Plk1 Inhibitors Research Articles

Integration of Machine Learning and Experimental Validation to Identify Anoikis-Related Prognostic Signature for Predicting the Breast Cancer Tumor Microenvironment and Treatment Response

Background/Objectives: Anoikis-related genes (ANRGs) are crucial in the invasion and metastasis of breast cancer (BC). The underlying role of ANRGs in the prognosis of breast cancer patients warrants further study. Methods: The anoikis-related prognostic signature (ANRS) was generated using a variety of machine learning methods, and the correlation between the ANRS and the tumor microenvironment (TME), drug sensitivity, and immunotherapy was investigated. Moreover, single-cell analysis and spatial transcriptome studies were conducted to investigate the expression of prognostic ANRGs across various cell types. Finally, the expression of ANRGs was verified by RT-PCR and Western blot analysis (WB), and the expression level of PLK1 in the blood was measured by the enzyme-linked immunosorbent assay (ELISA). Results: The ANRS, consisting of five ANRGs, was established. BC patients within the high-ANRS group exhibited poorer prognoses, characterized by elevated levels of immune suppression and stromal scores. The low-ANRS group had a better response to chemotherapy and immunotherapy. Single-cell analysis and spatial transcriptomics revealed variations in ANRGs across cells. The results of RT-PCR and WB were consistent with the differential expression analyses from databases. NU.1025 and imatinib were identified as potential inhibitors for SPIB and PLK1, respectively. Additionally, findings from ELISA demonstrated increased expression levels of PLK1 in the blood of BC patients. Conclusions: The ANRS can act as an independent prognostic indicator for BC patients, providing significant guidance for the implementation of chemotherapy and immunotherapy in these patients. Additionally, PLK1 has emerged as a potential blood-based diagnostic marker for breast cancer patients.

PLK1 inhibitor onvansertib demonstrates antitumorigenic effects in serous uterine cancer cell lines

PLK1 inhibitor onvansertib demonstrates antitumorigenic effects in serous uterine cancer cell lines

Single-cell landscape of innate and acquired drug resistance in acute myeloid leukemia

Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research.

PLK1 Inhibitor Onvansertib Enhances the Efficacy of Alpelisib in PIK3CA-Mutated HR-Positive Breast Cancer Resistant to Palbociclib and Endocrine Therapy: Preclinical Insights.

Endocrine therapy (ET) combined with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) is the preferred first-line treatment for hormone receptor-positive (HR+)/HER2- metastatic breast cancer. Although this is beneficial, acquired resistance leads to disease progression, and patients harboring PIK3CA mutations are treated with targeted therapies such as the PI3Kα inhibitor, alpelisib, alongside ET. Drug-associated resistance mechanisms limit the efficacy of alpelisib, highlighting the need for better combination therapies. This study aimed to evaluate the efficacy of combining alpelisib with a highly specific PLK1 inhibitor, onvansertib, in PIK3CA-mutant HR+ breast cancer preclinical models. We assessed the effect of the alpelisib and onvansertib combination on cell viability, PI3K signaling pathway, cell cycle phase distribution and apoptosis in PI3K-activated HR+ breast cancer cell lines. The antitumor activity of the combination was evaluated in three PIK3CA-mutant HR+ breast cancer patient-derived xenograft (PDX) models, resistant to ET and CDK4/6 inhibitor palbociclib. Pharmacodynamics studies were performed using immunohistochemistry and Simple Western analyses in tumor tissues. The combination synergistically inhibited cell viability, suppressed PI3K signaling, induced G2/M arrest and apoptosis in PI3K-activated cell lines. In the three PDX models, the combination demonstrated superior anti-tumor activity compared to the single agents. Pharmacodynamic studies confirmed the inhibition of both PLK1 and PI3K activity and pronounced apoptosis in the combination-treated tumors. Our findings support that targeting PLK1 and PI3Kα with onvansertib and alpelisib, respectively, may be a promising strategy for patients with PIK3CA-mutant HR+ breast cancer failing ET + CDK4/6i therapies and warrant clinical evaluation.

