Polo-like Kinase Research Articles

Chemical Probe for Imaging of Polo-like Kinase 4 and Centrioles.

Polo-like kinase (Plk4) is a serine/threonine-protein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy. Such direct labeling of Plk4 results in an increased resolution in STED imaging compared with using anti-Plk4 antibodies, underlining the importance of direct labeling strategies for super-resolution microscopy. We anticipate that Cen-TCO will become an important tool for investigating the biology of Plk4 and of centrioles.

Pentafuhalol-B, a Phlorotannin from Brown Algae, Strongly Inhibits the PLK-1 Overexpression in Cancer Cells as Revealed by Computational Analysis.

Polo-like kinase-1 (PLK-1) is an essential mitotic serine/threonine (Ser/Thr) kinase that belongs to the Polo-like kinase (PLK) family and is overexpressed in non-small cell lung cancer (NSCLC) via promotion of cell division. Therefore, PLK-1 may act as a promising target for the therapeutic cure of various cancers. Although a variety of anti-cancer drugs, both synthetic and naturally occurring, such as volasertib, onvansertib, thymoquinone, and quercetin, are available either alone or in combination with other therapies, they have limited efficacy, especially in the advanced stages of cancer. To the best of our knowledge, no anticancer agent has been reported from marine algae or microorganisms to date. Thus, the aim of the present study is a high-throughput virtual screening of phlorotannins, obtained from edible brown algae, using molecular docking and molecular dynamic simulation analysis. Among these, Pentafuhalol-B (PtB) showed the lowest binding energy (best of triplicate runs) against the target protein PLK-1 as compared to the reference drug volasertib. Further, in MD simulation (best of triplicate runs), the PtB-PLK-1 complex displayed stability in an implicit water system through the formation of strong molecular interactions. Additionally, MMGBSA calculation (best of triplicate runs) was also performed to validate the PtB-PLK-1 complex binding affinities and stability. Moreover, the chemical reactivity of PtB towards the PLK-1 target was also optimised using density functional theory (DFT) calculations, which exhibited a lower HOMO-LUMO energy gap. Overall, these studies suggest that PtB binds strongly within the pocket sites of PLK-1 through the formation of a stable complex, and also shows higher chemical reactivity than the reference drug volasertib. The present study demonstrated the inhibitory nature of PtB against the PLK-1 protein, establishing its potential usefulness as a small molecule inhibitor for the treatment of different types of cancer.

Crystal Structures of Plk1 Polo-Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2-Derived Peptide.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.

Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.

Design, synthesis, and biological evaluation of novel molecules as potent inhibitors of PLK1

Polo-like kinase 1 (PLK1) is an attractive therapeutic target for the treatment of tumors, as it is an essential cell-cycle regulator frequently overexpressed in tumor tissues. PLK1 can promote tumor invasion and metastasis, and is often associated with poor prognosis in cancer patients. However, no PLK1 inhibitor has been granted marketing approval until now. Therefore, more potentially promising PLK1 inhibitors need to be investigated. In this study, a series of novel inhibitors targeting PLK1 was designed and optimized derived from a new scaffold. After synthesis and characterization, we obtained the structure–activity relationship and led to the discovery of the most promising compound 30e for PLK1. The antiproliferative activity against HCT116 cells (IC50 = 5 nM versus 45 nM for onvansertib) and the cellular permeability and efflux ratio were significantly improved (PappA→B = 2.03 versus 0.345 and efflux ratio = 1.65 versus 94.7 for 30e and onvansertib, respectively). Further in vivo studies indicated that 30e had favorable antitumor activity with 116.2% tumor growth inhibition (TGI) in comparison with TGI of 43.0% for onvansertib. Furthermore, 30e improved volume of tumor tissue distribution in mice as compared to onvansertib. This initial study on 30e holds promise for further development of an antitumor agent.

Structural Basis for Variations in Polo-like Kinase 1 Conformation and Intracellular Stability Induced by ATP-Competitive and Novel Noncompetitive Abbapolin Inhibitors.

