Kinase Plk1 Research Articles

Cell polarity-dependent centrosome separation in the C. elegans embryo.

In animal cells, faithful chromosome segregation depends on the assembly of a bipolar spindle driven by the timely separation of the two centrosomes. Here we took advantage of the highly stereotypical cell divisions in Caenorhabditis elegans embryos to identify new regulators of centrosome separation. We find that at the two-cell stage, the somatic AB cell initiates centrosome separation later than the germline P1 cell. This difference is strongly exacerbated by the depletion of the kinesin-13 KLP-7/MCAK, resulting in incomplete centrosome separation at NEBD in AB but not P1. Our genetic and cell biology data indicate that this phenotype depends on cell polarity via the enrichment in AB of the mitotic kinase PLK-1, which itself limits the cortical localization of the dynein-binding NuMA orthologue LIN-5. We postulate that the timely separation of centrosomes is regulated in a cell type-dependent manner.

Dual Inhibition of TAF1 and BET Bromodomains from the BI-2536 Kinase Inhibitor Scaffold.

Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.

Mild replication stress causes chromosome mis-segregation via premature centriole disengagement

Replication stress, a hallmark of cancerous and pre-cancerous lesions, is linked to structural chromosomal aberrations. Recent studies demonstrated that it could also lead to numerical chromosomal instability (CIN). The mechanism, however, remains elusive. Here, we show that inducing replication stress in non-cancerous cells stabilizes spindle microtubules and favours premature centriole disengagement, causing transient multipolar spindles that lead to lagging chromosomes and micronuclei. Premature centriole disengagement depends on the G2 activity of the Cdk, Plk1 and ATR kinases, implying a DNA-damage induced deregulation of the centrosome cycle. Premature centriole disengagement also occurs spontaneously in some CIN+ cancer cell lines and can be suppressed by attenuating replication stress. Finally, we show that replication stress potentiates the effect of the chemotherapeutic agent taxol, by increasing the incidence of multipolar cell divisions. We postulate that replication stress in cancer cells induces numerical CIN via transient multipolar spindles caused by premature centriole disengagement.

Targeted Polo-like Kinase Inhibition Combined With Aurora Kinase Inhibition in Pediatric Acute Leukemia Cells.

Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.

Oncogenic MYC amplifies mitotic perturbations

The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.

Switching of INCENP paralogs controls transitions in mitotic chromosomal passenger complex functions

ABSTRACTA single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, Oikopleura dioica, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate O. dioica.Abbreviations: CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single α-helix domain

Differential Requirements for Centrioles in Mitotic Centrosome Growth and Maintenance

Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C.elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C.elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM.

Abstract 2551: Inhibition of mTOR abrogates mitosis progression

Abstract The mammalian target of rapamycin (mTOR) mediates the coordination of cell growth and cell cycle progression in response to nutrient supply. It is frequently activated in cancers and confers resistance to anti-cancer drugs. Accumulated studies have demonstrated that inhibition of mitotic kinases (PLK1 or aurora kinase A) enhances mTOR activities and the mTOR activation is associated with cell survival to anti-mitotic drugs. In this study, whether mTOR signaling is involved in mitosis progression is explored. Treatment of cells with mTOR inhibitors for 24 h resulted in the accumulation of G2 and mitotic cells and increased the percentage of mitotic cells with spindle abnormalities and chromosome missegregation. In addition, mTOR inhibitors sensitized cancer cells to taxol by enhancing apoptosis. Our results indicate that mTOR signaling pathway may be required for faithful mitosis progression and represent a potential resistance mechanism to the use of anti-mitotic drugs. Citation Format: Hsiao-Hui Kuo, Chieh-Ting Fang, Ling-Huei Yih. Inhibition of mTOR abrogates mitosis progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2551.

Abstract 4712: Selective polo-like kinase 1 PBD inhibitor

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitorshave advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD. We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy. Citation Format: Danda P. Chapagai, Merissa Baxter, Gurusankar Ramamoorthy, Campbell McInnes, Michael Wyatt. Selective polo-like kinase 1 PBD inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4712.

An order-to-disorder structural switch activates the FoxM1 transcription factor.

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.

Copy number analysis to identify tumor suppressor genes associated with enzalutamide (Enza) resistance and poor prognosis in metastatic castration-resistant prostate cancer (mCRPC) patients.

