Polo-like Kinase 1 Inhibitor BI2536 Research Articles

Designing Effective Multi-Target Drugs and Identifying Biomarkers in Recurrent Pregnancy Loss (RPL) Using In Vivo, In Vitro, and In Silico Approaches

Recurrent pregnancy loss (RPL) occurs in approximately 5% of women. Despite an abundance of evidence, the molecular mechanism of RPL's pathology remains unclear. Here, we report the protective role of polo-like kinase 1 (PLK1) during RPL. We aimed to construct an RPL network utilizing GEO datasets and identified hub high-traffic genes. We also investigated whether the expressions of PLK1 were altered in the chorionic villi collected from women with RPL compared to those from healthy early pregnant women. Gene expression differences were evaluated using both pathway and gene ontology (GO) analyses. The identified genes were validated using in vivo and in vitro models. Mice with PLK1-overexpression and PLK1-knockdown in vitro models were produced by transfecting certain plasmids and si-RNA, respectively. The apoptosis in the chorionic villi, mitochondrial function, and NF-κB signaling activity was evaluated. To suppress the activation of PLK1, the PLK1 inhibitor BI2536 was administered. The HTR-8/SVneo and JEG-3 cell lines were chosen to establish an RPL model in vitro. The NF-κB signaling, Foxo signaling, PI3K/AKT, and endometrial cancer signaling pathways were identified via the RPL regulatory network. The following genes were identified: PLK1 as hub high-traffic gene and MMP2, MMP9, BAX, MFN1, MFN2, FOXO1, OPA1, COX15, BCL2, DRP1, FIS1, TRAF2, and TOP2A. Clinical samples were examined, and the results demonstrated that RPL patients had tissues with decreased PLK1 expression in comparison to women with normal pregnancies (p < 0.01). In vitro, PLK1 knockdown induced the NF-κB signaling pathway and apoptosis activation while decreasing cell invasion, migration, and proliferation (p < 0.05). Furthermore, the in vivo model proved that cell mitochondrial function and chorionic villi development are both hampered by PLK1 suppression. Our findings revealed that the PLK1/TRAF2/NF-κB axis plays a crucial role in RPL-induced chorionic villi dysfunction by regulating mitochondrial dynamics and apoptosis and might be a potential therapeutic target in the clinic.

Abstract PS2-01: Plk1 expression &amp; efficacy of palbociclib in advanced hormonal receptor-positive breast cancer patients from PEARL study (GEICAM 2012-03)