BRCA1 orchestrates the response to BI-2536 and its combination with alisertib in MYC-driven small cell lung cancer

PLK1 is currently at the forefront of mitotic research and has emerged as a potential target for small cell lung cancer (SCLC) therapy. However, the factors influencing the efficacy of PLK1 inhibitors remain unclear. Herein, BRCA1 was identified as a key factor affecting the response of SCLC cells to BI-2536. Targeting AURKA with alisertib, at a non-toxic concentration, reduced the BI-2536-induced accumulation of BRCA1 and RAD51, leading to DNA repair defects and mitotic cell death in SCLC cells. In vivo experiments confirmed that combining BI-2536 with alisertib impaired DNA repair capacity and significantly delayed tumor growth. Additionally, GSEA analysis and loss- and gain-of-function assays demonstrated that MYC/MYCN signaling is crucial for determining the sensitivity of SCLC cells to BI-2536 and its combination with alisertib. The study further revealed a positive correlation between RAD51 expression and PLK1/AURKA expression, and a negative correlation with the IC50 values of BI-2536. Manipulating RAD51 expression significantly influenced the efficacy of BI-2536 and restored the MYC/MYCN-induced enhancement of BI-2536 sensitivity in SCLC cells. Our findings indicate that the BRCA1 and MYC/MYCN-RAD51 axes govern the response of small cell lung cancer to BI-2536 and its combination with alisertib. This study propose the combined use of BI-2536 and alisertib as a novel therapeutic strategy for the treatment of SCLC patients with MYC/MYCN activation.

RETRACTION: "Effect of PLK1 Inhibition on Cisplatin-resistant Gastric Cancer Cells".

Z. Chen, Y. Chai, T. Zhao, P. Li, L. Zhao, F. He, Y. Lang, J. Qin, H. Ju,"Effect of PLK1 Inhibition on Cisplatin-resistant Gastric Cancer Cells," Journal of Cellular Physiology 234, no. 5 (2019): 5904-5914, https://doi.org/10.1002/jcp.26777. The above article, published online on 29 November 2018 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors; the journal Editor-in-Chief, Alexander Hutchison; and Wiley Periodicals LLC. The retraction has been agreed upon the authors' request. After the publication of their article, the authors discovered that the cell lines BGC-823 and SGC-7901 used in their study have been reported as contaminated [1, 2]. The authors have contacted the journal requesting the retraction of their article as these issues compromise the reliability of the research presented. The retraction is warranted, as the editors concur with the authors that the data is unreliable and the conclusions are invalid. REFERENCES [1] F. Ye, C. Chen, J. Qin, J. Liu, C. Zheng, "Genetic Profiling Reveals an Alarming Rate of Cross-contamination among Human Cell Lines Used in China," The FASEB Journal 29, no. 10 (2015): 4268-4272, https://doi.org/10.1096/fj.14-266718 [2] X. Bian, Z. Yang, H. Feng, H. Sun, Y. Liu, "A Combination of Species Identification and STR Profiling Identifies Cross-contaminated Cells from 482 Human Tumor Cell Lines," Scientific Reports 7, no. 1 (2017): 9774, https://doi.org/10.1038/s41598-017-09660-w.

Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

O-008 Sperm-derived centrioles play a key role in ensuring correct chromosome segregation in the first mitotic division of mammalian zygotes

