Polo-like Kinase 1 Expression Research Articles

Application of PLK1 and HOXA13 Gene Expression Levels in Urine in the Diagnosis of Non-muscle Invasive Bladder Cancer.

Bladder cancer is the most common urinary tract neoplasm, affecting many people annually. Current diagnostic and surveillance methods for bladder cancer are frequently invasive and lack sensitivity and specificity. This study aimed to develop an accurate and non-invasive urine-based gene expression assay, including fibroblast growth factor receptor 3 (FGFR3), homeobox A13 (HOXA13), and polo-like kinase 1 (PLK1), to diagnose non-muscle-invasive bladder cancer (NMIBC) at stages Ta and T1. The samples were acquired from 62 patients with NMIBC, 31 control individuals, and 31 patients with non-cancerous genitourinary tract diseases. The expression levels of three relevant genes were determined using quantitative RT-PCR. In addition, the sensitivity and specificity of the data for these genes were computed. Our results showed that PLK1, HOXA13, and FGFR3 expressions of genes were significantly elevated in patients compared to the control groups (p = 0.0001; p = 0.039). The sensitivity and specificity for the FGFR3 gene were 55% and 76%, respectively (p = 0.39). These parameters for HOXA13 were 100% and 93% (p = 0.0001) and for PLK1 were 100% and 86% (p = 0.0001) for diagnosing and monitoring NMIBC. HOXA13 and PLK 1 exhibited adequate specificity and sensitivity for diagnosis. The results of this research showed that despite the higher expression of these genes in urine, only HOXA13 and PLK1 had sufficient and proper specificity and sensitivity, so the urinary expression of these two genes can be used in future studies for diagnosis and monitoring in cancer bladder.

Methylation of PLK-1 Potentially Drives Bendamustine Resistance in Leukemia Cells.

Drug resistance remains a significant impediment in leukemia treatment. While Bendamustine hydrochloride (BH) stands out as a promising therapeutic agent for non-Hodgkin's lymphoma and mantle cell lymphoma, the mechanisms of resistance to BH are not yet fully understood. Our study focuses on elucidating the mechanisms behind bendamustine resistance in leukemia cells, with a specific emphasis on epigenetics. Bendamustine-resistant cells were cultivated from human B cell lymphoblastic leukemia cell lines through systematic and sustained exposure to bendamustine, using the limiting dilution method. Gene expression was assessed via real-time polymerase chain reaction, while the expression of the multidrug resistance protein 1 (MDR1) was evaluated using flow cytometry. Bendamustine-resistant leukemia cells exhibited a decreased RNA expression level for Polo-like kinase-1 (PLK-1). Notably, after treatment with the demethylating agent 5-aza-2'-deoxycytidine, PLK-1 gene expression surged significantly, enhancing bendamustine's cytotoxicity in the resistant leukemia cells. However, MDR1 expression, as determined by flow cytometry, remained consistent between parental and bendamustine-resistant leukemia cells. Our findings indicate that the methylation of the PLK-1 gene plays a pivotal role in modulating PLK-1 expression and is central to the development of bendamustine resistance in leukemia cells.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

Abstract 3482: Single-cell transcriptome analysis reveals that PLK4 signature genes are highly associated with cancer proliferation and hotspot mutations in non-small cell lung cancer (NSCLC)

