Polo-like Kinase 1 Overexpression Research Articles

Targeted PLK1 suppression through RNA interference mediated by high-fidelity Cas13d mitigates osteosarcoma progression via TGF-β/Smad3 signalling.

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Overexpression of polo-like kinase 1 (PLK1) is frequent in osteosarcoma and drives disease progression and metastasis, making it a promising therapeutic target. In this study, we explored PLK1 knockdown in osteosarcoma cells using RNA interference mediated by high-fidelity Cas13d (hfCas13d). PLK1 was found to be significantly upregulated in osteosarcoma tumour tissues compared to normal bone. sgRNA-mediated PLK1 suppression via hfCas13d transfection inhibited osteosarcoma cell proliferation, induced G2/M cell cycle arrest, promoted apoptosis, reduced cell invasion and increased expression of the epithelial marker E-cadherin. Proximity labelling by TurboID coupled with co-immunoprecipitation identified novel PLK1 interactions with Smad3, a key intracellular transducer of TGF-β signalling. PLK1 knockdown impaired Smad2/3 phosphorylation and modulated TGF-β/Smad3 pathway inactivation. Finally, invivo delivery of hfCas13d vectors targeting PLK1 substantially attenuated osteosarcoma xenograft growth in nude mice. Taken together, this study highlights PLK1 as a potential therapeutic target and driver of disease progression in osteosarcoma. It also demonstrates the utility of hfCas13d-mediated gene knockdown as a strategy for targeted therapy. Further optimization of PLK1 suppression approaches may ultimately improve clinical outcomes for osteosarcoma patients.

PLK1 and PARP positively correlate in Middle Eastern breast cancer and their combined inhibition overcomes PARP inhibitor resistance in triple negative breast cancer.

Despite advancements in treatment approaches, patients diagnosed with aggressive breast cancer (BC) subtypes typically face an unfavorable prognosis. Globally, these cancers continue to pose a significant threat to women's health, leading to substantial morbidity and mortality. Consequently, there has been a significant struggle to identify viable molecular targets for therapeutic intervention in these patients. Polo-like Kinase-1 (PLK1) represents one of these molecular targets currently undergoing rigorous scrutiny for the treatment of such tumors. Yet, its role in the pathogenesis of BC in Middle Eastern ethnicity remains unexplored. We investigated the expression of PLK1 protein in a cohort of more than 1500 Middle Eastern ethnicity BC cases by immunohistochemistry. Association with clinicopathological parameters and prognosis were performed. In vitro studies were conducted using the PLK1 inhibitor volasertib and the PARP inhibitor olaparib, either alone or in combination, in PTC cell lines. Overexpression of PLK1 was detected in 27.4% of all BC cases, and this was notably correlated with aggressive clinicopathological markers. PLK1 was enriched in the triple-negative breast cancer (TNBC) subtype and exhibited poor overall survival (p = 0.0347). Notably, there was a positive correlation between PLK1 and PARP overexpression, with co-expression of PLK1 and PARP observed in 15.7% of cases and was associated with significantly poorer overall survival (OS) compared to the overexpression of either protein alone (p = 0.0050). In vitro, we studied the effect of PLK1 and PARP inhibitors either single or combined treatments in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and persuaded apoptosis in TNBC cell lines. Moreover, our findings indicate that inhibition of PLK1 can reinstate sensitivity in PARP inhibitor (PARPi) resistant TNBC cell lines. Our results shed light on the role of PLK1 in the pathogenesis and prognosis of Middle Eastern BC and support the potential clinical development of combined inhibition of PLK1 and PARP, a strategy that could potentially broaden the use of PLK1 and PARP inhibitors beyond BC cases lacking BRCA.

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.

A simple Turing reaction-diffusion model explains how PLK4 breaks symmetry during centriole duplication and assembly.