Abstract Study question What is the role of the centrosomes during mitotic division in the mammalian zygote? Does the atypical structure of sperm-derived centrioles affect their function? Summary answer Correct centrosome positioning guarantees spindle stability and correct chromosome segregation. Centrosome malposition or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. What is known already The centrosomes of somatic cells comprise two centrioles surrounded by pericentriolar material, and are major microtubule-organizing centres with an essential role in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and, although two centrioles are introduced by the spermatozoon at fertilization, they are structurally different to the canonical centrioles found in somatic cells. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, when they are often mal-positioned (not at the spindle poles). However, the causes and consequences of abnormal centrosome positioning in zygotes are not known. Study design, size, duration We combined systematic immunofluorescence of bovine zygotes, fixed at different stages, with real-time imaging of live zygotes to investigate the implications of centrosome mal-positioning during zygotic mitosis on the fidelity of chromosome segregation and cytokinesis. To study the functionality of the sperm-derived centrioles alone, we inhibited the formation of daughter centrioles using the PLK4 inhibitor (PLK4i), Centrinone. Bovine zygotes were used because the structure of bovine and human sperm-derived centrioles are very similar. Participants/materials, setting, methods Bovine oocytes were retrieved from slaughterhouse ovaries and matured in vitro. Matured oocytes were fertilized in vitro with semen from a bull of proven fertility and, either fixed at 27-29h post-fertilization, or used for time-lapse light-sheet imaging after injection of mRNA for microtubule and chromatin reporters. Fixed bovine zygotes were immunostained for chromatin, alpha-tubulin, centriolar and pericentriolar components, and imaged with a confocal microscope. Microtubule nucleation ability was evaluated using a microtubule regrowth assay. Main results and the role of chance Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malposition, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation. Centrosome malposition and inactivity induced chromosome segregation errors in 44% and 82% of the zygotes respectively. PLK4i treatment successfully inhibited centriole duplication, since the volume of core-centriole protein (POC5) in treated zygotes was half that in non-treated zygotes. When centriole duplication was prevented, the sperm-derived centrioles showed a similar ability to recruit pericentrosomal material (NEDD1) and nucleate microtubules. However, live imaging showed that 82% (14/17) of PLK4i treated zygotes the spindle, although bipolar, was unusually elongated and chromosomes either failed to align correctly on the metaphase plate or showed anaphase lag. Moreover, in treated zygotes where both centrioles were positioned at the same spindle pole, the spindle failed to anchor correctly to the cell membrane and resulted in rotational behavior of the zygote and an increase of cytokinesis failure from up to 47% (8/17). Therefore we conclude that centrosomes are likely to play a key role in correct chromosome segregation in mammalian zygotes. Limitations, reasons for caution For ethical reasons, we could not manipulate and study human zygotes. Instead, we chose an animal model with a rate of embryonic aneuploidy similar to man. It is, however, possible that zygotic centrosome function differs between these two species. Wider implications of the findings A better understanding of the key regulators of centrosome function might lead to the development of interventions to reduce the risk of malfunction, thereby reducing the incidence of embryonic aneuploidy. Moreover, our study unveiled an mechanism by which impaired PLK4 function induces aneuploidy in mammalian embryos. Trial registration number not applicable

MYC and HSF1 Cooperate to Drive PLK1 inhibitor Sensitivity in High Grade Serous Ovarian Cancer.

Ovarian cancer is a deadly female cancer with high rates of recurrence. The primary treatment strategy for patients is platinum-based therapy regimens that almost universally develop resistance. Consequently, new therapeutic avenues are needed to overcome the plateau that current therapies have on patient outcomes. We describe a gene amplification involving both HSF1 and MYC, wherein these two genes on chromosome 8q are co-amplified in over 7% of human tumors that is enriched to over 30% of patients with ovarian cancer. We further found that HSF1 and MYC transcriptional activity is correlated in human tumors and ovarian cancer cell lines, suggesting they may cooperate in ovarian cancer cells. CUT&RUN for HSF1 and MYC in co-amplified ovarian cancer cells revealed that HSF1 and MYC have overlapping binding at a substantial number of locations throughout the genome where their binding peaks are near identical. Consistent with these data, a protein-protein interaction between HSF1 and MYC was detected in ovarian cancer cells, implying these two transcription factors have a molecular cooperation. Further supporting their cooperation, growth of HSF1-MYC co-amplified ovarian cancer cells were found to be dependent on both HSF1 and MYC. In an attempt to identify a therapeutic target that could take advantage of this dependency on both HSF1 and MYC, PLK1 was identified as being correlated with HSF1 and MYC in primary human tumor specimens, consistent with a previously established effect of PLK1 on HSF1 and MYC protein levels. Targeting PLK1 with the compound volasertib (BI-6727) revealed a greater than 200-fold increased potency of volasertib in HSF1-MYC co-amplified ovarian cancer cells compared to ovarian cancer cells wild-type HSF1 and MYC copy number, which extended to several growth assays, including spheroid growth. Volasertib, and other PLK1 inhibitors, have not shown great success in clinical trials and this study suggests that targeting PLK1 may be viable in a precision medicine approach using HSF1-MYC co-amplification as a biomarker for response.