Abstract Background: Polo-like kinase 4 (PLK4) is a member of the polo-like kinase family known for its precise regulation during the cell cycle and its key role in centriole duplication, which is essential for proper cell division. Dysregulation of PLK4 is associated with abnormal centriole number, genomic instability and cancer development. In our previous study, we proposed PLK4 as a potential therapeutic target and biomarker for LUAD patients with TP53 mutations by bulk RNA-seq analysis and confirmed its association with drug resistance. In this study, we analyzed the expression of PLK4 and its association with cell cycle at the cellular level by scRNA-seq in NSCLC data. Methods: In a previous study, we analyzed NSCLC data from The Cancer Genome Atlas (TCGA) database using the UCSC Xena data portal. In this study, we analyzed E-MTAB-6653 dataset from the single cell expression atlas(https://www.ebi.ac.uk/gxa/sc/home). Results: In the previous study, we demonstrated that PLK4 expression is higher in primary tumors compared to normal tissues in NSCLC. Higher PLK4 expression in patients is associated with an unfavorable prognosis. Using genes highly correlated with PLK4, we confirmed a significant association between PLK4 and TP53 mutation by statistical analysis. Using the Drug Gene Interaction database, we observed an association between the drug resistance-related genes PBK and BIRC5 with PLK4, and these genes were also correlated with TP53 mutation. In this study, we investigated the association between PLK4 and the cell cycle using single cell RNA-seq. First, we performed data pre-processing to selectively isolate NSCLC cells. Using a set of 87 PLK4 signature genes obtained from previous research, we stratified the samples into PLK4 high and low groups based on the expression of these genes. We then analyzed cell cycle related genes in both groups and observed a higher expression of G2/M phase related genes in the PLK4 high group compared to the PLK4 low group. We also examined the expression of PLK4 signature genes based on pathological differences and found higher expression in tumor tissue compared to normal tissue. There was also an increasing expression gradient from the tumor margin to the tumor core. Finally, using Gene Ontology analysis of both groups, we confirmed the upregulation of genes associated with transcription and translation, as indicated by the GO terms obtained. Conclusion: In a previous study, the association between PLK4 expression, TP53 mutation and drug resistance was identified by bulk RNA-seq. In the current study, we used single cell RNA-seq to verify the correlation between PLK4 and cell cycle in NSCLC. In addition, we observed that the expression of PLK4 signature genes increased towards the tumor core based on pathological differences. Citation Format: Youngtaek Kim, Joon Yeon Hwang, YoungJoon Park, Dong Kwon Kim, Kwangmin Na, Seul Lee, Sujeong Baek, Seong-san Kang, Seung Min Yang, Mi Hyun Kim, Heekyung Han, Chai Young Lee, Yu Jin Han, Min Hee Hong, Jii Bum Lee, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Single-cell transcriptome analysis reveals that PLK4 signature genes are highly associated with cancer proliferation and hotspot mutations in non-small cell lung cancer (NSCLC) [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 3482.

Abstract 5726: The potential anti-cancer activity of dual TTK/PLK1 inhibitor, BAL0891, in bladder cancer

Abstract Background: BAL0891 is a novel dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) which affects mitotic spindle assembly checkpoint integrity resulting in aberrant mitotic progression of cancer cells. Since unlike other organs, bladder urothelium is highly regenerative in certain circumstances, there are high chances of cell cycle inhibitors to have anti-cancer activity over bladder cancer. In this study, we investigated the therapeutic potential of BAL0891 in bladder cancer compared to single TTK or PLK1 inhibitor and discovered the best therapeutic combination partner for BAL0891. Methods: We performed meta-analysis of the mRNA expression levels of bladder cancer using Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Representative bladder cancer cell lines (253J, 253J-BV, J82, RT4, T24, J82, HT1197, and HT1376) were treated with the dual protein kinase inhibitors, BAL0891, in comparison with TTK inhibitor (BAY1217389) or PLK1 inhibitor (onvansertib). BAL0891 was combined with CDK4/6 inhibitor, another cell cycle inhibitor working at different point from BAL0891. Cell viability was assessed using CCK-8 assay during 72 hours after treatment. Expression levels of TTK and PLK1 were measured by immunoblotting. Cell cycle analysis and cell apoptosis detection were performed on T24 cell. Results: The mRNA expression levels of both TTK (16.7 times) and PLK1 (10.0 times) were significantly upregulated only in bladder cancers compared to normal bladder tissues (P<0.05). Compared to TTK or PLK1 inhibitor, BAL0891 was sensitive in 75% of bladder cancer cell lines (IC50 <0.1 uM) and significantly reduced proliferation of bladder cancer cell lines depending on the dosage. TTK and PLK1 expression levels of cancer cells were associated with the sensitivity to BAL0891. BAL0891 significantly induced necrosis and increased in polyploidy and G2/M arrest in T24 cell. The anti-cancer activity of BAL0891 was further enhanced in certain cell lines (RT4, and J82) by combination of CDK4/6 inhibitor. Conclusion: This is the first study to suggest the potent anti-cancer activity of BAL0891 in bladder cancer. BAL0891 effectively worked on bladder cancer by two tracks which not only induced G2/M arrest but also increased in polyploidy. Furthermore, BAL0891 in combination with CDK4/6 inhibitor promoted anti-cancer effects. Lastly, this study suggests the use of TTK and PLK1 as the predictive biomarker for BAL0891. These data support further investigation of BAL0891 for the treatment of bladder cancer patients. Citation Format: Jee Soo Park, Ji-eun Lee, Myung Eun Lee, Jongchan Kim, Won Sik Jang, Jae Jung Lee, Jun Seung Lee, Minkyung Kang, Won Sik Ham. The potential anti-cancer activity of dual TTK/PLK1 inhibitor, BAL0891, in bladder 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 5726.