Centrioles duplicate when a mother centriole gives birth to a daughter that grows from its side. Polo-like-kinase 4 (PLK4), the master regulator of centriole duplication, is recruited symmetrically around the mother centriole, but it then concentrates at a single focus that defines the daughter centriole assembly site. How PLK4 breaks symmetry is unclear. Here, we propose that phosphorylated and unphosphorylated species of PLK4 form the 2 components of a classical Turing reaction-diffusion system. These 2 components bind to/unbind from the surface of the mother centriole at different rates, allowing a slow-diffusing activator species of PLK4 to accumulate at a single site on the mother, while a fast-diffusing inhibitor species of PLK4 suppresses activator accumulation around the rest of the centriole. This "short-range activation/long-range inhibition," inherent to Turing systems, can drive PLK4 symmetry breaking on a either a continuous or compartmentalised Plk4-binding surface, with PLK4 overexpression producing multiple PLK4 foci and PLK4 kinase inhibition leading to a lack of symmetry-breaking and PLK4 accumulation-as observed experimentally.

Mitotic Errors Increase Radiation Sensitivity in Multiple Cancer Cell Types and in Laryngeal Cancer Patients

Chromosomal instability (CIN) arises from continual mitotic errors over the course of multiple cell divisions and is common in cancer. When CIN is increased beyond a maximally tolerated threshold it leads to cell death due to loss of both copies of one or more essential chromosomes. Combining two independent insults that each cause tolerable levels of CIN results in high CIN, cell death and tumor suppression. Because radiation causes CIN, we hypothesize that pre-existing CIN sensitizes tumor cells to radiation therapy. We characterized CIN in 4 HPV+ and 4 HPV- head and neck (HNC), 4 cervical, and 2 breast cancer cell lines before and 24 hours after 2 Gy of radiation. CIN was quantified by scoring lagging, bridge, and misaligned chromosomes, centrosome amplification, and multipolar spindles using immunofluorescence microscopy. To test our hypothesis, we created isogenic CIN and non-CIN HeLa and FaDu cells by knocking down the mitotic checkpoint protein Mad1, which induces lagging chromosomes, and by using MCF10A cells expressing tet-inducible polo-like kinase 4 (PLK4) to induce centrosome amplification. Clonogenic assays and murine tumor growth curves were used to determine radiation sensitivity in vitro and in vivo respectively. 6-chromosome FISH was used to quantify CIN and test if it was associated with local recurrence (LR) in a cohort of 29 laryngeal cancer patients treated with definitive (chemo)radiation. Significant differences were determined using a two-tailed student's t-test or Fisher's exact test. A total of 2 Gy of radiation significantly increased lagging, bridge, and misaligned chromosomes and induced centrosome amplification in all cancer cell types in vitro and in vivo. Most abnormal chromosomes after radiation were acentric fragments, which are known to missegregate. Mad1 knockdown in HeLa and FaDu cells increased CIN and led to significantly increased radiation sensitivity compared to their wild-type counterparts. PLK4 overexpression in MCF10A cells led to centrosome amplification and multipolar spindles, which also increased radiation sensitivity. Additionally, mitotic errors were significantly correlated with radiation response in both HPV+ and HPV- patient derived xenograft (PDX) HNC tumors. Finally, patients with laryngeal tumors with CIN below the median of the cohort had a 31% rate of LR after therapy, while tumors with CIN greater than or equal to the median had a LR rate of only 6%. Cancer cells with higher CIN are more sensitive to radiation therapy in vitro and in vivo. This was true for different types of mitotic errors and in different cancer models implying this is likely tumor type agnostic. Importantly, laryngeal tumors with higher baseline levels of CIN had an improved response to definitive radiation therapy, implying increased radiation sensitivity. These studies have potential implications for personalization of radiation dose.

Polo-like kinase-1 mediates hepatitis C virus-induced cell migration, a drug target for liver cancer.

Polo-like kinase 1 (PLK1) is a regulator of cell mitosis and cytoskeletal dynamics. PLK1 overexpression in liver cancer is associated with tumour progression, metastasis, and vascular invasion. Hepatitis C virus (HCV) NS5A protein stimulates PLK1-mediated phosphorylation of host proteins, so we hypothesised that HCV-PLK1 interactions might be a mechanism for HCV-induced liver cancer. We used a HCV cell-culture model (Jc1) to investigate the effects of virus infection on the cytoskeleton. In HCV-infected cells, a novel posttranslational modification in β-actin was observed with phosphorylation at Ser239. Using in silico and in vitro approaches, we identified PLK1 as the mediating kinase. In functional experiments with a phosphomimetic mutant form of β-actin, Ser239 phosphorylation influences β-actin polymerization and distribution, resulting in increased cell motility. The changes were prevented by treating cells with the PLK1 inhibitor volasertib. In HCV-infected hepatocytes, increased cell motility contributes to cancer cell migration, invasion, and metastasis. PLK1 is an important mediator of these effects and early treatment with PLK1 inhibitors may prevent or reduce HCC progression, particularly in people with HCV-induced HCC.