Abstract A022: Synthetic lethality of ERBB2 and CCND1 in breast cancer at scale

Abstract Introduction: Synthetically lethal mutations offer unique molecular targets for oncologic therapy. At scale, synthetic lethality (SL) is observed when alterations to one gene alone do not correspond with worse overall survival (OS), but simultaneous expression with another gene does correspond with worse OS. Mutually exclusive gene expression is not a requirement for SL. Existing breast cancer (BC) literature suggests synthetic lethality between CKS1B and PLK1, as well as BRCA1/BRCA2 and PARP1. Unaltered RB1 and altered CCND1 are also known to exhibit SL in the HER2-deficient environment of triple-negative BC. CCND1 and ERBB2 are typically amplified in invasive ductal carcinoma, with ERBB2 amplifications correlating to larger tumor size. This relationship is not observed in invasive lobular carcinoma, although expression of E2F1, a downstream transcription factor of CCND1, is inversely correlated with tumor grade. Methods: Our in silico approach investigated SL between ERBB2 and CCND1 in breast invasive carcinoma without distinction to lobular or ductal origin (TCGA, PanCancer Atlas 2018). We identified 17 genes of interest based on molecular alterations present in >10% of the sample (n=1084) and common mentions in BC literature. SL was determined if alterations to one gene alone did not significantly worsen OS, while alterations to two genes corresponded with worse OS. Results: Twenty-three SL interactions were observed. CCND1 and ERBB2 were selected because neither gene significantly worsened OS alone (p=0.915 & p=0.0951 respectively). However, patients with alterations to both genes had significantly worse OS compared to those with alterations to one gene alone. CCND1 and ERBB2 alterations were present in 14.9% and 13.7% of the sample, respectively. HR=2.568 (p=0.0138) for SL patients compared to those with CCND1 alterations alone; HR=2.244 (p=0.0497) for SL patients compared to those with ERBB2 alterations alone. Mutual exclusivity was not significantly observed (p=0.084). Multivariate analysis was performed using Cox regression models; neither age (older or younger than 55 years) nor race (White, Black, Asian) confounded our results due to p>0.05. Conclusion: Our findings support potential therapeutic use of CCND1 targets in HER2+ BC, specifically for patients with SL between CCND1 and ERBB2. These include PLK1 inhibitors, commonly used in estrogen receptor-positive patients with CDK4/6 inhibitor resistance, along with HER2 inhibitors. A combination of MAP2K and PIK3CA inhibitors should also be investigated in HER2+ BC patients with CCND1 amplification, since HER2+ colorectal cancer patients have been demonstrated to fare better with this treatment. Citation Format: Rishi Nair, Evan P. White, Roy Khalife, Anthony M. Magliocco. Synthetic lethality of ERBB2 and CCND1 in breast cancer at scale [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A022.

PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer originating from the epithelial cells within the bile duct and ranks as the second most prevalent form of liver cancer in Thailand. Polo-like kinase 1 (PLK1), a protein serine/threonine kinase, regulates a number of steps in cell mitosis and is upregulated in several types of cancer, including CCA. Our previous study identified PLK1 as a biomarker of the C1 subtype, correlating with poor prognosis in intrahepatic CCA. The present study aimed to examine the effect of PLK1 inhibition on CCA cells. Different CCA cell lines developed from Thai patients, HuCCA1, KKU055, KKU100 and KKU213A, were treated with two PLK1 inhibitors, BI2536 and BI6727, and were transfected with small interfering RNA, followed by analysis of cell proliferation, cell cycle distribution and cell apoptosis. It was discovered that BI2536 and BI6727 inhibited cell proliferation and caused G2/M-phase arrest in CCA cells. Furthermore, the number of total apoptotic cells was increased in PLK1 inhibitor-treated CCA cells. The expression levels of mitotic proteins, aurora kinase A, phosphorylated PLK1 (T210) and cyclin B1, were augmented in PLK1-inhibited CCA cells. Additionally, inhibition of PLK1 led to increased DNA damage, as determined by the upregulated levels of γH2AX and increased cleavage of poly (ADP-ribose) polymerase, an apoptotic marker. These results suggested that inhibiting PLK1 prolonged mitotic arrest and subsequently triggered cell apoptosis. Validation of the antiproliferative effects of PLK1 inhibition was accomplished through silencing of the PLK1 gene. In conclusion, targeting PLK1 provided promising results for further study as a potential candidate for targeted therapy in CCA.

Phosphorylation of LZTS2 by PLK1 activates the Wnt pathway

Lung adenocarcinoma (LUAD), responsible for nearly half of lung cancer cases, is one of the most prevalent and lethal malignant tumors globally. There is increasing evidence suggesting that the oncoprotein PLK1 plays a role in the onset and advancement of different types of cancer, including LUAD. Nonetheless, the precise mechanism by which PLK1 promotes tumorigenesis remains unclear. In this study, we demonstrate the upregulation of PLK1 in LUAD samples, which leads to a poor prognosis for LUAD patients. Intriguingly, PLK1 enables to bind to LZTS2 and promote its phosphorylation without affecting LZTS2 degradation. Furthermore, we identify that Ser451 is a key phosphorylation site in LZTS2 protein. LZTS2 exerts an anti-tumor effect by restricting the translocation of the transcription factor β-Catenin into the nucleus, thereby suppressing the Wnt pathway. PLK1 disrupts the interaction between LZTS2 and β-Catenin, resulting in the nuclear accumulation of β-Catenin and the activation of the Wnt pathway. Additionally, we reveal that LZTS2 inhibits the proliferation and migration of LUAD cells, which is rescued by PLK1. Finally, PLK1 inhibitors exhibit a dose-dependent suppression of LUAD cell proliferation and migration. Collectively, this study uncovers the pro-tumorigenic mechanism of PLK1, positioning it as a promising therapeutic target for Wnt-related LUAD.

Therapeutic targeting of PLK1 in TERT promoter-mutant hepatocellular carcinoma.

Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.

A polo-like kinase 1 inhibitor enhances erastin sensitivity in head and neck squamous cell carcinoma cells in vitro

BackgroundPolo-like kinase 1 (PLK1) is a critical therapeutic target in the treatment of head and neck squamous cell carcinoma (HNSCC). The objective of this study was to investigate the therapeutic effect of the combination of BI 2536, a PLK1 inhibitor, and erastin, a ferroptosis inducer, in HNSCC.MethodsThe proliferation, invasion, and migration abilities of Tu177 and FaDu cells upon exposure to BI 2536 and erastin, used in combination or alone, were tested. Fe2+, glutathione (GSH), and malondialdehyde (MDA) detection kits were used to determine whether the addition of BI 2536 enhanced the accumulation of Fe2+ and MDA, along with the depletion of GSH. Quantitative real-time PCR, western blot analyses were performed to investigate whether BI 2536 further altered the mRNA and expression level of ferroptosis genes. Furthermore, si PLK1 was used to investigate whether targeting PLK1 gene promoted erastin-induced ferroptosis.ResultsThe combination of BI 2536 and erastin exerted a stronger cytotoxicity than treatment with a single agent. Compared with erastin treatment alone, the combination of BI 2536 and erastin lowered the ability of tumor cells to self-clone, invade, and migrate. BI 2536 enhanced the accumulation of Fe2+ and MDA, and the depletion of GSH. BI 2536 increased erastin-induced changes in ferroptosis-related gene mRNA and expression. Importantly, targeting PKL1 enhanced the anti-cancer effect of erastin.ConclusionBI 2536 enhanced the sensitivity of HNSCC cells to erastin, which provides a new perspective for cancer treatment.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 606.