PLK4 as a potential target to enhance radiosensitivity in triple-negative breast cancer

Radioresistance is one of the barriers to developing more effective therapies against the most aggressive, triple-negative, breast cancer (TNBC) subtype. In our previous studies, we showed that inhibition of Polo-like Kinase 4 (PLK4) by a novel drug, CFI-400945 significantly enhances the anticancer effects of radiotherapy (RT) compared to single treatment alone. Here we further investigate the role of PLK4 in enhancing radiation effects in TNBC and explore mechanisms of PLK4 inhibition and radiation combinatorial antiproliferative effects. To assess cellular proliferation in response to treatments, we used colony formation assays in TNBC cell lines and patient-derived organoids (PDOs). Downregulation of PLK4 expression was achieved using siRNA silencing in TNBC cell lines. Immunofluorescence against centrin was used to assess the alteration of centriole amplification in response to treatments. We observed that inhibition of PLK4 by CFI-400945 or Centrinone B or its downregulation by siRNA, when combined with RT, resulted in a significant increase in antiproliferative effect in TNBC cells lines and PDOs compared to untreated or single-treated cells. Anticancer synergy was observed using a response matrix in PDOs treated with CFI-400945 and RT. We show that the overamplification of centrioles might be involved in the combined antiproliferative action of RT and PLK4 inhibition. Our data suggest that PLK4 is a promising target for enhancing the anticancer effects of RT in TNBC that, at least in part, is modulated by the overamplification of centrioles. These results support further mechanistic and translational studies of anti-PLK4 agents and RT as an anticancer combination treatment strategy.

Role of PLK1/NUMB/NOTCH in epithelial-mesenchymal transition in human melanoma

Polo-like kinase 1 (PLK1), a serine/threonine kinase, is overexpressed in melanoma and its expression has been associated with poor disease prognosis. PLK1 has been shown to interact with NUMB, a NOTCH antagonist. However, the exact role of PLK1, NUMB, and NOTCH signaling in epithelial-mesenchymal transition (EMT) in melanoma progression is unclear. In this study, Affymetrix microarray analysis was performed to determine differentially expressed genes following shRNA-mediated knockdown of PLK1 in human melanoma cells that showed significant modulations in EMT and metastasis-related genes. Using multiple PLK1-modulated melanoma cell lines, we found that PLK1 is involved in the regulation of cell migration, invasion, and EMT via its kinase activity and NOTCH activation. In vitro kinase assay and mass spectrometry analysis demonstrated a previously unknown PLK1 phosphorylation site (Ser413) on NUMB. Overexpression of non-phosphorylatable (S413A) and phosphomimetic (S413D) mutants of NUMB in melanoma cells implicated the involvement of NUMB-S413 phosphorylation in cell migration and invasion, which was independent of NOTCH activation. To determine the clinical relevance of these findings, immunohistochemistry was performed using melanoma tissue microarray, which indicated a strong positive correlation between PLK1 and N-cadherin, a protein required for successful EMT. These findings were supported by TCGA analysis, where expression of high PLK1 with low NUMB or high NOTCH or N-cadherin showed a significant decrease in survival of melanoma patients. Overall, these results suggest a potential role of PLK1 in EMT, migration, and invasion of melanoma cells. Our findings support the therapeutic targeting of PLK1, NUMB, and NOTCH for melanoma management.

Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients.

Invasive breast carcinoma (BRCA) is associated with poor prognosis and high risk of mortality. Therefore, it is critical to identify novel biomarkers for the prognostic assessment of BRCA. The expression data of polo-like kinase 1 (PLK1) in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases. PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Single sample gene set enrichment analysis (ssGSEA) was performed to evaluate immune infiltration in the BRCA microenvironment, and the random forest (RF) and support vector machine (SVM) algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore (IPS). The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration. Finally, a prognostic nomogram was constructed with the risk score and pathological stage, and its clinical potential was evaluated by plotting calibration charts and DCA curves. The application of the nomogram was further validated in an immunotherapy cohort. PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort. Furthermore, PLK1 expression level, age and stage were identified as independent prognostic factors of BRCA. While the IPS was unaffected by PLK1 expression, the TMB and MATH scores were higher in the PLK1-high group, and the TIDE scores were higher for the PLK1-low patients. We also identified 6 immune cell types with high infiltration, along with 11 immune cell types with low infiltration in the PLK1-high tumors. A risk score was devised using PLK1 expression and hub immune cells, which predicted the prognosis of BRCA patients. In addition, a nomogram was constructed based on the risk score and pathological staging, and showed good predictive performance. PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

Increased hsa-miR-100-5p Expression Improves Hepatocellular Carcinoma Prognosis in the Asian Population with PLK1 Variant rs27770A>G.