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.

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.

GSK461364A suppresses proliferation of gastric cancer cells and induces apoptosis

Polo-like kinase 1 (PLK1) is crucial in regulating cell division and has been shown to have an oncogenic function in several cancers. Since PLK1 overexpression is closely related to tumorigenesis and has been correlated with poor clinical outcomes, specific inhibition of PLK1 in cancer cells is a promising approach for developing new anticancer drugs. In this context, the aim of the present study was to evaluated the potential cytotoxic effects of GSK461364A, a competitive inhibitor for PLK1, in gastric cancer cell line SNU-1 cells and explored its cytotoxic mechanism. The cells were exposed to GSK461364A at different concentrations ranging from 1 to 40 μM for 24 h, and it showed considerable cytotoxicity with an IC50 value of 4.34 μM. The treatment of SNU-1 cells with GSK461364A results in cell cycle arrest at the G2/M phase, decreased mitochondrial membrane potential, and increased apoptosis as indicated by Annexin V binding assay. In addition, GSK461364A treatment significantly increased the total oxidant (TOS) level, a signal of oxidative stress, and increased cleaved PARP and 8-oxo-dG levels as an indicator of DNA damage. ELISA experiments evaluating Bax, BCL-2, and cleaved caspase 3 also confirmed the apoptotic effect of GSK461364A. Current findings suggest that GSK461364A may be a chemotherapeutic agent in patients with gastric cancer. Nevertheless, more research is needed to evaluate GSK461364A as a cancer treatment drug.

Abstract 2655: Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma

Abstract Previously, we have shown that overexpression of Polo-like kinase (PLK)-1 is associated with poor clinical outcomes in patients with aggressive B cell lymphomas. PLK-1 selective inhibitor, Volasertib (BI 6727), has a potent anti-tumor effect in aggressive B cell lymphomas. Unfortunately, Volasertib was withdrawn in Phase III clinical trials due to severe myelosuppression. In this study, we developed a Volasertib antibody-drug conjugate (V-ADC) using -CD19 antibody Inebilizumab to increase the targeting specificity for B-cell lymphoma cells and minimize the side effects of Volasertib. However, we found that V-ADC treatment exhibited little cytotoxic effects, comparing to free Volasertib treatment in Z138 cells, a mantle cell lymphoma (MCL) cell line. We hypothesized that the expression level/density of CD19 on B-lymphoma cells as well as its gene mutations may affect V-ADC binding affinity required for its endocytosis. We, therefore, established Z138 cell lines with overexpression of either wild-type (WT) or mutant (Mut) CD19. The cell viability at 48h post 10nM V-ADC treatment was determined to be 90%, 61%, and 69% in parent, WT, and Mut CD19-overexpressing Z138 cells, respectively. In comparison, the cell viability with 10nM free volasertib treatment ranged from 3-30% in these cell lines. Therefore, other factors, in addition to CD19 expression, may have also played an important role in V-ADC induced cytotoxicity. Upon endocytosis, the V-ADC will enter the endolysosome where cathepsin, a lysosomal enzyme, cleaves the linker between Volasertib and Inebilizumab and releases free Volasertib into the cells. Our previous works have shown that a starvation condition (culture media with 1% FBS) increased lysosomal cathepsin activity, comparing to the culture media with 10% FBS. Herein, we found that upon treatment with 10nM V-ADC, CD19-overexpressing Z138 cells cultured in media with 1% FBS exhibited markedly increased apoptotic cell death comparing to cells in media with 10% FBS (64% vs 41%, respectively). Furthermore, WT and Mut CD19-overexpressing Z138 cells exhibited 2.8- and 2.4-folds of apoptotic cell death compared to control Z138 cells at 48h post 10nM V-ADC treatment with 1% FBS. The cell apoptosis occurred via upregulation of caspase 3, but not caspase 9, with induction of cleavage of poly (ADP-ribose) polymerase (PARP) and reduction of mitochondria membrane potential. In conclusion, starvation and lysosomal cathepsin activation increased V-ADC-induced apoptotic cell death in CD19 overexpression Z138 cell lines. We are actively investigating the in vivo therapeutic effects of V-ADC in patient-derived xenograft (PDX) animal models of MCL and other aggressive B-cell lymphomas. Citation Format: Chieko Saito, Chengfeng Bi, Kai Fu. Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2655.