Polo-like kinase 1 (PLK1) is an essential protein kinase with multiple roles in mitotic progression. PLK1 consists of a kinase domain (KD) and a phosphopeptide-binding polobox domain (PBD), which is responsible for substrate recognition and subcellular localization. The regulation of PLK1 involves an autoinhibitory conformation in which KD and PBD interact. Our previous work identified PBD-binding molecules termed abbapolins that inhibit the cellular phosphorylation of a PLK1 substrate and induce the loss of intracellular PLK1. Here, we describe a comparison of the abbapolin activity with that of KD inhibitors to gain insight into conformational features of PLK1. As measured by a cellular thermal shift assay, abbapolins produce ligand-induced thermal stabilization of PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggesting that catalytic-site binding causes a less thermally stable PLK1 conformation. Binding measurements with full-length PLK1 and a KD inhibitor also demonstrated a conformational change. Interestingly, the cellular consequences of KD versus PBD engagement contrast as KD binding causes the accumulation of intracellular PLK1, whereas PBD binding produces a striking loss of nuclear PLK1. These data are consistent with the relief of autoinhibited PLK1 by KD binders; an explanation for these observations is presented using structures for the catalytic domain and full-length PLK1 predicted by AlphaFold. Collectively, the results highlight an underappreciated aspect of targeting PLK1, namely, conformational perturbations induced by KD versus PBD binding. In addition to their significance for PBD-binding ligands, these observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 noncatalytic functions, which may explain their lack of clinical efficacy to date.

Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.

BI6727 and GSK461364A, potent PLK1 inhibitors induce G2/M arrest and apoptosis against cholangiocarcinoma cell lines

Polo-like kinase 1 (PLK1) is an essential mitotic checkpoint protein that plays a key role in cell cycle division. Overexpression of PLK1 has been associated with poor prognosis in various cancers. Cholangiocarcinoma (CCA) is a lethal bile duct cancer and the current treatments in inoperable patients have not been satisfactory. In order to develop novel targeted therapies, we investigated the efficacy of BI6727 (volasertib) and GSK461364A, polo-like kinase 1 (PLK1) inhibitors in KKU-100 and KKU-213A CCA cell lines. PLK1 expression was significantly up-regulated in CCA cases compared with normal tissues based on the results derived from GEPIA. Western blot results exhibited PLK1 protein expression in both CCA cell lines. Molecular dynamics simulations and free energy calculations based on MM/GBSA method revealed that BI6727-PLK1 and GSK461364A-PLK1 complexes were stable in an aqueous environment, and their complexation was mainly driven by Van der Waals interaction. BI6727 and GSK461364A clearly suppressed CCA cell proliferation and induced G2/M arrest, accompanied with upregulation of cyclin B1 and phosphorylated Histone H3 at Ser10 (pS10H3), specific markers of mitosis. Furthermore, both compounds triggered mitotic catastrophe followed by cell apoptosis via activation of PARP and Caspase 3, as well as downregulation of Mcl-1 anti-apoptotic protein in both CCA cell lines. In conclusion, pharmacologic PLK1 inhibition by BI6727 and GSK461364A blocked survival of CCA cells by several mechanisms. Our study provides evidence that BI6727 and GSK461364A could be alternative drugs and have potential implications at the clinical level for CCA therapy.

Study on the Mechanism of Astragalus Polysaccharides on Cervical Cancer Based on Network Pharmacology.

Astragalus polysaccharides (APS) is a natural phytochemical which has been extensively utilized for anti-tumor therapy over the past few years. However, its impact on cervical cancer (CC) has rarely been studied. To clarify the exact mechanism of anti-cancer effects of Astragalus polysaccharides (APS) on Cervical Cancer (CC), we screened differentially expressed genes (DEGs) from The Cancer Genome Atlas (TCGA) to construct the cancer network. Then we performed functional enrichment analysis with gene ontology (GO) and KEGG pathway analyses, constructed protein-protein interaction (PPI) network, and performed molecular docking (MD) analysis to identify the key gene for docking with APS. Further, we observed the effects of APS on cell proliferation, cell cycle, and apoptosis experiments in HeLa cells. qRT-PCR and western blot were used to detect the expression of target genes. A total of 793 DEGs were screened using criteria, which included 541 genes that were upregulated and 251 genes that were down-regulated. Using topological attributes for identifying critical targets, molecular docking (MD), and survival analyses, this study predicted the APS targets: POLO-like kinase 1(PLK1), Cyclin-cell division 20(CDC20), and Cyclin-dependent kinase 1 (CDK1), which regulated HeLa cells. The results of cell proliferation, cell cycle, and apoptosis experiments concluded that APS inhibited the development of HeLa cells in a concentrationdependent manner. Also, qRT-PCR and western blot experiments demonstrated that APS could significantly down-regulate the expression of PLK1, CDC20, and CDK1 in the CC cells. The result revealed that APS might have a therapeutic potential in treating CC and might permit intervention with treatments targeting PLK1, CDC20, and CDK1.