Hepatocellular carcinoma (HCC) has the highest incidence and mortality in the Asian population, and race is an independent risk factor affecting survival time in liver cancer. Micro RNAs (miRNAs) are remarkably dysregulated in HCC and closely associated with HCC prognosis. Recent studies show that genetic variability between ethnic groups may result in differences in the specificity of HCC miRNA biomarkers. Here, we reveal a high expression level of hsa-miR-100-5p, an HCC prognosis-related miRNA, which improves HCC prognosis in the Asian Population with Polo-like kinase 1 (PLK1) variant rs27770A>G. In this study, we discovered that hsa-miR-100-5p was downregulated in various HCC cell lines. While mimics transient transfection and mouse liver cancer model confirmed the interaction between hsa-miR-100-5p and PLK1, a stratified analysis based on the Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) data suggest both low hsa-miR-100-5p expression level and high PLK1 expression level associated with poor HCC prognosis, especially in the Asian population. According to the 1000 Genomes Project database, the SNP rs27770 located in 3'UTR of PLK1 had a significantly higher G allele frequency in the East Asian population. Bioinformatics analysis suggested that rs27770 A>G affects PLK1 mRNA secondary structure and alters the hsa-miR-100-5p/PLK1 interaction by forming an additional seedless binding site. This racial variation caused PLK1 to be more vulnerable to hsa-miR-100-5p inhibition, resulting in hsa-miR-100-5p being more favorable for HCC prognosis in the Asian population.

Tangeretin attenuates acute lung injury in septic mice by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis.

NLRP3 inflammasome-mediated pyroptosis of macrophage acts essential roles in the progression of sepsis-induced acute lung injury (ALI). Tangeretin (TAN), enriched in citrus fruit peel, presents anti-oxidative and anti-inflammatory effects. Here, we aimed to explore the potentially protective effect of TAN on sepsis-induced ALI, and the underlying mechanism of TAN in regulating NLRP3 inflammasome. The effect of TAN on sepsis-induced ALI and NLRP3 inflammasome-mediated pyroptosis of macrophage were examined in vivo and in vitro using a LPS-treated mice model and LPS-induced murine macrophages, respectively. The mechanism of TAN regulating the activation of NLRP3 inflammasome in sepsis-induced ALI was investigated with HE staining, Masson staining, immunofluorescent staining, ELISA, molecular docking, transmission electron microscope detection, qRT-PCR, and western blot. TAN could evidently attenuate sepsis-induced ALI in mice, evidenced by reducing pulmonary edema, pulmonary congestion and lung interstitial fibrosis, and inhibiting macrophage infiltration in the lung tissue. Besides, TAN significantly suppressed inflammatory cytokine IL-1β and IL-18 expression in the serum or bronchoalveolar lavage fluid (BALF) samples of mice with LPS-induced ALI, and inhibited NLRP3 inflammasome-mediated pyroptosis of macrophages. Furthermore, we found TAN inhibited ROS production, preserved mitochondrial morphology, and alleviated excessive mitochondrial fission in LPS-induced ALI in mice. Through bioinformatic analysis and molecular docking, Polo-like kinase 1 (PLK1) was identified as a potential target of TAN for treating sepsis-induced ALI. Moreover, TAN significantly inhibited the reduction of PLK1 expression, AMP-activated protein kinase (AMPK) phosphorylation, and Dynamin related protein 1 (Drp1) phosphorylation (S637) in LPS-induced ALI in mice. In addition, Volasertib, a specific inhibitor of PLK1, abolished the protective effects of TAN against NLRP3 inflammasome-mediated pyroptosis of macrophage and lung injury in the cell and mice septic models. TAN attenuates sepsis-induced ALI by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis, and TAN is a potentially therapeutic candidate against ALI through inhibiting pyroptosis.

A novel L-shaped ortho-quinone analog as PLK1 inhibitor blocks prostate cancer cells in G2 phase

Prostate cancer is the most common malignant tumor among men worldwide. Currently, the main treatments are radical prostatectomy, radiotherapy, chemotherapy, and endocrine therapy. However, most of them are poorly effective and induce side effects. Polo-like kinase 1 (PLK1) regulates cell cycle and mitosis. Its inhibitor BI2536 promotes the therapeutic effect of nilotinib in chronic myeloid leukemia, enhances the sensitivity of neural tube cell tumors to radiation therapy and PLK1 silencing enhances the sensitivity of squamous cell carcinoma to cisplatin. Therefore, the aim of this study was to evaluate the effect of the PLK1 inhibitor L-shaped ortho-quinone analog TE6 on prostate cancer. In vitro on prostate cancer cells showed that TE6 inhibited PLK1 protein expression and consequently cell proliferation by blocking the cell cycle at G2 phase. In vivo on a subcutaneous tumor model in nude mice confirmed that TE6 effectively inhibited tumor growth in nude mice, inhibited PLK1 expression and regulated the expression of cell cycle proteins such as p21, p53, CDK1, Cdc25C, and cyclinB1. Thus, PLK1 was identified as the target protein of TE6, these results reveal the critical role of PLK1 in the growth and survival of prostate cancer and point out the ability of TE6 on targeting PLK1, being a potential drug for prostate cancer therapy.