Abstract 1934: The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts

Abstract Background: Cetuximab and panitumumab are monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), that provide clinical benefit to metastatic colorectal cancer (mCRC) patients with RAS wild-type (RASWT) tumors. Unfortunately, intrinsic or acquired resistance to these therapies limits their clinical effectiveness, necessitating the development of more efficient therapeutic strategies. Onvansertib is an oral, small molecule, selective inhibitor of the PLK1 kinase, currently in clinical development for KRAS-mutant mCRC. The aim of this study was to assess the anti-tumor activity of onvansertib monotherapy and in combination with cetuximab in RASWT CRC patient-derived xenograft (PDX) models. Methods: Twenty RASWT CRC PDXs were selected and engrafted in nude mice. Once tumors reached 200-350-mm3, mice were treated with vehicle, onvansertib (60mg/kg, QD), cetuximab (20mg/kg, BIW) or the combination for 18 days. PDX models were chosen based on their sensitivity to cetuximab, resulting in a selection of 7 cetuximab-sensitive (CetuxS) and 13 cetuximab-resistant (CetuxR) PDXs, including 7 with intrinsic resistance and 6 with acquired-resistance. Tumor volume change from baseline (TVC) was calculated as 100%x(Vt-Vo)/Vo and tumor growth inhibition (TGI) as 100%x(TVCcontrol-TVCtreated)/TVCcontrol. Tumor regression was defined as TVCD18<0 and tumor stasis 0≤TVCD18<100. Results: Cetuximab sensitivity was confirmed in the selected models, cetuximab induced tumor regression in all the CetuxS PDXs, while no or limited activity was observed in the resistant models (median TGI=29%, IQR 16-61). Onvansertib exhibited potent anti-tumor activity in 17 (85%) PDXs, resulting in tumor regression (n=11) or tumor stasis (n=6). Onvansertib TGI at Day 18 was not significantly different in CetuxS PDXs (median TGI 102, IQR 76-103) compared to CetuxR PDXs (median TGI 108, IQR 74-124), supporting that onvansertib anti-tumor activity is independent of the sensitivity/resistance to cetuximab. The combination of onvansertib and cetuximab induced tumor regression in 18 (90%) PDXs. The combination showed significantly improved efficacy compared to individual therapies in some of the models. Conclusions: Onvansertib monotherapy displayed potent anti-tumor activity in RASWT CRC PDX models, independently of their sensitivity to cetuximab. Additionally, onvansertib combined with cetuximab exhibited either comparable or superior anti-tumor activity than the monotherapies. Collectively, this data supports the clinical development of the PLK1 inhibitor onvansertib for RASWT mCRC. Citation Format: Maya Ridinger, Preeti Kanikarla Marie, Fengqin Gao, Zhensheng Liu, Giulia Maddalena, David Menter, Alexey Sorokin, Tod Smeal, Scott Kopetz. The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1934.