5011 Background: Although enza prolongs life in mCRPC pts, the development of drug resistance and subsequent disease progression is nearly universal. Seeking to clarify molecular mechanisms that underlie enza resistance, we analyzed whole genome sequencing (WGS) and RNA sequencing (seq) of tumors obtained from patients with enza-naive or -resistant mCRPC. Methods: One hundred and one men with mCRPC who underwent image-guided biopsy and subsequent WGS were included (n = 64 with enza-naive and n = 37 with enza-resistant mCRPC). The differential copy number alteration (CNA) events enriched in enza-resistant vs. naïve samples were determined, and the prognostic significance of differential CNAs was assessed. RNA-seq data were evaluated to confirm that CNAs correlated with changes in gene expression of relevant loci and to identify potentially druggable targets selectively activated in tumors with specific CNAs. Results: Copy number loss was more common than gain in enza-resistant tumors. Specifically, we identified 123 protein-coding genes that were more commonly lost in enza-resistant samples—eight of which were previously described tumor suppressor genes. There was a strong concordance of copy number loss and reduced mRNA expression of these genes. We identified one gene from this list of eight genes whose copy number loss was associated with poor overall survival (median overall survival from date of CRPC was 19.1 months in tumors with gene loss vs. 42.0 months in intact tumors, hazard ratio 3.8 [1.46–9.8], log-rank p = 0.003). Finally, Master Regulator analysis determined that tumors with copy number loss of this poor prognosis gene had activation of several potentially targetable factors, including the kinases Akt and PLK1. Conclusions: Copy number loss of specific tumor suppressor genes is associated with enza resistance in mCRPC patients. Previously unappreciated molecular subsets of enza-resistant CRPC were identified, including one subset associated with poor clinical outcome.

Design, synthesis and biological evaluation of novel 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as potential BRD4 inhibitors.

Recently, diverse kinase inhibitors were reported having interaction with BRD4. It provided a strategy for developing a new structural framework for the next-generation BRD4-selective inhibitors. Starting from PLK1 kinase inhibitor BI-2536, we designed 18 compounds by modifying dihydropteridine core. Compound 23 showed potent BRD4 inhibitory activities with IC50 of 79 nM and no inhibitory activities for PLK1. Cell antiproliferation assay was performed and potent inhibitory activity against MV4;11 with IC50 of 1.53 μM. Cell apoptosis and western blotting indicated compound 23 induced apoptosis by down-regulating c-Myc. These novel selective BRD4 inhibitors provided new lead compounds for further drug development.

Corrigendum to "A PLK1 kinase inhibitor enhances the chemosensitivity of cisplatin by inducing pyroptosis in oesophageal squamous cell carcinoma" [EBioMedicine 41 (2019) 244-255

Corrigendum to "A PLK1 kinase inhibitor enhances the chemosensitivity of cisplatin by inducing pyroptosis in oesophageal squamous cell carcinoma" [EBioMedicine 41 (2019) 244-255

Non‐canonical cMet regulation by vimentin mediates Plk1 inhibitor–induced apoptosis

To address the need for improved systemic therapy for non–small‐cell lung cancer (NSCLC), we previously demonstrated that mesenchymal NSCLC was sensitive to polo‐like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cMet was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal cells. Constitutively active cMet abrogates Plk1 inhibitor–induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor–induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cMet and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cMet phosphorylation via β1‐integrin. This research defines a heretofore unknown mechanism of ligand‐independent activation of cMet downstream of Plk1 and an effective combination therapy.

A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair.

Polo-like kinase 1 (Plk1) is a crucial regulator of cell cycle progression; but the mechanism of regulation of Plk1 activity is not well understood. We present evidence that Plk1 activity is controlled by a balanced methylation and phosphorylation switch. The methyltransferase G9a monomethylates Plk1 at Lys209, which antagonizes phosphorylation of T210 to inhibit Plk1 activity. We found that the methyl-deficient Plk1 mutant K209A affects DNA replication, whereas the methyl-mimetic Plk1 mutant K209M prolongs metaphase-to-anaphase duration through the inability of sister chromatids separation. We detected accumulation of Plk1 K209me1 when cells were challenged with DNA damage stresses. Ablation of K209me1 delays the timely removal of RPA2 and RAD51 from DNA damage sites, indicating the critical role of K209me1 in guiding the machinery of DNA damage repair. Thus, our study highlights the importance of a methylation-phosphorylation switch of Plk1 in determining its kinase activity and functioning in DNA damage repair.