Metformin Potentiates Gilteritinib Sensitivity Via Targeting PLK1 Signaling: A Strategy to Improve Outcomes and Reduce Costs in Treating FLT3-Mutated Acute Myeloid Leukemia

FLT3 mutations (including FLT3-ITD and FLT3-TKD), occurring in ~30% of acute myeloid leukemia (AML) patients, signal poor prognosis due to elevated relapse risk. Albeit FLT3 tyrosine kinase inhibitors (FLT3 TKI) demonstrated efficacy in treating FLT3-mutated AML in the clinic, there are issues such as high cost, side effects, and high rate of drug resistance/relapse. Hence, an affordable, efficient treatment with lower side effects is needed. Metformin (MET), a cost-effective anti-diabetic drug with minimal side effects, has demonstrated anti-tumor effects (Dowling R et al. Cancer Res. 2007). Here we aimed to determine if combining MET with FLT3 TKI, like Gilteritinib (GLT), could enhance efficacy, reduce side effects and costs in treating FLT3-mutated AML. To this end, we first assessed the IC50 values of MET and GLT across various AML cell lines including those carrying FLT3/ITD (MOLM13, MV4-11, and MA9.3-ITD), FLT3 TKI resistant cells (MOLM13-RES) and those carrying FLT3 wild-type (FLT3/WT) (THP1, NOMO1 and Kasumi-1). MET's IC50 S were consistent (7.284 -12.76 mM) (Fig. 1A), while GLT's varied, with lower values in FLT3/ITD cells (10 nM) and higher in FLT3 TKI resistant (241 nM) and FLT3/WT cells (334-1,053 nM) (Fig. 1B). Using the Chou-Talalay Method, a Combination Index (CI) below 1 in FLT3/ITD and MOLM13-RES cells indicated synergy, whereas it showed antagonism (CI>1) in FLT3/WT cells (Fig. 1C). Both MET and GLT induced cell cycle arrest and apoptosis, with Combo1 (GLT 5 nM + MET 5 mM) matching GLT 10 nM effects; Combo2 (GLT 10 nM + MET 10 mM) amplified these effects (Fig. 1D and E). We next evaluated the potential synergistic effect of MET and GLT in treating TKI-resistance AML in vivo using NRGS mice xeno-transplanted with MOLM13-RES cells. Both bioluminescence imaging and flow cytometry assays showed each treatment alone reduced leukemia burden, and their combination had a synergistic effect, reducing AML burden dramatically (Fig. 1F). While MET or GLT alone doubled overall survival compared to vehicle treatment (56-62 days vs. 26 days), ~90% of the mice treated with the Combo survived over 120 days (Fig. 1G) and their leukemia burden was still minimal, highlighting the synergistic effect and the promising therapeutic potential of this combination in treating FLT3-mutated AML (even with TKI-resistance). To understand the underlying molecular mechanism(s), we performed RNA-seq with vehicle- or drug-treated MOLM13-RES cells, followed by pathway analysis and validation/mechanistic studies, we identified Polo-like kinase 1 (PLK1) as a key target of MET and GLT (Fig. 1H). PLK1's expression is notably suppressed by both treatments (Fig. 1I). Mimicking MET treatment, PLK1 knockdown enhanced GLT sensitivity, reducing IC50 from 259.2 nM to 47-60 nM (Fig. 1J). Conversely, PLK1 overexpression increased GLT's IC50 7-fold (13.07 nM to 95.94 nM) (Fig. 1K), largely reversing MET's effect (data not shown). PLK1, a crucial serine/threonine-protein kinase, influences multiple signaling pathways such as MAPK and mTOR pathways. Upon treating MOLM13 or MOLM13-RES cells with MET and GLT alone or combined, the PLK1 levels and phosphorylation levels of FLT3, STAT5, ERK, and mTOR were decreased, with the most significant decrease occurred upon the combined treatment (Fig. 1L). Depletion and overexpression of PLK1 decreased and increased these phosphorylation levels, respectively (data not shown). Together, our findings suggest that the synergistic therapeutic effect of MET and GLT in treating FLT3-mutated AML is likely through synergistically targeting PLK1 and its downstream pathways. Meanwhile, we executed a retrospective clinical study of FLT3/ITD AML patients who were also diagnosed with diabetes. Metformin users showed a notably longer median OS (13.2 months) than non-MET users (4.5 months, P=0.0197) (Fig. 1M). Despite FLT3/ITD status, FLT3 TKIs were seldom used in China due to high cost. Additionally, PLK1 level was dramatically higher in FLT3/ITD AML cases than in both healthy and FLT3/WT cases (P<0.001) (Fig. 1N), implying PLK1's role in FLT3/ITD's poor prognosis. In summary, our findings suggest a potential synergistic approach in treating FLT3-mutated AML using MET and GLT. This approach could reduce side effects and costs (crucial for developing countries) and improve outcomes. Larger clinical trials are needed to confirm these promising initial findings.