Abstract 1111: Poor radiation sensitivity and potential radiosensitizers for betel-nuts related head and neck squamous cell carcinoma in Taiwan

Abstract Background: Betel nut chewing might contribute to strong invasion & treatment refractoriness in HNSCC from Taiwan. Betel-nuts exposed HNSCC cell line, TW2.6, had high PDL1, defective p53 mutation, p16 loss, & BCL2 overexpression. In our studies, PI3K/AKT/mTOR inhibitors, anti-angiogenesis therapies, CDK4/6 inhibitors, DDR interventions, epigenetic modifications, and immunotherapy-containing regimens will be future combination options. The genomic signature of TW2.6 has been figured out in our study(AACR2020 & 2023), too, mainly with PIK3CA H1047R mutation, high TMB(8.42 muts/Mb)/MSS, VEGF-A amplification, p53/MYC/HRAS/DDR2/PDGFRbeta/EPHB1/ATM mutations, FAT1 loss, amplification of TERT/FGF10/CCND3/SOX9/IL-7R/SDHA/RICTOR/FLCN, CDK12/PTPRD loss of function, CDK8 mutation, & deletions of STK11/ARID1B/MITF/TNFAIP3 and molecularly-guided novel interventions will be designed. Purpose & Methods: Polo-like kinase 1 inhibitor(volasertib), gluconate(blocking citrate transport & glucose metabolism), IAP inhibitor(Debio1143), glutaminase 1 inhibitor(CB839) were tested on TW2.6 to evaluate synergistic effects with radiation. Results: TW2.6 was resistant to volasertib, gluconate, or radiation alone; but sensitive to Debio1143 and CB839. Volasertib significantly enhanced radiation. The flow cytometry revealed volasertib and radiation resulted in a significant G2/M arrest. Western blotting showed volasertib and radiation increased p-histone3, p-CDK1, & cyclin B levels and induced apoptosis. Gluconate, CB839, or Debio1143 each had significant synergistic effects with radiation. The radiosensitization efficacy would be volasertib>gluconate>CB839>Debio1143. Conclusions: In Taiwan, patients with R/M HNSCC progressing within 3 months after CCRT could be rescued by early ICIs. Recently, sequential CCRT followed by ICI or Debio-1143 with CCRT showed potential survival benefits. In our studies, novel agents such as PLK inhibitor, gluconate, and CB839 had even better radiosensitization compared with Debio1143. Sequential CCRT with novel agents followed by ICI will be the future trend, esp. in biology-aggressive betel-nuts related locally advanced HNSCC in Taiwan. Citation Format: Jo-Pai Chen, Jui-Ying Chang, Ruey-Long Hong. Poor radiation sensitivity and potential radiosensitizers for betel-nuts related head and neck squamous cell carcinoma in Taiwan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1111.

Abstract 7205: PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative breast cancer

Abstract Patients with aggressive breast cancer (BC) subtypes usually do not have favorable prognosis despite improvements in treatment modalities. These cancers remain a major cause of morbidity and mortality in women globally. This has fostered a major effort to discover actionable molecular targets to treat these patients. Polo-like Kinase (PLK1) is one of these molecular targets that are under comprehensive investigation for the treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not been explored. Therefore, we examined the expression of PLK1 protein in a large cohort of more than 1500 Middle Eastern ethnicity BC by immunohistochemistry. Correlation with clinico-pathological parameters and the prognosis was performed. PLK1 overexpression was observed in 27.4% of all BC and was significantly associated with aggressive clinico-pathological markers. The antitumor effect of PLK1 and PARP inhibitors either alone or in combination was tested in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and induced apoptosis in TNBC cell lines. Furthermore, our data suggest that PLK1 inhibition overcomes PARP inhibitor resistance in TNBC cell lines. These findings would pave the way to explore the future use of PLK1 inhibitors in the clinical setting of PARPi-resistant patients. Citation Format: Abdul K. Siraj, Pratheesh Kumar Poyil, Divya Padmaja, Sandeep Kumar Parvathareddy, Khadija Alobaisi, Rafia Begum, Osama Almalik, Fouad Al-Dayel, Khawla S. Al-Kuraya. PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7205.