A PLK1 kinase inhibitor enhances the chemosensitivity of cisplatin by inducing pyroptosis in oesophageal squamous cell carcinoma.

BackgroundTargeting PLK1 has recently been proven as a viable therapeutic strategy against oesophageal squamous cell carcinom (ESCC). Therefore, this study aimed to explore whether the PLK1 inhibitor BI2536 is able to sensitize ESCC cells to cisplatin (DDP) and determine the underlying mechanisms.MethodsViability, clonogenicity, cell cycle distribution and apoptosis were assessed in ESCC cells treated with BI2536 or DDP alone or in combination. Checkpoint activation was examined by immunoblotting and immunohistochemistry. Xenograft model was used to assess the efficacy of the co-treatment. The expression level of GSDME in tissue samples were examined by immunohistochemistry.FindingsWe found that the combination of BI2536 and DDP was synergistic in ESCC cells, which induced pyroptosis in ESCC cells at low doses. Mechanistic studies revealed that BI2536 significantly induced DNA damage and impaired the DNA damage repair pathway in DDP-treated cells both in vitro and in vivo. Interestingly, we found that co-treatment with BI2536 and DDP induced pyroptosis in ESCC cells depending on the caspase-3/GSDME pathway. Importantly, our study found that GSDME was more highly expressed in tumour tissue than that in normal adjacent tissues, and could serve as a prognostic factor.InterpretationBI2536 sensitizes ESCC cells to DDP by inhibiting the DNA damage repair pathway and inducing pyroptosis, which provides new information for understanding pyroptosis. Our study also reveals that the PLK1 inhibitor BI2536 may be an attractive candidate for ESCC targeted therapy, especially when combined with DDP for treating the GSDME overexpression subtype.FundNational 973 Program and National Natural Science Fundation of China.

A PLK1 Kinase Inhibitor Enhances the Chemosensitivity of Cisplatin by Inducing Pyroptosis in Esophageal Squamous Cell Carcinoma

Objective/Background: Esophageal squamous cell carcinoma (ESCC) urgently requires more effective chemotherapies because of its relatively low overall survival rate. Targeting PLK1 has recently been proven as a viable therapeutic strategy against ESCC. Therefore, this study aimed to explore whether the PLK1 inhibitor BI2536 is able to sensitize ESCC cells to cisplatin (DDP) and determine the underlying mechanisms. Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in KYSE150 and KYSE510 cell lines treated with BI2536 or DDP alone or in combination. DNA damage was assessed by phospho-histone H2AX (γH2AX) and RAD51 foci, and checkpoint activation was examined by Western blotting and immunohistochemistry. A mouse xenograft model was used to assess the efficacy of the co-treatment. GSDME expression and prognostic significance were assessed by a staining tissue microarray that included more than 100 samples of esophageal and esophageal cancer tissues. Results: We found that the combination of BI2536 and DDP was synergistic in ESCC cells. BI2536 enhanced the anti-proliferative and pro-apoptotic effects of DDP and blocked the cell cycle at the G2/M phase. The combination treatment induced pyroptosis in ESCC cells at low doses. Moreover, mechanistic studies revealed that BI2536 significantly induced DNA damage and impaired the DNA damage repair pathway in DDP-treated cells both in vitro and in vivo. Interestingly, we found that co-treatment with BI2536 and DDP caused pyroptosis in a subset of cells via the BAX/caspase-3/GSDME pathway but not via the caspase-1/GSDMD pathway. Importantly, our study found that GSDME was more highly expressed in tumor tissue than that seen in normal esophageal mucosa tissue, and it may serve as a prognostic factor. Conclusions: In conclusion, BI2536 is able to sensitize ESCC cells to DDP by inhibiting the DNA damage repair pathway and inducing pyroptosis, suggesting new information for understanding pyroptosis. Our study also reveals that the PLK1 inhibitor BI2536 might be an attractive candidate for ESCC targeted therapy, especially when combined with DDP for treating the GSDME overexpression subtype. Funding Statement: This work is supported by the National 973 Program (2015CB553904), National Natural Fund of China (81490753, 81830086 and 81802780), China Postdoctoral Science Foundation (2017M620010), National Postdoctoral Program for Innovative Talent, Science Foundation of Peking University Cancer Hospital (2017-27). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: Tissue microarrays (TMAs) of ESCC specimens were obtained from Shanghai Outdo Biotech Co., Ltd. (SOBC), with the approval of the Institutional Review Board. Detailed clinicopathological characteristics for all specimens are summarized in Supplementary Table S1. Written informed consent was obtained from all patients prior to the study. The use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee.

Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote.

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3′s response to upstream polarity cues.

Identification of Compounds That Target Acute Myeloid Leukemia Stem Cells Using a Scalable Next-Generation Screening Platform

▪The main barrier to curing acute myeloid leukemia (AML) is disease relapse, which occurs due to therapy resistance and persistence of leukemic stem cells (LSCs) after conventional induction chemotherapy. LSCs possess stem cell properties such as quiescence and self-renewal, as well as a transcriptional signature that resembles that of normal hematopoietic stem and progenitor cells. While patient-derived xenograft (PDX) models are the most stringent way to examine the properties of LSCs in human AML, this labor-intensive assay is not amenable to high throughput approaches to identify druggable vulnerabilities.Although current therapies effectively reduce tumor burden, achieving lasting event-free survival remains a challenge in the management of patients with AML. Current drug discovery efforts are focused on finding agents that eradicate bulk tumor, but not necessarily LSCs. Here, we report a next-generation screening strategy as a novel paradigm that allows the direct elucidation of molecules which antagonize LSC properties. This screening workflow is based on two scalable models of LSCs. The first is the gene expression profile of a core set of 104 genes (LSC104) that are differentially expressed between LSC+ and LSC‒ fractions obtained from primary AML samples and validated in xenotransplantation assays (Ng et al, Nature 2016). This LSC gene expression signature is strongly associated with overall survival and chemotherapy response in independent cohorts of AML patients comprising all subtypes. The second is a continuous AML culture system derived from a patient with relapsed AML that maintains, and allows the prospective enrichment of, a rare population of quiescent, self-renewing LSCs restricted to the CD34+CD38‒ fraction. These LSCs, but not the bulk tumor cells in this system, demonstrate leukemia-initiating capacity both in vitro and in PDX models. Similarly, the LSC fraction of this system, but not the bulk cells, has a LSC104 expression profile that correlates strongly with that of LSC-enriched fractions from patient samples. Together, these components form a clinically-relevant scalable model system to identify compounds with activity against LSC stemness properties, based on alterations in the proportion of LSCs in the continuous AML culture system as well as in the core LSC104 gene expression profile. To this end, we assembled a collection of 1200 curated bioactive small molecules, including 150 metabolic inhibitors and 35 targeted epigenetic probes, and performed a high-dimensional throughput screen using flow-cytometry to examine the effect of these compounds on the LSC-enriched CD34+CD38‒ fraction in our AML culture system. Candidate hits were then tested for their effects on the LSC104 profile. Rigorous analyses unveiled a number of candidate compounds with the potential to antagonize LSC properties, including several already in clinical use or in clinical trials against AML, such as cytarabine, and inhibitors of FLT3, CDK, PLK1, and aurora kinase, as well as several classes of compounds not previously described against AML, targeting NAMPT, BRPF1B, CHK1, and KSP, among others.In conclusion, we describe a next-generation scalable throughput approach that integrates stem cell biology to uncover modulators of stemness in AML. This multi-parametric screening strategy serves as a platform for the deeper understanding of druggable vulnerabilities of LSCs, and serves as a starting point to achieve lasting event-free survival in AML patients. DisclosuresChan:AbbVie: Research Funding; Celgene: Research Funding; Genentech: Research Funding.

Selective Degradation of Polo‐like Kinase 1 by a Hydrophobically Tagged Inhibitor of the Polo‐Box Domain

AbstractHydrophobic tagging (HT) of bioactive compounds can induce target degradation via the proteasomal pathway. The first application of hydrophobic tagging to an existing inhibitor of protein–protein interactions is now presented. We developed Poloxin‐2HT by fusing an adamantyl tag to Poloxin‐2, an inhibitor of the polo‐box domain of the protein kinase Plk1, which is a target for tumor therapy. Poloxin‐2HT selectively reduced the protein levels of Plk1 in HeLa cells and had a significantly stronger effect on cell viability and the induction of apoptosis than the untagged PBD inhibitor Poloxin‐2. The change in cellular phenotype associated with the addition of the hydrophobic tag to Poloxin‐2 demonstrated that Poloxin‐2HT targets Plk1 in living cells. Our data validate hydrophobic tagging of selective inhibitors of protein–protein interactions as a novel strategy to target and destroy disease‐relevant proteins.