HDAC Inhibitor Synergizes with GL-V9, a Derivative of Wogonin, to Suppress Acute Myeloid Leukemia By Downregulating ESPL1 and PLK4

Background Developing more effective non-chemotherapy options is emerging needed for acute myeloid leukemia (AML). GL-V9, a newly synthetic flavonoid derivative, exhibits anticancer activities with low toxicity as its nature features, however, the anti-leukemia effect and molecular mechanism against AML remain elusive. HDACs inhibitors are promising effective therapies for cancers. Chidamide (CS055/HBI-8000) is a benzamide histone deacetylase (HDAC) inhibitor that induces growth inhibition, cell cycle arrest, and apoptosis in MDS and AML. Here, we sought to determine the potential synergistic anti-leukemia effect of Chidamide combined with GL-V9 in AML. Methods Cell proliferation assays were measured by Cell Counting Kit-8(CCK-8) in AML cells; CalcuSyn was used for synergy analysis. Cell cycle and apoptosis were measured by flow cytometry analysis. Transcriptome was analyzed by RNA-seq in U937 cells treated with 10μM Chidamide and in MV4-11 treated with 4μM GL-V9. qPCR was utilized for gene expression; R2 genomics analysis and visualization application were used for comparison of the expression difference and the Kaplan-Meier overall survival analysis was conducted by the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Results GL-V9 had a dose-dependent effect on cell proliferation arrest in U937 and MV4-11 cells. Chidamide significantly sensitized this effect of GL-V9 versus single-drug controls, and CalcuSyn analysis showed the synergistic effect of the combination (Fig.1A). The combination also significantly increased the % of apoptotic cells versus single-drug controls (p<0.0001) (Fig1.B). Chidamide induced G0/G1-phase arrest and GL-V9 induced G2/M arrest, whereas the combination significantly decreased the % of S-phase cells (Fig.1C). These data indicate that Chidamide+GL-V9 combination results in the synergistic effect on cell growth inhibition, apoptosis and cell cycle arrest in AML. Next, the drug-induced change of the global transcriptome was analyzed. The significantly different expression genes (DEGs) were identified upon GL-V9 treatment and Chidamide treatment versus vehicle control. 1663 DEGs are identified upon GL-V9 treatment and 3756 DEGs upon Chidamide, in which 1405 were intersected regulated (1231 genes changed in the same direction but 174 changed in the opposite (Fig.2AB). The overlapped DEGs are mainly enriched in cell cycle regulation, DNA repair, chromosome segregation, spindle organization, DNA damage, etc. (Fig2.C). ESPL1 and PLK4 are at the top of the overlapped down-regulated DEGs; and qPCR results confirmed their mRNA levels are significantly downregulated by GL-V9+Chidamide, GL-V9, and Chidamide versus the vehicle control (p<0.0001) (Fig. 2D). ESPL1 encoded protein, Separase is an enzyme resolving sister chromatid cohesion, and plays a pivotal role in cell division and oncogenesis. PLK4 (polo-like kinase 4) is a key regulator of centrosome duplication and the substrate of histone acetyltransferases, and PLK4 overexpression leads to centrosome amplification in human cancer and contributes to multipolar spindle assembly. Our results not only indicated that the combination exerts its anti-tumor effect through down-regulation of ESPL1 and PLK4 expression but also reveal their oncogenic roles in AML. Indeed, the mRNA levels of ESPL1 and PLK4 are significantly high expressed in AML patients versus normal BM controls, and ESPL1 high expression is correlated with poor prognosis (Log-rank p=0.0039) (Fig2.E). In addition, our data showed that the expression of the anti-apoptosis genes BCL2 and BCL2L1 are significantly decreased, and the early apoptosis indicators, Caspase-3 and Casepase-9 increased by the combination versus the single-drug controls in U937 cells (Fig2.F). The proposed mechanism model is summarized in Fig. 2G. Conclusion Our data indicate that HDAC inhibitor Chidamide significantly synergizes GL-V9-induced cell growth inhibition, apoptosis, and cell cycle arrest in AML, and the underlying mechanism by suppressing the expression of ESPL1 and PLK4, the key regulators of cell division and proliferation. Our results also provide a potential chemotherapy-free option for AML patients.

