination with full dose gemcitabine. Best response was at least stable disease in a majority of pts with PR observed in 9% of this heavily pretreated group of patients. Most grade ≥3 TRAEs were hematologic. Results of PK studies will also be reported. Clinical trial information: NCT01924260 .

Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent—or independent—on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

The anaphase-promoting complex: A key mitotic regulator associated with somatic mutations occurring in cancer.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that helps control chromosome separation and exit from mitosis in many different kinds of organisms, including yeast, flies, worms, and humans. This review represents a new perspective on the connection between APC/C subunit mutations and cancer. The complex nature of APC/C and limited mutation analysis of its subunits has made it difficult to determine the relationship of each subunit to cancer. In this work, cancer genomic data were examined to identify APC/C subunits with a greater than 5% alteration frequency in 11 representative cancers using the cBioPortal database. Using the Genetic Determinants of Cancer Patient Survival database, APC/C subunits were also studied and found to be significantly associated with poor patient prognosis in several cases. In comparing these two kinds of cancer genomics data to published large-scale genomic analyses looking for cancer driver genes, ANAPC1 and ANAPC3/CDC27 stood out as being represented in all three types of analyses. Seven other subunits were found to be associated both with >5% alteration frequency in certain cancers and being associated with an effect on cancer patient prognosis. The aim of this review is to provide new approaches for investigators conducting in vivo studies of APC/C subunits and cancer progression. In turn, a better understanding of these APC/C subunits and their role in different cancers will help scientists design drugs that are more precisely targeted to certain cancers, using APC/C mutation status as a biomarker.

Inhibition of Aurora A enhances radiosensitivity in selected lung cancer cell lines

BackgroundIn mammalian cells, Aurora serine/threonine kinases (Aurora A, B, and C) are expressed in a cell cycle-dependent fashion as key mitotic regulators required for the maintenance of chromosomal stability. Aurora-A (AURKA) has been proven to be an oncogene in a variety of cancers; however, whether its expression relates to patient survival and the association with radiotherapy remains unclear in non-small cell lung cancer (NSCLC).MethodsHere, we first analyzed AURKA expression in 63 NSCLC tumor samples by immunohistochemistry (IHC) and used an MTS assay to compare cell survival by targeting AURKA with MLN8237 (Alisertib) in H460 and HCC2429 (P53-competent), and H1299 (P53-deficient) cell lines. The radiosensitivity of MLN8237 was further evaluated by clonogenic assay. Finally, we examined the effect of combining radiation and AURKA inhibition in vivo with a xenograft model and explored the potential mechanism.ResultsWe found that increased AURKA expression correlated with decreased time to progression and overall survival (p = 0.0447 and 0.0096, respectively). AURKA inhibition using 100 nM MLN8237 for 48 h decreases cell growth in a partially P53-dependent manner, and the survival rates of H460, HCC2429, and H1299 cells were 56, 50, and 77%, respectively. In addition, the survival of H1299 cells decreased 27% after ectopic restoration of P53 expression, and the radiotherapy enhancement was also influenced by P53 expression (DER H460 = 1.33; HCC2429 = 1.35; H1299 = 1.02). Furthermore, tumor growth of H460 was delayed significantly in a subcutaneous mouse model exposed to both MLN8237 and radiation.ConclusionsTaken together, our results confirmed that the expression of AURKA correlated with decreased NSCLC patient survival, and it might be a promising inhibition target when combined with radiotherapy, especially for P53-competent lung cancer cells. Modulation of P53 function could provide a new option for reversing cell resistance to the AURKA inhibitor MLN8237, which deserves further investigation.

PLK1 and EGFR targeted nanoparticle as a radiation sensitizer for non-small cell lung cancer.

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR+NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR+cancer types such as colorectal and breast cancer is also demonstrated.

Cdc14 activation requires coordinated Cdk1-dependent phosphorylation of Net1 and PP2A-Cdc55 at anaphase onset.

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2ACdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2ACdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2ACdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.

PP2A-B55/SUR-6 collaborates with the nuclear lamina for centrosome separation during mitotic entry.

Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope–based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6–regulated nuclear envelope–based dynein pulling forces and centrosome–nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

Regulating a key mitotic regulator, polo-like kinase 1 (PLK1).

During cell division, duplicated genetic material is separated into two distinct daughter cells. This process is essential for initial tissue formation during development and to maintain tissue integrity throughout an organism's lifetime. To ensure the efficacy and efficiency of this process, the cell employs a variety of regulatory and signaling proteins that function as mitotic regulators and checkpoint proteins. One vital mitotic regulator is polo‐like kinase 1 (PLK1), a highly conserved member of the polo‐like kinase family. Unique from its paralogues, it functions specifically during mitosis as a regulator of cell division. PLK1 is spatially and temporally enriched at three distinct subcellular locales; the mitotic centrosomes, kinetochores, and the cytokinetic midbody. These localization patterns allow PLK1 to phosphorylate specific downstream targets to regulate mitosis. In this review, we will explore how polo‐like kinases were originally discovered and diverged into the five paralogues (PLK1‐5) in mammals. We will then focus specifically on the most conserved, PLK1, where we will discuss what is known about how its activity is modulated, its role during the cell cycle, and new, innovative tools that have been developed to examine its function and interactions in cells.

WAC Promotes Polo-like Kinase 1 Activation for Timely Mitotic Entry

The key mitotic regulator Polo-like kinase 1 (Plk1) isactivated during G2 phase by Aurora A kinase (AurkA)-mediated phosphorylation of its activation loop, which is important for timely mitotic entry. The mechanism for Plk1 activation remains incompletely understood. Here, we report that the activation of Plk1 requires WAC, a WW domain-containing adaptor protein with a coiled-coil region that predominantly localizes to the nucleus in interphase. Cyclin-dependent kinase 1 (Cdk1) phosphorylates WAC, priming its direct interaction with the polo-box domain of Plk1. Knockdown of WAC compromises Plk1 activity and delays mitotic entry. These defects are rescued by exogenous expression of wild-type WAC, but not the Plk1-binding-deficient mutant. WAC also binds AurkA and can enhance Plk1 phosphorylation by AurkA invitro. Taken together, these results indicate an important role for WAC in promoting Plk1 activation and the timely entry into mitosis.