USP1 promotes the aerobic glycolysis and progression of T-cell acute lymphoblastic leukemia via PLK1/LDHA axis.

The effect of aerobic glycolysis remains elusive in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Increasing evidence has revealed that dysregulation of deubiquitination is involved in glycolysis, by targeting glycolytic rate-limiting enzymes. Here, we demonstrated that upregulated deubiquitinase ubiquitin-specific peptidase 1 (USP1) expression correlated with poor prognosis in pediatric primary T-ALL samples. USP1 depletion abolished cellular proliferation and attenuated glycolytic metabolism. Invivo experiments showed that USP1 suppression decreased leukemia progression in nude mice. Inhibition of USP1 caused a decrease in both mRNA and protein levels in lactate dehydrogenase A (LDHA), a critical glycolytic enzyme. Moreover, USP1 interacted with and deubiquitinated polo-like kinase 1 (PLK1), a critical regulator of glycolysis. Overexpression of USP1 with upregulated PLK1 was observed in most samples of patients with T-ALL. In addition, PLK1 inhibition reduced LDHA expression and abrogated the USP1-mediated increase of cell proliferation and lactate level. Ectopic expression of LDHA can rescue the suppressive effect of USP1 silencing on cell growth and lactate production. Pharmacological inhibition of USP1 by ML323 exhibited cell cytotoxicity in human T-ALL cells. Taken together, our results demonstrated that USP1 may be a promising therapeutic target in pediatric T-ALL.

DYRK1A-mediated PLK2 phosphorylation regulates the proliferation and invasion of glioblastoma cells.

Polo-like kinases (PLKs) are a family of serine-threonine kinases that exert regulatory effects on diverse cellular processes. Dysregulation of PLKs has been implicated in multiple cancers, including glioblastoma (GBM). Notably, PLK2 expression in GBM tumor tissue is lower than that in normal brains. Notably, high PLK2 expression is significantly correlated with poor prognosis. Thus, it can be inferred that PLK2 expression alone may not be sufficient for accurate prognosis evaluation, and there are unknown mechanisms underlying PLK2 regulation. In the present study, it was demonstrated that dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) interacts with and phosphorylates PLK2 at Ser358. DYRK1A-mediated phosphorylation of PLK2 increases its protein stability. Moreover, PLK2 kinase activity was markedly induced by DYRK1A, which was exemplified by the upregulation of alpha-synuclein S129 phosphorylation. Furthermore, it was found that phosphorylation of PLK2 by DYRK1A contributes to the proliferation, migration and invasion of GBM cells. DYRK1A further enhances the inhibition of the malignancy of GBM cells already induced by PLK2. The findings of the present study indicate that PLK2 may play a crucial role in GBM pathogenesis partially in a DYRK1A-dependent manner, suggesting that PLK2 Ser358 may serve as a therapeutic target for GBM.

RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling.

Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra‑ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27‑specific small interfering RNA and proliferation was assessed invitro and invivo using proliferation assays, fluorescence‑activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing‑induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo‑like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M‑associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere‑forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere‑forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next‑generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

PLK4 inhibitor exhibits antitumor effect and synergizes sorafenib via arresting cell cycle and inactivating Wnt/β-catenin pathway in anaplastic thyroid cancer