Abstract 5782: Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors

Abstract Polo Like Kinase 1 (PLK1), a sought-after target for drug discovery, is an essential mitotic kinase consisting of a catalytic domain (KD) and a polobox domain (PBD), the latter of which plays roles in substrate recognition, subcellular localization and further acting as an autoinhibitory regulatory domain. PLK1 can exist either in an inhibited closed conformation or an open active state allowing interaction with and phosphorylation of substrates. PLK1 has also been shown to be regulated by interchange between monomeric and dimeric forms through binding to the Bora protein and by activation loop phosphorylation. We have described PBD-binding molecules termed abbapolins that inhibit cellular phosphorylation of a PLK1 substrate and induce loss of intracellular PLK1. In addition, due to their engagement of the cryptic pocket (ligand induced) of the PBD, we proposed that abbapolins would interfere with formation of PLK1 dimers. Here we provide evidence further supporting the ability of abbapolins to block PLK1 cellular dimerization and that this is related to their mechanism of anti-proliferative activity. In a separate study, using a cellular thermal shift assay, KD inhibitors were shown to decrease soluble PLK1, indicating that catalytic-site binding results in less stable PLK1 suggestive of an open conformation. We have extended these observations using a sensitive readout of cellular target engagement employing a Microtag overexpressed PLK1. In this format, we observe heat-induced destabilization of PLK1 upon nanomolar engagement by two lead KD inhibitors, BI2536 and onvansertib, currently being clinically evaluated. Furthermore, cyclapolin 9, a low molecular weight kinase inhibitor that, unlike the other two KD inhibitors does not protrude from the active site, did not induce significant destabilization. We have also carried out experiments using a fluorescently labeled version of BI2536 to probe the conformational effects of PBD binding upon the KD. These data solidify the observations that relief of autoinhibited PLK1 is induced by certain KD binders and highlight the conformational perturbations from KD versus PBD binding. These observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 non-catalytic functions, which may explain their lack of clinical efficacy to date. Furthermore, the demonstration that the abbapolins are mechanistically unique in terms of blocking the dimerization of PLK through the PBD provides impetus for their development as next generation PLK1 inhibitors. Citation Format: Mourad Sanhaji, Monika Raab, George Merhej, Chintada Nageswara Rao, Ivan Babic, Elmar Nurmemmedov, Klaus Strebhardt, MIchael Wyatt, Campbell McInnes. Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5782.

Abstract 4611: The co-targeting of PLK1 and FoxM1 contributes to synergistic antitumor effects in PTC

Abstract Polo-like Kinase 1 (PLK1) plays a key role in cell proliferation with vital regulatory functions in mitosis and cell survival. We investigated the expression of PLK1 protein in a large cohort of PTC patient’s samples and explored its functional role in PTC cell lines in vitro and in vivo. PLK1 overexpression was observed in 54% of all PTC and was significantly associated with aggressive clinico-pathological parameter and it was found to be independent prognostic marker for recurrence-free survival. PLK1 inhibition indeed inhibited cell proliferation and induced cell cycle arrest and apoptosis in PTC cell lines. Importantly, silencing of PLK1 decreases the self-renewal ability of spheroids generated from PTC cells. Immunoprecipitation analysis shows that PLK1 binds to FoxM1 and vice versa in vitro. Mechanistically, PLK1 knockdown suppresses FoxM1 expression, whereas inhibition of FoxM1 does not affect PLK1 expression, which suggests that PLK1 acts through the FoxM1 pathway. The combined inhibition of PLK1 with volasertib and FoxM1 with thiostrepton synergistically attenuated PTC cell growth in vitro and in vivo. Our findings suggest that co-targeting of PLK1 and FoxM1 could be a viable therapeutic strategy for treating patients with an aggressive subtype of PTC. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Divya Padmaja, Sandeep Kumar Parvathareddy, Saravanan Thangavel, Saif S. Al-Sobhi, Fouad Al-Dayel, Khawla S. Al-Kuraya. The co-targeting of PLK1 and FoxM1 contributes to synergistic antitumor effects in PTC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4611.