Abstract 4997: PLK1 promotes the metastasis and drug resistance in melanoma

Abstract Melanoma is one of the most frequently diagnosed cancers in the caucasian population of both genders. The survival rate of advanced melanoma patients will be exceedingly poor, as low as 27.3%. BRAF-V600E is the most common mutation found clinically, resulting in the constitutive activation of the MAPK signaling pathway. Vemurafenib, a specific inhibitor of mutant BRAF, has shown an impressive response in phase 3 clinical trials and was approved by FDA in 2011. However, due to the rapid development of resistance, the duration of response under a single treatment is frequently short, highlighting the urgent requirement for novel therapy in melanoma treatment. Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, participates in multiple mitotic processes, including centrosome maturation, mitotic entry, spindle assembly, sister chromatid segregation, mitotic exit, and cytokinesis. Compared to normal tissues, the expression level of PLK1 significantly upregulates in multiple cancers. More importantly, the expression level of PLK1 negatively correlates with the melanoma patients’ survival rate based on the TCGA database. Besides, PLK1 is identified as an oncogene to promote proliferation, motility, and resistance to various drugs. In the present study, we found that overexpression of PLK1 promotes tumor growth, metastasis, metabolic reprogramming, and vemurafenib resistance in the BrafCA/Ptenloxp mouse melanoma model. Through phosphorylation, overexpressed PLK1 stabilizes BACH1, a critical transcriptional factor of multiple metastatic and metabolic genes, and protects it from degradation. Subsequently, a higher level of BACH1 leads to increasing metastasis, upregulation of glycolysis, and suppression of OXPHOs. Besides, we have validated the strong synergy between PLK1 inhibitor volasertib and vemurafenib in treating melanoma carrying BRAF-V600E. Our in vitro and in vivo experiments have shown an improved efficacy of this combined therapy on inhibition of cell proliferation, induction of cell death, and suppression of cell metastasis compared to mono-treatment. In short, our data has indicated PLK1 as a potent and promising target in cancer treatment. Citation Format: Fengyi Mao, Yifan Kong, Chaohao Li, Yanquan Zhang, Xiongjian Rao, Qiongsi Zhang, Xinyi Wang, Zhiguo Li, Xiaoqi Liu. PLK1 promotes the metastasis and drug resistance in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4997.

Polo-like kinase-1 as a potential prognostic marker of prostate cancer utilizing ORIEN data.

384 Background: Polo-Like Kinase-1(PLK1) is one of the key regulators in G2/M cell cycle. Preclinical studies have shown that PLK1 might be involved in treatment resistance in prostate cancer (PCa). PLK1 overexpression by immunohistochemistry is associated with higher Gleason grade and thus PLK1 could play a role in PCa tumorigenesis. In this study, we utilized a different methodology by using RNA-seq PLK1 expression and correlated with clinical outcomes. Methods: Patients with all stages of prostate adenocarcinoma diagnosed between 2014 and 2020 from nine participating members of the Oncology Research Information Exchange Network (ORIEN) were included in the study. Tumor RNA-seq was analyzed for PLK1 expression and associated with clinical data obtained from ORIEN AVATAR. PLK1 expression was stratified into high expression (defined as ≥75th percentile of PLK1 mRNA expression) vs. low expression (≤75th percentile). Univariate and multivariate Cox proportional hazard model were used to compare overall survival between patients with low vs high PLK1 expression adjusting for age, race, PSA and stage. Results: A total of 452 tumors were evaluated for PLK1 expression. Of those, 241 (53.3%) had high PLK1 and 211 (46.7%) had low PLK1 expression. Median age at diagnosis for high and low PLK1 expression was 64.5 and 63.2 years respectively. The majority of patients were Caucasian in both groups. In high PLK1 group, 93 (65%) had localized high risk/very high risk disease, 27 (19%) had localized intermediate risk, 20 (14%) had metastatic vs. 97 (63%), 38 (25%) and 18(12%) respectively in low PLK1 group. Gleason ≥ 8 and PSA <20 were seen slightly more in high PLK1 compared to low PLK1; 40% vs 33% and 96% vs.94% respectively. The median follow-up time was 2.74 years in low PLK1 group and 2.71 years in high PLK1 group. There was no difference between PLK1 low vs. high expression for age, race, stage, PSA and Gleason grade. There was no difference in overall survival between low PLK1 and high PLK1 expression by univariate analysis (p=0.86; HR 1.09) or after adjusting for multivariate analysis (p=0.85; HR 1.12). Conclusions: In our study, we did not find RNA-seq PLK1 expression as a potential prognostic marker for prostate cancer. There was no survival difference between low PLK1 vs. high PLK1 expression.