ABSTRACT The anti-tumor effect of polo-like kinase 4 (PLK4) inhibitor has been explored in several solid carcinomas, while its application in anaplastic thyroid cancer (ATC) remains scarce. Hence, the current study aimed to investigate the effect of PLK4 inhibitor on the malignant behaviors of ATC cell lines and its synergistic antitumor effect with sorafenib. C643 and 8305c cells were cultured in various concentrations of centrinone (PLK4 inhibitor) with or without sorafenib. Meanwhile, the cell viability, cell apoptosis, cell cycle and expressions of glycogen synthetase kinase beta (GSK3β), p-GSK3β, β-catenin were determined. PLK4 mRNA and protein expressions were higher in most ATC cell lines than the normal thyroid epithelial cell line (all P < .05). Centrinone decreased cell viability, induced cell apoptosis, arrested cell cycle at G2/M phase and inactivated Wnt/β-catenin signaling with dose-dependent manners in C643 and 8305c cells (all P < .05). Interestingly, centrinone plus sorafenib further improved antitumor effect (P < .05 at most concentrations), with the highest combination index at 5 nM centrinone plus 4 μM sorafenib in C643 cells, then 4 nM centrinone plus 4 μM sorafenib in C643 cells. Subsequently, centrinone plus sorafenib reduced cell viability, promoted cell apoptosis, facilitated cell cycle at G2/M phase and repressed Wnt/β-catenin signaling more effectively compared with centrinone or sorafenib monotherapy in C643 and 8305c cells (all P < .05). PLK4 inhibitor exhibits antitumor effect and synergizes sorafenib via arresting cell cycle and inactivating Wnt/β-catenin pathway in ATC.

The intermediate filament protein nestin serves as a molecular hub for smooth muscle cytoskeletal signaling

BackgroundThe recruitment of the actin-regulatory proteins cortactin and profilin-1 (Pfn-1) to the membrane is important for the regulation of actin cytoskeletal reorganization and smooth muscle contraction. Polo-like kinase 1 (Plk1) and the type III intermediate filament protein vimentin are involved in smooth muscle contraction. Regulation of complex cytoskeletal signaling is not entirely elucidated. The aim of this study was to evaluate the role of nestin (a type VI intermediate filament protein) in cytoskeletal signaling in airway smooth muscle.MethodsNestin expression in human airway smooth muscle (HASM) was knocked down by specific shRNA or siRNA. The effects of nestin knockdown (KD) on the recruitment of cortactin and Pfn-1, actin polymerization, myosin light chain (MLC) phosphorylation, and contraction were evaluated by cellular and physiological approaches. Moreover, we assessed the effects of non-phosphorylatable nestin mutant on these biological processes.ResultsNestin KD reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Moreover, contractile stimulation enhanced nestin phosphorylation at Thr-315 and the interaction of nestin with Plk1. Nestin KD also diminished phosphorylation of Plk1 and vimentin. The expression of T315A nestin mutant (alanine substitution at Thr-315) reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Furthermore, Plk1 KD diminished nestin phosphorylation at this residue.ConclusionsNestin is an essential macromolecule that regulates actin cytoskeletal signaling via Plk1 in smooth muscle. Plk1 and nestin form an activation loop during contractile stimulation.

Electrostatic Attractive Self-Delivery of siRNA and Light-Induced Self-Escape for Synergistic Gene Therapy.

Small interfering RNA (siRNA) holds immense promise for suppressing gene expression and treating various life-threatening diseases, including cancer. However, efficient delivery and lysosomal escape remain critical challenges that hinder the therapeutic effectiveness of siRNA. Herein, cationic photosensitizer (NB-Br) is grafted onto polo-like kinase 1 (PLK1) siRNA to form an amphiphilic siRNA-photosensitizer conjugate (siPLK1-NB), which can self-assemble into nanoparticles (siPLK1-NB NPs) via electrostatic attraction. Notably, siPLK1-NB NPs exhibit rapid and efficient cell endocytosis, as well as outstanding tumor-targeting property in multiple tumor-bearing mice models. When siPLK1-NB NPs are located inside tumor cell lysosomes, the generated reactive oxygen species (ROS) after photoactivation can disrupt the lysosome membrane structure and facilitate siRNA escape from lysosomes. Under light irradiation, siPLK1-NB NPs can downregulate PLK1 expression and induce photodynamic killing, effectively inhibiting tumor cell growth both in vitro and in vivo. Consequently, this study provides a novel design strategy for carrier-free siRNA delivery systems. As far as it is known, this is the first report of a carrier-free siRNA delivery system based on electrostatic attraction.

Discovery of the First Potent, Selective, and In Vivo Efficacious Polo-like Kinase 4 Proteolysis Targeting Chimera Degrader for the Treatment of TRIM37-Amplified Breast Cancer.