Preliminary Results from a Phase 1b/2 Open-Label, Multicenter, Dose Optimization Clinical Study of the Safety, Tolerability, and Pharmacokinetic (PK) and Pharmacodynamic (PD) Profiles of Cfi-400945 As a Single Agent or in Combination with Azacitidine in Patients (Pts) with Acute Myeloid Leukemia, Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia (TWT-202)

Background: CFI-400945 is a potent, selective, orally administered, first-in-class inhibitor of the serine/threonine kinase Polo-like kinase 4 (PLK4), a unique Polo-like kinase family member, that is a conserved upstream regulator of centriole duplication. Dysregulation of PLK4 expression causes loss of centrosome numeral integrity, promotes genomic instability and also potentially enables cancer cells to tolerate genomic instability (Mason et al, Cancer Cell 2014). Its inhibition likely causes mitotic catastrophe. An investigator-initiated study (IIS) demonstrated clinical activity in relapsed/refractory AML with adverse cytogenetics. Dose limiting toxicity was colitis, sepsis and the RP2D 96 mg, three CR's (9 evaluable pts) across 2 dose levels were observed [Data on File, Murphy et al]. We report updated data on a company sponsored study (TWT-202; NCT04730258). Study Design and Methods: TWT-202 is a study of CFI-400945 with 3+3 design in escalation and Simon 2 stage in expansion. For Part 1 (CFI-400945), pts with relapsed and/or refractory AML, MDS, or CMML after >1 prior therapy will be included. Pts with MDS or CMML must have progressed or no response after 4 cycles of hypomethylating agents. For Part 2, (CFI-400945 with azacitidine), pts should have relapsed and/or refractory AML, untreated MDS or CMML. Untreated pts ineligible for intensive therapy may be included. The maximum tolerated dose (MTD) is defined as the dose where dose-limiting toxicities (DLTs) are <1 out of 6 at or below the maximally administered dose. Pharmacokinetics (PK) and pharmacodynamic (PD) markers will be assessed. Results: At 2May2023 cut, 26 pts were enrolled into Part 1. Eighteen (69%) pts had AML, 4 (15%) MDS, 4 (15%) CMML. The median number of prior therapies was 3 (range 1-8), 7 (27%) pts had received a previous stem cell transplant. Twenty (77%) were male, the median age 68 years (range 22, 85). Approximately 80% pts with AML were adverse risk by ELN 2022 criteria. Six pts with AML had TP53 mutations. At 21 days on/7 days off schedule, 5 pts received 32mg, 4 pts 48mg, 3 pts 64mg, 5 pts 80mg, 9 pts 96mg daily. The 28 days on/0 days off schedule was introduced at 96mg, 3 pts enrolled, with 0 DLTs to date. There was 1 DLT at 96mg on the 21/7 schedule (fungemia, gastroparesis, septic shock, enteritis). There have been 23 (89%) SAEs with the following occurring in >3 pts (febrile neutropenia (13 pts), sepsis (5 pts), diarrhea (4 pts), pneumonia (4 pts)). Common treatment emergent adverse events (TEAE) in > 5 pts, all grades, all doses; febrile neutropenia-15 pts (58%), nausea-11 pts (42%), diarrhea, vomiting-both 10pts (39%), hypokalemia- 9 pts (35%), dsypnea, hypophosphatemia-8pts each (31%), anemia-7pts (27%), and abdominal pain, fatigue, hypomagnesemia, pneumonia -6 pts each (23%). 31 % (33 events, 8 pts) ≥grade 3 TEAE's were considered related to CFI-400945 . Three pts (50%) with AML (all with prior venetoclax based therapies) at the 96mg dose level (21/7) from 6 evaluable achieved a response after 1 cycle. One pt (85M) with ELN2022 adverse genetics: complex karyotype, TP53 mutation, achieved CRi, and remains in remission after 3 cycles. One pt (70M) with AML (ELN2022 adverse genetics, complex cytogenetics, TP53 mutation), 2 prior regimens, achieved an MLFS and is pending count recovery. Third pt (51M) had AML normal cytogenetics, ELN 2022 adverse genetics (RUNX1, IDH2, SRSF2), and had relapsed/refractory disease after 5 regimens. The pt achieved an MLFS, however medical complications not related to study drug resulted in dose reduction, therapy hold and subsequent relapse. Pharmacokinetic (PK) evaluations indicated mean terminal-life ranged from 7 - 12 hours. Low accumulation was observed after 21d (AR <2-fold). Exposure in this study is overlapped with that observed in the IIS (Data on File, Murphy et al) study. PD studies evaluating the effect of CFI-400945 on markers of mitosis, ploidy and centriole function are ongoing. Conclusion: CFI-400945 has been generally well tolerated in this difficult to treat patient population, including patients whose disease progressed on or following venetoclax based therapies. Three of 6 evaluable patients with AML achieved a response (MLFS=2, CRi=1) at the 96mg dose. PK characteristics support daily dosing of CFI-400945 and PD studies are ongoing. Dose expansions are planned and a combination with azacitidine will open shortly. Murphy et al, Data on File, PMCC Mason et al Cancer Cell 26, 163-176