PLK1 protects intestinal barrier function in sepsis: A translational research.

Sepsis and its related complications are very challenging in the intensive care unit, among which intestinal barrier injury is a general manifestation. Polo-like kinase 1 (PLK1) is widely studied in cancer, while its role in sepsis is poorly understood. In this study, the efficiency of PLK1 as a marker of intestinal barrier function as well as a predictor of mortality in sepsis was evaluated. The level of serum PLK1 was measured in septic patients (n=51) and controls (n=20); subsequently, its correlation with serum diamine oxidase (DAO), d-lactate, and endotoxin levels and its ability topredict mortality were analysed. The survival rate and barrier injury degree were also assessed in septic mice. Serum PLK1 levels were elevated in septic patients, were negatively correlated with serum DAO, d-lactate, and endotoxin levels, and had a high predictive value for 28-day mortality in patients. The serum PLK1 level in non-survivors was lower. The expression of PLK1 in the intestine was decreased in septic mice, and overexpression or inhibition of PLK1 alleviated or aggravated intestinal barrier injury, respectively, as evaluated by Chiu's score, serum levels of DAO and d-lactate, and expression of tight junction proteins. Overexpressing PLK1 also decreased the 72-hour death rate of septic mice. Further study also revealed the negative correlation of PLK1 and IL-6 in patients, and increasing or interfering with PLK1 expression reduced or increased the serum IL-6 level in mice. PLK1 plays a critical role in intestinal barrier function during sepsis, providing a novel perspective for sepsis therapy in the clinic.

3D-QSAR Studies, Molecular Docking, Molecular Dynamic Simulation, and ADMET Proprieties of Novel Pteridinone Derivatives as PLK1 Inhibitors for the Treatment of Prostate Cancer.

Overexpression of polo-like kinase 1 (PLK1) has been found in many different types of cancers. With its essential role in cell proliferation, PLK1 has been determined to be a broad-spectrum anti-cancer target. In this study, 3D-QSAR, molecular docking, and molecular dynamics (MD) simulations were applied on a series of novel pteridinone derivatives as PLK1 inhibitors to discover anti-cancer drug candidates. In this work, three models-CoMFA (Q² = 0.67, R² = 0.992), CoMSIA/SHE (Q² = 0.69, R² = 0.974), and CoMSIA/SEAH (Q² = 0.66, R² = 0.975)-of pteridinone derivatives were established. The three models that were established gave Rpred2 = 0.683, Rpred 2= 0.758, and Rpred 2= 0.767, respectively. Thus, the predictive abilities of the three proposed models were successfully evaluated. The relations between the different champs and activities were well-demonstrated by the contour chart of the CoMFA and CoMSIA/SEAH models. The results of molecular docking indicated that residues R136, R57, Y133, L69, L82, and Y139 were the active sites of the PLK1 protein (PDB code: 2RKU), in which the more active ligands can inhibit the enzyme of PLK1. The results of the molecular dynamic MD simulation diagram were obtained to reinforce the previous molecular docking results, which showed that both inhibitors remained stable in the active sites of the PLK1 protein (PDB code: 2RKU) for 50 ns. Finally, a check of the ADME-Tox properties of the two most active molecules showed that molecular N° 28 could represent a good drug candidate for the therapy of prostate cancer diseases.