Polo-like kinase 4 (PLK4) is a master regulator of centriole replication and has been proposed as a therapeutic target for multiple cancers, especially TRIM37-amplified breast cancer. The development of novel and effective therapeutic strategies for TRIM37-amplified breast cancer therapy is challenging and extremely desirable. Herein, a structure-activity relationship (SAR) study with an emphasis on exploring different linker lengths and compositions was performed to report the discovery and characterization of SP27 as the first selective PLK4 proteolysis targeting chimera (PROTAC) degrader. SP27 exhibited effective PLK4 degradation, more potent inhibition of cell growth, and more efficient precision-therapeutic effect in the TRIM37-amplified MCF-7 cell line than conventional inhibitor CZS-035. Moreover, SP27 showed 149% bioavailability after intraperitoneal administration in PK studies and potent antitumor efficacy in vivo. The discovery of SP27 demonstrated the practicality and importance of PLK4 PROTAC and paved the way for studying PLK4-dependent biological functions and treat TRIM37-amplified breast cancer.

Selective BD2 Inhibitor Exerts Anti-Fibrotic Effects via BRD4/FoxM1/Plk1 Axis in Orbital Fibroblasts From Patients With Thyroid Eye Disease.

We investigated the therapeutic potential of ABBV744, a bromodomain and extra-terminal (BET) inhibitor with selectivity for the second bromodomain (BD2) in thyroid eye disease (TED). The anti-fibrotic effects of ABBV744 and its underlying mechanism were explored in cultured orbital fibroblasts (OFs) from patients with TED. Immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) assays were conducted on orbital connective tissues from TED and controls. RT-qPCR, Western blot, Cell-counting Kit-8 (CCK-8), and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assays were conducted on OFs isolated from patients with TED. The expression of BRD4 was upregulated in the orbital tissues of patients with TED relative to controls and in TED OFs stimulated with TGF-β1. Further, we showed that BRD4 modulated the profibrotic process through the interaction with Forkhead Box M1 (FoxM1) and its downstream molecule Polo-like kinase 1 (Plk1) in cultured TED OFs. Inhibition of BRD4 both by BD2 selective inhibitor ABBV744 and pan-BET inhibitor JQ1 exerted anti-fibrotic effects, whereas ABBV744 displayed superior anti-fibrotic effects and acceptable safety compared to JQ1. We conclude that BDR4 may modulate the profibrotic process in OFs of patients with TED via the FoxM1/Plk1 axis, and that selectively targeting BD2 domain of BRD4 may therefore be a potential therapeutic option for treating patients with TED.

A polo-like kinase inhibitor identified by computational repositioning attenuates pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with few effective therapeutic options. Recently, drug repositioning, which involves identifying novel therapeutic potentials for existing drugs, has been popularized as a new approach for the development of novel therapeutic reagents. However, this approach has not yet been fully utilized in the field of pulmonary fibrosis.MethodsThe present study identified novel therapeutic options for pulmonary fibrosis using a systematic computational approach for drug repositioning based on integration of public gene expression signatures of drug and diseases (in silico screening approach).ResultsAmong the top compounds predicted to be therapeutic for IPF by the in silico approach, we selected BI2536, a polo-like kinase (PLK) 1/2 inhibitor, as a candidate for treating pulmonary fibrosis using an in silico analysis. However, BI2536 accelerated mortality and weight loss rate in an experimental mouse model of pulmonary fibrosis. Because immunofluorescence staining revealed that PLK1 expression was dominant in myofibroblasts while PLK2 expression was dominant in lung epithelial cells, we next focused on the anti-fibrotic effect of the selective PLK1 inhibitor GSK461364. Consequently, GSK461364 attenuated pulmonary fibrosis with acceptable mortality and weight loss in mice.ConclusionsThese findings suggest that targeting PLK1 may be a novel therapeutic approach for pulmonary fibrosis by inhibiting lung fibroblast proliferation without affecting lung epithelial cells. In addition, while in silico screening is useful, it is essential to fully determine the biological activities of candidates by wet-lab validation studies.