PLK1 inhibitors as a new targeted treatment for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options. Polo-like kinase 1 (PLK1) is a promising drug target; PLK1 inhibitors (PLK1i) have been investigated in solid cancers and are more effective in TP53-mutated cases. We evaluated PLK1 expression in ACC samples and the efficacy of two PLK1i in ACC cell lines with different genetic backgrounds. PLK1 protein expression was investigated by immunohistochemistry in tissue samples and correlated with clinical data. The efficacy of rigosertib (RGS), targeting RAS/PI3K, CDKs and PLKs, and poloxin (Pol), specifically targeting the PLK1 polo-box domain, was tested in TP53-mutated NCI-H295R, MUC-1, and CU-ACC2 cells and in TP53 wild-type CU-ACC1. Effects on proliferation, apoptosis, and viability were determined. PLK1 immunostaining was stronger in TP53-mutated ACC samples vs wild-type (P = 0.0017). High PLK1 expression together with TP53 mutations correlated with shorter progression-free survival (P= 0.041). NCI-H295R showed a time- and dose-dependent reduction in proliferation with both PLK1i (P< 0.05at 100 nM RGS and 30 µM Pol). In MUC-1, a less pronounced decrease was observed (P< 0.05at 1000 nM RGS and 100 µM Pol). 100 nM RGS increased apoptosis in NCI-H295R (P< 0.001), with no effect on MUC-1. CU-ACC2 apoptosis was induced only at high concentrations (P < 0.05 at 3000 nM RGS and 100 µM Pol), while proliferation decreased at 1000 nM RGS and 30 µM Pol. CU-ACC1 proliferation reduced, and apoptosis increased, only at 100 µM Pol. TP53-mutated ACC cell lines demonstrated better response to PLK1i than wild-type CU-ACC1. These data suggest PLK1i may be a promising targeted treatment of a subset of ACC patients, pre-selected according to tumour genetic signature.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n=76). In contrast, <5% of classic Hodgkin lymphoma cases (n=70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis MethodsIn this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. ResultsAfter PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. ConclusionIn ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML

AbstractAcute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the “don’t-eat-me” signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

Polo-like Kinase 4: A Multifaceted Marker Linking Tumor Aggressiveness and Unfavorable Prognosis, and Insights into Therapeutic Strategies.

(1) Background: This study investigated whether polo-like kinase 4 (PLK4) is a suitable therapeutic target or biomarker for lung adenocarcinoma (LUAD). (2) Methods: We acquired LUAD data from The Cancer Genome Atlas (TCGA) database through the UCSC Xena data portal. Gene expression, clinical, survival, and mutation data from multiple samples were analyzed. Gene enrichment analysis, unsupervised clustering of PLK4-related pathways, and differential gene expression analyses were performed. Additionally, correlations, t-tests, survival analyses, and statistical analyses were performed. (3) Results: PLK4 expression was higher in LUAD tissues than in normal tissues and was associated with poor prognosis for both overall and progression-free survival in LUAD. PLK4 was highly correlated with cell-proliferation-related pathways using Gene Ontology (GO) biological process terms. PLK4 expression and pathways that were highly correlated with PLK4 expression levels were upregulated in patients with LUAD with the TP53 mutation. (4) Conclusions: PLK4 expression affects the survival of patients with LUAD and is a potential therapeutic target for LUAD with TP53 mutations.

Polo-like kinase 1 promotes pulmonary hypertension

BackgroundPulmonary hypertension (PH) is a lethal vascular disease with limited therapeutic options. The mechanistic connections between alveolar hypoxia and PH are not well understood. The aim of this study was to investigate the role of mitotic regulator Polo-like kinase 1 (PLK1) in PH development.MethodsMouse lungs along with human pulmonary arterial smooth muscle cells and endothelial cells were used to investigate the effects of hypoxia on PLK1. Hypoxia- or Sugen5416/hypoxia was applied to induce PH in mice. Plk1 heterozygous knockout mice and PLK1 inhibitors (BI 2536 and BI 6727)-treated mice were checked for the significance of PLK1 in the development of PH.ResultsHypoxia stimulated PLK1 expression through induction of HIF1α and RELA. Mice with heterozygous deletion of Plk1 were partially resistant to hypoxia-induced PH. PLK1 inhibitors ameliorated PH in mice.ConclusionsAugmented PLK1 is essential for the development of PH and is a druggable target for PH.