Cell cycle kinases (AUKA, CDK1, PLK1) are prognostic biomarkers and correlated with tumor-infiltrating leukocytes in HBV related HCC

Hepatocellular carcinoma (HCC) is one of the high incidence cancers and third leading cause of cancer-related mortality. HBV is the top most risk factor accounting for 50–80% of the HCC cases. Kinases: Aurora kinase A (AURKA), cyclin-dependent kinase (CDK1) and Polo-like kinase 1 (PLK1), the key regulators of cell mitosis are overexpressed in varieties of cancers including HCC. However, the exact role of these genes in prognosis of HCC is not fully unveiled. In addition, there is no such an accurate prognostic biomarker for HBV-related HCC. To address this issue, we performed a multidimensional analysis of AURKA, CDK1 and PLK1 with a series of publicly available databases in multiple cancers and with experimental validation in HBV-related HCC tissues. Overexpression of AURKA, CDK1 and PLK1 was found in multiple cancers including HCC. Elevated expression of these genes could result from lowered DNA methylation and genomic alterations. Transcriptional overexpression was significantly correlated with poor prognosis of HCC patients. The expression levels were also significantly positively associated with tumor grades and stages. Furthermore, the expression levels of these genes had a strong correlation with infiltration of immune cells. Our analysis shows that AURKA, CDK1 and PLK1 are correlated with immune infiltration and are the prognostic biomarkers for HBV-induced HCC. Communicated by Ramaswamy H. Sarma

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

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

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

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

Interleukin 17 Receptor C, but not Polo-like Kinase 4, Plays a Critical Role in the Development of Skin Papilloma and Psoriasis in Mouse Models

Abstract Introduction/Objective Hyperproliferation of epidermis is a histopathological hallmark of skin cancers and psoriasis. As a master regulator of centriole replication, overexpression of Polo-like kinase 4 (PLK4) has been identified in skin cancers. The aberrant proliferation of epidermal keratinocytes provoked by interleukin 17 (IL17) leads to psoriasis. Thus, targeting centriole replication and IL17 signaling simultaneously has been speculated as a potential therapeutic strategy. We hypothesized that inhibition of centriole duplication enhances the blockade of epidermal proliferation through Il17rc knockout. Methods/Case Report To test our hypothesis, 37 mice were used to imbed a two-stage model of skin carcinogenesis using wild-type (WT), IL17 receptor A (T779A) knock-in (Il17ra(T779A)-KI), and IL17 receptor C knock-out (Il17rc-KO) C57BL/6J mouse strains. Furthermore, an imiquimod-induced psoriasis model was established using 69 mice with the same strains. Results (if a Case Study enter NA) In our two-stage skin carcinogenesis model, Il17ra(T779A)-KI mice showed significantly decreased tumor incidence, tumor multiplicity, and tumor volume compared to the WT mice. Il-17rc-KO mice didn’t develop any skin papilloma. The skin papilloma formed never progressed into squamous carcinoma. However, centrinone, a selective inhibitor of PLK4, didn’t affect skin papilloma formation or epidermal thickening. In our psoriasis model, the epidermis thickness of Il-17rc-KO mice was dramatically decreased compared to WT and Il17ra(T779A)-KI mice. There was no significant difference between WT and Il17ra(T779A)-KI mice in terms of skin lesion and thickening of the epidermis. Centrinone didn’t stall the thickening of the epidermis in the psoriasis model. IHC staining showed significantly increased Ki67+ basal keratinocytes in the untreated skin of Il17ra(T779A)-KI male mice compared to WT mice. In imiquimod-treated skin, the percentage of Ki67+ basal keratinocytes significantly decreased in the order from WT, Il17ra(T779A)-KI to Il17rc-KO mice. Conclusion Our data suggest that the proliferation of keratinocytes is not stalled by centrinone but is inhibited by Il17rc-KO. Il17ra(T779A)-KI significantly inhibits skin papilloma formation, but slightly decreases epidermal thickening in the psoriasis model. However, Il17ra(T779A)-KI increases keratinocyte proliferation based on Ki67 staining in the untreated normal skin.

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

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