Abstract B019: A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer

Abstract Despite substantial improvements in the treatment landscape of prostate cancer, the evolution of hormone therapy-resistant and metastatic prostate cancer remains a major cause of cancer-related death globally. The mainstay of treatment for advanced prostate cancer is targeting of androgen receptor signaling, including androgen deprivation therapy plus second-generation androgen receptor blockade (e.g., enzalutamide, apalutamide, darolutamide), and/or androgen synthesis inhibition (abiraterone). While these agents have significantly prolonged the lives of patients with advanced prostate cancer, the evolution of resistance to these treatments in nearly universal. This therapy resistance is mediated by diverse mechanisms, including both androgen receptor-dependent mechanisms, such as androgen receptor mutations, amplifications, alternatively spliced isoforms, and structural rearrangements, as well as non-androgen receptor-mediated mechanisms, such as lineage plasticity toward neuroendocrine-like or epithelial-mesenchymal transition (EMT)-like lineages. Our prior work identified the EMT transcriptional regulator Snail as critical to hormonal therapy resistance and commonly detected in human metastatic prostate cancer. In the current study, we sought to interrogate the actionable landscape of EMT-mediated hormone therapy-resistant prostate cancer to identify synthetic lethality and collateral sensitivity approaches to treating this aggressive disease state. Using a combination of high-throughput drug screens and multi-parameter phenotyping by confluence imaging, ATP production, and phenotypic plasticity reporters of EMT, we identified candidate synthetic lethalities to Snail-mediated EMT in prostate cancer. These analyses identified multiple actionable targets, such as XPO1, PI3K/mTOR, aurora kinases, c-MET, polo-like kinases, and JAK/STAT as synthetic lethalities in Snail+ prostate cancer. We validated these targets in a subsequent validation screen in an LNCaP-derived model of resistance to sequential androgen deprivation and enzalutamide. This follow-up screen provided validation of inhibitors of JAK/STAT and PI3K/mTOR as therapeutic vulnerabilities for Snail+ and enzalutamide-resistant prostate cancer. Citation Format: Kathryn E. Ware, Beatrice C. Thomas, Pelumi Olawuni, Maya U. Sheth, Nathan Hawkey, Yeshwanth M, Brian C. Miller, Katherine J. Vietor, Mohit Kumar Jolly, So Young Kim, Andrew J. Armstrong, Jason A. Somarelli. A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B019.

A pilot, open study to assess efficacy and safety of ON-01910 (rigosertib) in patients with recessive dystrophic epidermolysis bullosa associated locally advanced/metastatic squamous cell carcinoma.

TPS9604 Background: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an extremely rare, severe blistering disease driven by mutations in collagen seven (COL7A1). Besides the skin and mucosal blistering, this genodermatosis is also characterized by aggressive cutaneous squamous cell carcinomas (cSCCs) arising from patients’ chronic wounds. The cumulative risk of death from these cSCCs is 70% by age 45. There are limited effective treatment options for advanced (unresectable/metastatic) RDEB-associated cSCCs. While the standard of care for any advanced cSCC is immune checkpoint inhibitors, only half of the treated patients had responses in clinical trials. Their use for RDEB-associated SCCs been limited to case reports, and our own experience has not been positive. Conventional chemotherapy in RDEB-associated cSCC is another proposed treatment strategy, however, case reports demonstrated limited response rates without lasting effects. Currently it is recommended palliatively, although there are concerns about adverse effects. Other existing options are tyrosine kinase inhibitors, resulting in mixed responses to treatment for advanced cSCCs of any type, and epidermal growth factor receptor inhibitors, demonstrating limited effectiveness in a small number of case reports on RDEB-associated cSCCs. More effective therapeutic modalities for advanced/metastatic RDEB-associated cSCCs are needed. We previously demonstrated that RDEB SCC keratinocytes are specifically affected by polo-like kinase-1 (PLK-1) siRNA. Eight PLK-1 inhibitors were screened for cytotoxicity. Of the 8 candidates, ON-01910 was superior as it demonstrated the largest therapeutic window for distinguishing between tumor versus normal cells. The ON-01910 clinical trial is in progress in patients with RDEB with advanced/metastatic cSCCs. Methods: ON-01910 is being evaluated in both oral and intravenous formulations in an open-label, non-randomized phase 2 study in RDEB patients with advanced, treatment resistant cSCC. The trial explores the anti-tumor activity of ON-01910 and evaluates its safety and tolerability. Secondary objectives are to assess the impact on quality of life and to perform biomarker analysis on patient tissue. This study began in August 2021 and is ongoing. One patient is currently enrolled in the oral arm at Thomas Jefferson University in the USA. Safety is assessed according to the CTCAE v5. Tumor response in lymph nodes is determined per the RECIST 1.1 and in skin via clinical examination with serial tumor measurements. Key eligibility criteria include 1) diagnosis of RDEB and associated measurable, advanced cSCC confirmed histologically 2) failure to respond to standard of care including excision for localized disease 3) no concomitant cancer therapies. Clinical trial information: NCT04177498 .