Abstract Background: CDK 4/6 inhibitors (CDK 4/6i) with endocrine therapy (ET) combination therapy have improved outcomes in patients (pts) with hormonal receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-) advanced breast cancer (ABC). However, most pts eventually develop resistance to these drugs, and one third never respond. Aside from HR positivity, predictive markers of clinical benefit from CDK 4/6i remains elusive. We aimed to identify biomarkers of response to palbociclib (PAL) and analyze potential therapeutic targets to reverse resistance. Methods: PEARL trial is a multicenter phase 3 study that assigned 601 postmenopausal HR+/HER2- ABC pts, whose disease progressed on aromatase inhibitors (AIs), to receive PAL + ET vs capecitabine (CAPE). We performed a differential gene expression analysis in pre-treatment tumors in extreme responders to PAL using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Inc.), containing 2534 cancer related genes. Samples were subset in 2 categories: refractory (progressive disease as best response) vs sensitive (progression-free survival (PFS) within the upper quartile). Cox regression and Significance Analysis of Microarrays (SAM) analysis adjusting for multiple comparisons were performed. Results: We analyzed 455 (75.7%) pts with pre-treatment tumors available [from them, PAL + ET arm: 229 (50.3%) pts; CAPE arm: 226 (49.7%) pts]. Fifty genes (false discovery rate (FDR)&amp;lt;0.05) were differentially expressed in pts sensitive vs refractory to PAL (E2F target genes, epithelial-to-mesenchymal transition (EMT) and cell cycle genes, mainly). Unsupervised hierarchical clustering of pts based on the expression of these genes revealed two clusters. Cluster 1 is composed mostly of resistant tumors, highly proliferative (Ki67≥20%: 70%) with a great proportion of luminal B (59%) and non-luminal tumors (19%). Cluster 2 is composed of sensitive, low proliferative (Ki67&amp;lt;20%: 58%), mostly luminal A tumors (75%). There was no difference in ESR1 mutations distribution between the two clusters (Table 1). Forty genes were up-regulated and associated with resistance, including CCNE1 and PLK1 (Polo Like Kinase 1). In the whole cohort, pts with high levels (&amp;gt; median) of PLK1 (PLK1-high) treated with PAL, had a worse PFS in a multivariate model (5.7 months (m) vs 9.3 m of median PFS in PLK1-High vs -Low; HR=1.64, 95% CI (1.25-2.34), p=0.0008; adjusted model for confounders: age, site of disease, sites of metastasis, prior chemotherapy and Ki67). There were no differences in population treated with CAPE (9.9 m vs 9.4 m, PLK1-High vs -Low; HR=0.82, 95% CI (0.56-1.21), p=0.3189). In the METABRIC cohort, PLK1-High was associated with worse overall survival in HR+/HER2- BC but not in triple negative nor in HER2+ tumors. Among HR+/HER2- tumors, PLK1 expression was higher in luminal B and HER2-enriched intrinsic subtypes. We interrogated DepMap database and found that in BC cells lines there was an inverse correlation between PLK1 expression and effect on cell viability of CDK4 CRISPR knock-out (Pearson correlation r:0.54, p=0.009), but not of CDK6 knock-out. Also, HR+/HER2-/High Ki67 BC cell lines (HCC1428, EFM19 and MCF7) showed resistance to PAL on cell proliferation assays but sensitivity to the PLK1 inhibitor BI-2536. Conclusion: High expression of PLK1 is associated with intrinsic resistance to PAL and ET, this might be overcome with PLK1 inhibition. Table 1PATIENT CHARACTERISTICSCluster 1Cluster 2ALLn=57n=47n=104RespondersSensitive42 (73.68%)14 (29.79%)56 (53.85%)Refractory15 (26.32%)33 (70.21%)48 (46.15%)ESR1Mutated9 (15.79%)13 (27.66%)22 (21.15%)Wild type45 (78.95%)34 (72.34%)79 (75.96%)Unknown3 (5.26%)0 (0%)3 (2.88%)PriorQTN42 (73.68%)31 (65.96%)73 (70.19%)Y15 (26.32%)16 (34.04%)31 (29.81%)SubtypeLumA43 (75.44%)10 (21.28%)53 (50.96%)LumB14 (24.56%)28 (59.57%)42 (40.38%)Non Luminal0 (0%)9 (19.15%)9 (8.65%)MetastasisOne21 (36.84%)15 (31.91%)36 (34.62%)Multiple36 (63.16%)32 (68.09%)68 (65.38%)KI67 20%KI67&amp;lt;2033 (57.89%)7 (14.89%)40 (38.46%)KI67≥2016 (28.07%)33 (70.21%)49 (47.12%)Unknown8 (14.04%)7 (14.89%)15 (14.42%)Objective ResponseComplete1 (1.75%)0 (0%)1 (0.96%)Partial16 (28.07%)6 (12.77%)22 (21.15%)Progressive15 (26.32%)33 (70.21%)48 (46.15%)Stable25 (43.86%)8 (17.02%)33 (31.73%) Citation Format: Angel Guerrero- Zotano, Christoph Zielinski, Miguel Gil-Gil, Manuel Ruiz-Borrego, Eva M. Ciruelos, Montserrat Munoz, Begoña Bermejo, Mireia Margeli, Antonio Antón, Tibor Csöszi, Andrés García-Palomo, Ana Santaballa, Jose Luis Alonso, Antonio Fernández, Massimo Corsaro, Jesús Herranz, Paula López, Rosalia Caballero, Christiane Thallinger, Miguel Martin. Plk1 expression &amp; efficacy of palbociclib in advanced hormonal receptor-positive breast cancer patients from PEARL study (GEICAM 2012-03) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-01.

PLK1 (polo like kinase 1)-dependent autophagy facilitates gefitinib-induced hepatotoxicity by degrading COX6A1 (cytochrome c oxidase subunit 6A1)

ABSTRACT Liver dysfunction is an outstanding dose-limiting toxicity of gefitinib, an EGFR (epidermal growth factor receptor)-tyrosine kinase inhibitor (TKI), in the treatment of EGFR mutation-positive non-small cell lung cancer (NSCLC). We aimed to elucidate the mechanisms underlying gefitinib-induced hepatotoxicity, and provide potentially effective intervention strategy. We discovered that gefitinib could sequentially activate macroautophagy/autophagy and apoptosis in hepatocytes. The inhibition of autophagy alleviated gefitinib-induced apoptosis, whereas the suppression of apoptosis failed to lessen gefitinib-induced autophagy. Moreover, liver-specific Atg7 +/− heterozygous mice showed less severe liver injury than vehicle, suggesting that autophagy is involved in the gefitinib-promoted hepatotoxicity. Mechanistically, gefitinib selectively degrades the important anti-apoptosis factor COX6A1 (cytochrome c oxidase subunit 6A1) in the autophagy-lysosome pathway. The gefitinib-induced COX6A1 reduction impairs mitochondrial respiratory chain complex IV (RCC IV) function, which in turn activates apoptosis, hence causing liver injury. Notably, this autophagy-promoted apoptosis is dependent on PLK1 (polo like kinase 1). Both AAV8-mediated Plk1 knockdown and PLK1 inhibitor BI-2536 could mitigate the gefitinib-induced hepatotoxicity in vivo by abrogating the autophagic degradation of the COX6A1 protein. In addition, PLK1 inhibition could not compromise the anti-cancer activity of gefitinib. In conclusion, our findings reveal the gefitinib-hepatotoxicity pathway, wherein autophagy promotes apoptosis through COX6A1 degradation, and highlight pharmacological inhibition of PLK1 as an attractive therapeutic approach toward improving the safety of gefitinib-based cancer therapy. Abbreviations: 3-MA: 3-methyladenine; AAV8: adeno-associated virus serotype 8; ATG5: autophagy related 5; ATG7: autophagy related 7; B2M: beta-2-microglobulin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CHX: cycloheximide; COX6A1: cytochrome c oxidase subunit 6A1; c-PARP: cleaved poly(ADP-ribose) polymerase; CQ: chloroquine; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; HBSS: Hanks´ balanced salt solution; H&E: hematoxylin and eosin; MAP1LC3/LC3: microtubule associated proteins 1 light chain 3; PLK1: polo like kinase 1; RCC IV: respiratory chain complex IV; ROS: reactive oxygen species; TUBB8: tubulin beta 8 class VIII

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Retinoid X receptor alpha (RXRα), a nuclear receptor of transcription factor, controls various physiological and pathological pathways including cellular growth, proliferation, differentiation, and apoptosis. Here, we report that RXRα is phosphorylated at its N-terminal A/B domain by cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis, triggering its translocation to the centrosome, where phosphorylated-RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) through its N-terminal A/B domain by a unique mechanism. The interaction promotes PLK1 activation, centrosome maturation, and mitotic progression. Levels of p-RXRα are abnormally elevated in cancer cell lines, during carcinogenesis in animals, and in clinical tumor tissues. An RXRα ligand XS060, which specifically inhibits p-RXRα/PLK1 interaction but not RXRα heterodimerization, promotes mitotic arrest and catastrophe in a tumor-specific manner. These findings unravel a transcription-independent action of RXRα at the centrosome during mitosis and identify p-RXRα as a tumor-specific vulnerability for developing mitotic drugs with improved therapeutic index.

Inhibition of Polo-Like Kinase 1 by BI2536 Reverses the Multidrug Resistance of Human Hepatoma Cells In Vitro and In Vivo.

Multi Drug Resistance (MDR) is the phenomenon that cancers develop resistance to majority of chemotherapy drugs and is a serious obstacle to the treatment for Hepatocellular Carcinoma (HCC). Polo-Like Kinase 1 (PLK1) is a serine/threonine kinase associated with tumor growth and clinical prognosis in HCC and BI2536 is its potent inhibitor with IC50 of 0.83nM. To test whether the down-regulation of PLK1 by its inhibitor BI2536 would have beneficial effects on the reversal of MDR in HCC cells. The CCK-8 assay was used to determine the viability of HepG2/ADM and SMMC7721/ADM cells and their parental cells treated with BI2536. Then animal model studies were performed. Cell invasion assay and wound healing assay were used to determine the invasion ability and motility. Flow cytometric was used to test the apoptosis induced by BI2536. Western blot and quantitative real-time PCR were performed to test the change of expression of MDR and apoptosis-related gene. BI2536 down-regulated the expression of PLK1 protein and mRNA specifically. BI2536 can significantly reduce IC50 for ADM and other drugs in ADM-resistant HCC cells. Meanwhile, it inhibited cell viability, proliferation, and invasion, and induced cell cycle arrest and apoptosis in HCC cells with MDR. Our results suggest that PLK1 inhibitor BI2536 can re-sensitize HCC cancer cell with MDR through induction of apoptosis. Thus, PLK1 inhibitor BI2536 may act as an effective chemotherapeutic drug in the clinical treatment of HCC patients with MDR.

Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication.

PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).

Comprehensive Biomarker Analyses in Patients with Advanced or Metastatic Non-Small Cell Lung Cancer Prospectively Treated with the Polo-Like Kinase 1 Inhibitor BI2536

Background: Polo like kinase 1 (PLK1) is frequently upregulated in tumors and is thus viewed as a promising therapeutic target in various cancers. Several PLK1 inhibitors have recently been developed and clinically tested in solid cancers, albeit with limited success. So far, no predictive biomarkers for PLK1 inhibitors have been established. To this end, we conducted a post-hoc biomarker analysis of tumor samples from non-small cell lung cancer (NSCLC) patients treated with the PLK1 inhibitor BI2536 in a phase II study. Methods: We analyzed formalin-fixed paraffin-embedded surplus tumor tissue from 47 study patients using immunohistochemistry (IHC) and DNA sequencing of KRAS, EGFR, BRAF, and PIK3CA. Results:KRAS-mutated patients showed numerically prolonged progression-free survival, but statistical significance was not established. Interestingly, when pathways rather than single genes were analyzed, a positive correlation between IHC staining of activated ERK (p-ERK) and mutated KRAS was detected, whereas KRAS mutation status was found to be negatively correlated with activated AKT (p-AKT). Conclusion: With this hypothesis-generating study in BI2531-treated patients, we could not establish a correlation between KRAS mutations and relevant clinical endpoints. Future clinical trials with concomitant systematic biosampling and comprehensive molecular analyses are required to identify biomarkers predictive for response to PLK1 inhibitors.

Dual Targeting of WEE1 and PLK1 by AZD1775 Elicits Single Agent Cellular Anticancer Activity.

Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775's anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.

Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan

Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic.

45 Polo-like-kinase-1 as a potential therapeutic target in merkel cell carcinoma

Background Merkel cell carcinoma (MCC) is a rare and aggressive skin malignancy. Late stage MCC treatment options include radio- and chemotherapy. The aim of this study was to assess the expression of polo-like-kinase-1 (PLK1) in MCC and whether the PLK1 inhibitor BI2536 is a potential therapeutic option in MCC. Material and methods To evaluate PLK1 expression in patient specimens a tissue microarray was constructed for immunohistochemical staining. The two MCC cell lines MCC13 and MCC26 were exposed to increasing doses of BI2536 in combination with cisplatin or irradiation. Suppression of PLK1 was measured by Western blot analysis. Clonogenic assays were performed after irradiation to evaluate long-term results. Apoptotic and necrotic fractions were measured via flow cytometry. Results BI2536 is capable of inhibiting cell proliferation with an IC50 of 12nM. Combination with cisplatin is synergistic and a partially synergistic effect in combination with irradiation was detected. Clonogenic survival is inhibited synergistically. BI2536 induces apoptosis in MCC cell lines. Conclusion PLK1 seems to be a promising target in the treatment of MCC. Treatment with BI2536, a potent inhibitor of PLK1, resulted in suppression of MCC growth in vitro. Our data warrant further investigations for the potential use of BI2536 in MCC.

SC-16 * DIVERGENT CELL CYCLE REGULATION IN GBM CELLS PROVIDES A UNIQUE POINT OF INTERVENTION IN POLARIZED CD133+ CELLS

Recent studies in distinct glioma types have separately identified phenotypically distinct populations of tumor propagating cells (TPCs). Enrichment of TPCs by cell surface marker expression (CD133 [PMID:17051156], NG2 [PMID: 21907924]) has revealed the possibility of multiple TPC populations, within a single tumor, that have distinct properties and therapy sensitivities. Here we identify and compare the coexisting populations of glioblastoma multiforme (GBM) cells expressing the stem marker CD133 and the progenitor marker NG2. We compare the proliferative properties of CD133+ and NG2+ cells, and investigate how polarity and mode of division may determine other functional properties including cell cycle regulation and therapy sensitivity. Established and primary GBM cell cultures were analyzed by immunofluorescence and flow cytometry for expression of CD133 and NG2, alongside markers of polarity and proliferation. We showed that CD133+ and NG2+ GBM cells coexist as predominantly separate subpopulations. CD133+ cells are highly polarized and, compared to NG2+ or marker-negative subpopulations, are slower dividing. Cell cycle and gene expression analyses showed that the increase in cell cycle length in CD133+ cells is due to an extended G2-M period. As expected CD133+ cells have increased expression of DNA-damage response genes, however paradoxically we also detected increased activation of polo-like kinase 1 (PLK1). CD133+ cells were uniquely sensitive to the PLK1 inhibitor BI2536 in vivo, and treatment of intracranial GBM xenografts with the BRAF inhibitor PLX4720 and BI2536 showed greater anti-tumor activity than either alone. Our studies identified two putative TPC subpopulations in GBM with distinct cell cycle dynamics, DNA damage response and cell division mode. Ongoing work will investigate the role of Plk1 activity in linking these divergent phenotypes. Understanding this phenotypic heterogeneity in adjacent TPC types will allow the design of treatment strategies that eradicate multiple putative TPC populations.

PLK1 Signaling in Breast Cancer Cells Cooperates with Estrogen Receptor-Dependent Gene Transcription

Polo-like kinase 1 (PLK1) is a key regulator of cell division and is overexpressed in many types of human cancers. Compared to its well-characterized role in mitosis, little is known about PLK1 functions in interphase. Here, we report that PLK1 mediates estrogen receptor (ER)-regulated gene transcription in human breast cancer cells. PLK1 interacts with ER and is recruited to ER cis-elements on chromatin. PLK1-coactivated genes included classical ER target genes such as Ps2, Wisp2, and Serpina3 and were enriched in developmental and tumor-suppressive functions. Performing large-scale phosphoproteomics of estradiol-treated MCF7 cells in the presence or absence of the specific PLK1 inhibitor BI2536, we identified several PLK1 end targets involved in transcription, including the histone H3K4 trimethylase MLL2, the function of which on ER target genes was impaired by PLK1 inhibition. Our results propose a mechanism for the tumor-suppressive role of PLK1 in mammals as an interphase transcriptional regulator.

Abstract 436: Enhanced induction of apoptosis by PLK1 inhibition and radiation is associated with the additive, but not supra-additive, killing effect of the combined treatment.

Abstract Chemoradiotherapy has been employed to improve the clinical outcomes of cancer patients. The rationale for the combined usage of chemotherapeutics and radiation is the benefits of the additive or synergistic (supra-additive) effect of the two agents, leading to better treatment efficacy. Recent studies have shown that inhibition of Polo-like kinase1 (PLK1) function can sensitize head-and-neck squamous cell carcinoma and medulloblastoma cells to radiation. PLK1 is an important regulator of mitotic functions that is over-expressed in many cancers. It also plays a role in DNA double-strand break repair through phosphorylation of Rad51 recombinase. Therefore, PLK1 presents as an attractive target and we set out to determine if PLK1 inhibition can be used as a general strategy for radiosensitization. The half-maximal inhibitory concentrations (IC50) of the PLK1 inhibitor BI2536 were first determined for various cancer cell lines as indicators of sensitivity using XTT assay. The IC50 values of six different cancer (breast, lungs, pancreas, and colon) cell lines were &amp;lt; 6 nM while that of primary human fibroblasts was ∼ 25 nM. Results of western blots suggest that the protein levels of PLK1 do not correlate with sensitivity. Clonogenic assays were performed to evaluate if synergistic effect (radiosensitization) exists with combined treatment of BI2536 and radiation. Cells were treated with BI2536 for 24 hr before irradiation with various dosages. Additive killing effect was observed with all the cell lines tested; however, radiosensitization (supra-additive) effect was observed in only some cell lines. To determine if the additive or supra-additive killing is associated with apoptosis, cells treated with BI2536 and radiation were incubated with a caspase 3/7 substrate that emits a fluorescent signal upon cleavage. The induction of the fluorescent signal was followed for 72 hrs and the results suggest that enhanced induction of apoptosis occur in all cell lines exposed to the combined treatment. Therefore, enhanced induction of apoptosis is associated with additive, but not supra-addictive, killing of the combined treatment.PLK1 inhibition causes synergistic radiosensitization in some, but not all, cancer cell lines. However, additive killing of the combined treatment was observed with all the cancer cells tested. Our current effort is to decipher why some cancer cells are radiosensitized synergistically by PLK1 inhibition while some are not. We hope the results will give further insights into the mechanism of radiosensitization and identify novel targets for chemoradiotherpy. Citation Format: Nelson Wong, Ripen Misri, Mohamed Khan. Enhanced induction of apoptosis by PLK1 inhibition and radiation is associated with the additive, but not supra-additive, killing effect of the combined treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 436. doi:10.1158/1538-7445.AM2013-436

Polo-Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo-like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1-high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110-470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G(2) /M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells.