Bajrami, I., Marlow, R., van de Ven, M., Brough, R., Pemberton, H.N., Frankum, J., Song, F., Rafiq, R., Konde, A., Krastev, D.B., et al.
(2018). E-Cadherin/ROS1 Inhibitor Synthetic Lethality in Breast Cancer. Cancer discov,
The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371..
Patel, N., Weekes, D., Drosopoulos, K., Gazinska, P., Noel, E., Rashid, M., Mirza, H., Quist, J., Brasó-Maristany, F., Mathew, S., et al.
(2018). Integrated genomics and functional validation identifies malignant cell specific dependencies in triple negative breast cancer. Nat commun,
Triple negative breast cancers (TNBCs) lack recurrent targetable driver mutations but demonstrate frequent copy number aberrations (CNAs). Here, we describe an integrative genomic and RNAi-based approach that identifies and validates gene addictions in TNBCs. CNAs and gene expression alterations are integrated and genes scored for pre-specified target features revealing 130 candidate genes. We test functional dependence on each of these genes using RNAi in breast cancer and non-malignant cells, validating malignant cell selective dependence upon 37 of 130 genes. Further analysis reveals a cluster of 13 TNBC addiction genes frequently co-upregulated that includes genes regulating cell cycle checkpoints, DNA damage response, and malignant cell selective mitotic genes. We validate the mechanism of addiction to a potential drug target: the mitotic kinesin family member C1 (KIFC1/HSET), essential for successful bipolar division of centrosome-amplified malignant cells and develop a potential selection biomarker to identify patients with tumors exhibiting centrosome amplification..
Woodward, H.L., Innocenti, P., Cheung, K.-., Hayes, A., Roberts, J., Henley, A.T., Faisal, A., Mak, G.W., Box, G., Westwood, I.M., et al.
(2018). Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722). J med chem,
Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate..
Koo, C.-., Giacomini, C., Reyes-Corral, M., Olmos, Y., Tavares, I.A., Marson, C.M., Linardopoulos, S., Tutt, A.N. & Morris, J.D.
(2017). Targeting TAO Kinases Using a New Inhibitor Compound Delays Mitosis and Induces Mitotic Cell Death in Centrosome Amplified Breast Cancer Cells. Mol cancer ther,
Thousand-and-one amino acid kinases (TAOK) 1 and 2 are activated catalytically during mitosis and can contribute to mitotic cell rounding and spindle positioning. Here, we characterize a compound that inhibits TAOK1 and TAOK2 activity with IC50 values of 11 to 15 nmol/L, is ATP-competitive, and targets these kinases selectively. TAOK inhibition or depletion in centrosome-amplified SKBR3 or BT549 breast cancer cell models increases the mitotic population, the percentages of mitotic cells displaying amplified centrosomes and multipolar spindles, induces cell death, and inhibits cell growth. In contrast, nontumorigenic and dividing bipolar MCF-10A breast cells appear less dependent on TAOK activity and can complete mitosis and proliferate in the presence of the TAOK inhibitor. We demonstrate that TAOK1 and TAOK2 localize to the cytoplasm and centrosomes respectively during mitosis. Live cell imaging shows that the TAOK inhibitor prolongs the duration of mitosis in SKBR3 cells, increases mitotic cell death, and reduces the percentages of cells exiting mitosis, whereas MCF-10A cells continue to divide and proliferate. Over 80% of breast cancer tissues display supernumerary centrosomes, and tumor cells frequently cluster extra centrosomes to avoid multipolar mitoses and associated cell death. Consequently, drugs that stimulate centrosome declustering and induce multipolarity are likely to target dividing centrosome-amplified cancer cells preferentially, while sparing normal bipolar cells. Our results demonstrate that TAOK inhibition can enhance centrosome declustering and mitotic catastrophe in cancer cells, and these proteins may therefore offer novel therapeutic targets suitable for drug inhibition and the potential treatment of breast cancers, where supernumerary centrosomes occur. Mol Cancer Ther; 16(11); 2410-21. ©2017 AACR..
Faisal, A., Mak, G.W., Gurden, M.D., Xavier, C.P., Anderhub, S.J., Innocenti, P., Westwood, I.M., Naud, S., Hayes, A., Box, G., et al.
(2017). Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy. Br j cancer,
BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850..
Dolly, S.O., Gurden, M.D., Drosopoulos, K., Clarke, P., de Bono, J., Kaye, S., Workman, P. & Linardopoulos, S.
(2017). RNAi screen reveals synthetic lethality between cyclin G-associated kinase and FBXW7 by inducing aberrant mitoses. Br j cancer,
BACKGROUND: F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours. METHODS: We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency. RESULTS: We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK. CONCLUSIONS: These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options..
Gurden, M.D., Anderhub, S.J., Faisal, A. & Linardopoulos, S.
(2016). Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis. Oncotarget,
Accurate chromosome segregation is dependent on the spindle assembly checkpoint (SAC). In current models, the key direct role of Aurora B in the SAC has been suggested to be to promote rapid kinetochore localisation of MPS1, allowing MPS1 to generate the checkpoint signal. However, Aurora B is also thought to play an indirect role in the SAC through the destabilisation of kinetochore-microtubule (KT-MT) attachments. Here, we demonstrate that Aurora B activity is not required for the kinetochore recruitment of the majority of SAC proteins. More importantly, we show that the primary role of Aurora B in the SAC is to prevent the premature removal of SAC proteins from the kinetochore, which is strictly dependent on KT-MT interactions. Moreover, in the presence of KT-MT interactions, Aurora B inhibition silences a persistent SAC induced by tethering MPS1 to the kinetochore. This explains the highly synergistic interaction between Aurora B and MPS1 inhibitors to override the SAC, which is lost when cells are pre-arrested in nocodazole. Furthermore, we show that Aurora B and MPS1 inhibitors synergistically kill a panel of breast and colon cancer cell lines, including cells that are otherwise insensitive to Aurora B inhibitors alone. These data demonstrate that the major role of Aurora B in SAC is to prevent the removal of SAC proteins from tensionless kinetochores, thus inhibiting premature SAC silencing, and highlights a therapeutic strategy through combination of Aurora B and MPS1 inhibitors..
Innocenti, P., Woodward, H.L., Solanki, S., Naud, S., Westwood, I.M., Cronin, N., Hayes, A., Roberts, J., Henley, A.T., Baker, R., et al.
(2016). Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach. J med chem,
Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model..
Bavetsias, V., Pérez-Fuertes, Y., McIntyre, P.J., Atrash, B., Kosmopoulou, M., O'Fee, L., Burke, R., Sun, C., Faisal, A., Bush, K., et al.
(2015). 7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent. Bioorg med chem lett,
Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217. .
Morra, F., Luise, C., Visconti, R., Staibano, S., Merolla, F., Ilardi, G., Guggino, G., Paladino, S., Sarnataro, D., Franco, R., et al.
(2015). New therapeutic perspectives in CCDC6 deficient lung cancer cells. Int j cancer,
Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC. .
Linardopoulos, S. & Blagg, J.
(2015). Aurora kinase inhibition: a new light in the sky?. J med chem,
The quest for potent and selective small molecule inhibitors of the Aurora kinases has been long and resource intensive with multiple agents progressed to the clinic. To definitively explore the potential for clinical efficacy at well-tolerated dosing schedules requires a well-characterized, selective inhibitor with pharmacokinetic properties, flexible dosing regimen, and suite of target engagement biomarkers suitable for clinical use. AMG900 is a promising opportunity to definitively test the clinical benefit of dual Aurora kinase A and B inhibition. .
Gurden, M.D., Westwood, I.M., Faisal, A., Naud, S., Cheung, K.-., McAndrew, C., Wood, A., Schmitt, J., Boxall, K., Mak, G., et al.
(2015). Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance. Cancer res,
Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells..
Bavetsias, V. & Linardopoulos, S.
(2015). Aurora Kinase Inhibitors: Current Status and Outlook. Front oncol,
The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions. .
Cheeseman, M.D., Faisal, A., Rayter, S., Barbeau, O.R., Kalusa, A., Westlake, M., Burke, R., Swan, M., van Montfort, R., Linardopoulos, S., et al.
(2014). Targeting the PPM1D phenotype; 2,4-bisarylthiazoles cause highly selective apoptosis in PPM1D amplified cell-lines. Bioorg med chem lett,
The metal-dependent phosphatase PPM1D (WIP1) is an important oncogene in cancer, with over-expression of the protein being associated with significantly worse clinical outcomes. In this communication we describe the discovery and optimization of novel 2,4-bisarylthiazoles that phenocopy the knockdown of PPM1D, without inhibiting its phosphatase activity. These compounds cause growth inhibition at nanomolar concentrations, induce apoptosis, activate p53 and display impressive cell-line selectivity. The results demonstrate the potential for targeting phenotypes in drug discovery when tackling challenging targets or unknown mechanisms. .
Drosopoulos, K., Tang, C., Chao, W.C. & Linardopoulos, S.
(2014). APC/C is an essential regulator of centrosome clustering. Nat commun,
Centrosome amplification has been extensively associated with cancer. Cancer cells with extra centrosomes have the ability to cluster the extra centrosomes and divide in a bipolar fashion. Although a number of proteins have been shown to be involved in centrosome clustering, a mechanistic understanding of how this process is coordinated is not yet well defined. Here, to reveal regulators of centrosome clustering, we perform small interfering RNA (siRNA) screens with multiple assay readouts in a human isogenic cellular model. We find that APC/C activity is essential for centrosome clustering. We show that the motor kinesin Eg5 is a substrate of APC/C-CDH1, and that inhibition of APC/C results in stabilization of Eg5. Increased Eg5 protein levels disturb the balance of forces on the spindle and prevent centrosome clustering. This process is completely reversed after a short treatment with the Eg5 inhibitor, monastrol. These data advance our understanding of the regulation of centrosome clustering. .
Aarts, M., Linardopoulos, S. & Turner, N.C.
(2013). Tumour selective targeting of cell cycle kinases for cancer treatment. Curr opin pharmacol,
The deregulation of the cell cycle and checkpoint machinery in cancer presents a highly attractive therapeutic strategy. We review here the strategies used to exploit the dysregulated cell cycle, both through targeting kinases required for cell cycle progression, and checkpoint kinases to inappropriately force cells through the cell cycle. Appropriate control of the cell cycle is critical for proliferating normal cells, and we discuss the importance of defining tumour specific vulnerabilities that could be targeted with cell cycle kinase inhibitors. Recent studies have shown that ER-positive breast cancers rely on CDK4 to promote proliferation. TP53 mutant cancer cell lines are sensitive to WEE1 and CHK1 inhibitors in combination with chemotherapy, while PTEN-deficient aneuploid cancer cell lines are sensitive to TTK inhibitors. .
Regan, J.L., Sourisseau, T., Soady, K., Kendrick, H., McCarthy, A., Tang, C., Brennan, K., Linardopoulos, S., White, D.E. & Smalley, M.J., et al.
(2013). Aurora A kinase regulates mammary epithelial cell fate by determining mitotic spindle orientation in a Notch-dependent manner. Cell rep,
Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA) in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland. .
Couty, S., Westwood, I.M., Kalusa, A., Cano, C., Travers, J., Boxall, K., Chow, C.L., Burns, S., Schmitt, J., Pickard, L., et al.
(2013). The discovery of potent ribosomal S6 kinase inhibitors by high-throughput screening and structure-guided drug design. Oncotarget,
The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases..
Bavetsias, V., Faisal, A., Crumpler, S., Brown, N., Kosmopoulou, M., Joshi, A., Atrash, B., Pérez-Fuertes, Y., Schmitt, J.A., Boxall, K.J., et al.
(2013). Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells. J med chem,
Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells. .
Naud, S., Westwood, I.M., Faisal, A., Sheldrake, P., Bavetsias, V., Atrash, B., Cheung, K.M., Liu, M., Hayes, A., Schmitt, J., et al.
(2013). Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of the mitotic kinase monopolar spindle 1 (MPS1). J med chem,
Drosopoulos, K. & Linardopoulos, S.
(2013). Integration of RNAi and small molecule screens to identify targets for drug development. Methods mol biol,
Cellular models for siRNA and small molecule high throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a two-fold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screening strategy..
Pintzas, A., Zhivotovsky, B., Workman, P., Clarke, P.A., Linardopoulos, S., Martinou, J.-., Lacal, J.C., Robine, S., Nasioulas, G. & Andera, L., et al.
(2012). Sensitization of (colon) cancer cells to death receptor related therapies: a report from the FP6-ONCODEATH research consortium. Cancer biol ther,
The objective of the ONCODEATH consortium [EU Research Consortium "ONCODEATH" (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 mo, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, Choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients..
Moore, A.S., Faisal, A., Gonzalez de Castro, D., Bavetsias, V., Sun, C., Atrash, B., Valenti, M., de Haven Brandon, A., Avery, S., Mair, D., et al.
(2012). Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns. Leukemia,
Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML..
Faisal, A., Naud, S., Schmitt, J., Westwood, I., Hayes, A., Gurden, M., Bavetsias, V., Berry, T., Mak, G., Innocenti, P., et al.
(2012). Characterisation of CCT251455, a novel, selective and highly potent Mps1 kinase inhibitor. Cancer research,
Drosopoulos, K., Scott, C., Tang, C. & Linardopoulos, S.
(2012). Integration of centrosomeassociated and kinase-related screens to identify centrosome amplification -related synthetic lethal interactions. Cancer research,
Bavetsias, V., Crumpler, S., Sun, C., Avery, S., Atrash, B., Faisal, A., Moore, A.S., Kosmopoulou, M., Brown, N., Sheldrake, P.W., et al.
(2012). Optimization of imidazo[4,5-b]pyridine-based kinase inhibitors: identification of a dual FLT3/Aurora kinase inhibitor as an orally bioavailable preclinical development candidate for the treatment of acute myeloid leukemia. J med chem,
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children..
Brough, R., Frankum, J.R., Sims, D., Mackay, A., Mendes-Pereira, A.M., Bajrami, I., Costa-Cabral, S., Rafiq, R., Ahmad, A.S., Cerone, M.A., et al.
(2011). Functional viability profiles of breast cancer. Cancer discov,
UNLABELLED: The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. SIGNIFICANCE: Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified..
Podesta, J.E., Sugar, R., Squires, M., Linardopoulos, S., Pearson, A.D. & Moore, A.S.
(2011). Adaptation of the plasma inhibitory activity assay to detect Aurora, ABL and FLT3 kinase inhibition by AT9283 in pediatric leukemia. Leuk res,
Non-invasive assessment of biomarker modulation is important for evaluating targeted therapeutics, particularly in pediatrics. The plasma inhibitory activity (PIA) assay is used clinically to assess FLT3 inhibition ex vivo and guide dosing. AT9283 is a novel Aurora kinase inhibitor with secondary activity against FLT3 and ABL. We adapted the PIA assay to simultaneously detect inhibition of Aurora and FLT3 in AML, and Aurora and ABL in CML by AT9283. Furthermore, we optimized the assay for children, where limited blood volumes are available for pharmacodynamic studies. Simultaneously detecting multiple kinase inhibition may identify important mechanisms of action for novel anti-leukemic drugs..
Faisal, A., Vaughan, L., Bavetsias, V., Sun, C., Atrash, B., Avery, S., Jamin, Y., Robinson, S.P., Workman, P., Blagg, J., et al.
(2011). The aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo. Mol cancer ther,
Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora A and B kinase overexpression has also been associated with various human cancers, and as such, they have been extensively studied as novel antimitotic drug targets. Here, we characterize the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC(50) values in both biochemical and cellular assays and exhibits antiproliferative activity against a wide range of human solid tumor cell lines. CCT137690 efficiently inhibits histone H3 and transforming acidic coiled-coil 3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumor cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy, and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 downregulation, and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumor growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN-amplified neuroblastoma..
Moore, A.S., Faisal, A., Bavetsias, V., Sun, C., Atrash, B., Valenti, M., Brandon, A.D., Avery, S., de Castro, D.G., Raynaud, F.I., et al.
(2011). CCT137690, a dual inhibitor of Aurora and FLT3 kinases, sensitizes FLT3-ITD positive acute myeloid leukemia and overcomes resistance to selective FLT3-inhibition. Cancer research,
Sourisseau, T., Maniotis, D., McCarthy, A., Tang, C., Lord, C.J., Ashworth, A. & Linardopoulos, S.
(2010). Aurora-A expressing tumour cells are deficient for homology-directed DNA double strand-break repair and sensitive to PARP inhibition. Embo mol med,
The protein kinase Aurora-A is a major regulator of the cell cycle that orchestrates mitotic entry and is required for the assembly of a functional mitotic spindle. Overexpression of Aurora-A has been strongly linked with oncogenesis and this has led to considerable efforts at therapeutic targeting of the kinase activity of this protein. However, the exact mechanism by which Aurora-A promotes oncogenesis remains unclear. Here, we show that Aurora-A modulates the repair of DNA double-strand breaks (DSBs). Aurora-A expression inhibits RAD51 recruitment to DNA DSBs, decreases DSB repair by homologous recombination and sensitizes cancer cells to PARP inhibition. This impairment of RAD51 function requires inhibition of CHK1 by Polo-like kinase 1 (PLK1). These results identify a novel function of Aurora-A in modulating the response to DNA DSB that likely contributes to carcinogenesis and suggest a novel therapeutic approach to the treatment of cancers overexpressing this protein..
Moore, A.S., Blagg, J., Linardopoulos, S. & Pearson, A.D.
(2010). Aurora kinase inhibitors: novel small molecules with promising activity in acute myeloid and Philadelphia-positive leukemias. Leukemia,
Aurora kinases are a family of protein kinases that have a key role in multiple stages of mitosis. Over-expression of Aurora kinases, particularly Aurora A, has been demonstrated in a number of solid tumors and hematological malignancies. Not surprisingly, these serine/threonine kinases have become attractive small molecule targets for cancer therapeutics, with several inhibitors currently in early-phase clinical trials. A small number of compounds developed to date are highly selective for either Aurora A or Aurora B, while the majority inhibit both Aurora A and Aurora B; many of these compounds exhibit 'off-target' inhibition of kinases such as ABL, JAK2 and FLT3. It is currently unclear whether the therapeutic activity of these compounds in leukemia is primarily due to selective Aurora or multi-kinase inhibition. The most promising application for Aurora kinase inhibitors to date appears to be in FLT3-mutated acute myeloid leukemia (AML) and imatinib-resistant chronic myeloid leukemia/Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia, particularly when caused by the T315I mutation. Here we review the growing body of evidence supporting the use of Aurora kinase inhibitors as effective agents for AML and Ph+ leukemias. Leukemia (2010) 24, 671-678; doi: 10.1038/leu.2010.15; published online 11 February 2010.
Lambros, M.B., Natrajan, R., Geyer, F.C., Lopez-Garcia, M.A., Dedes, K.J., Savage, K., Lacroix-Triki, M., Jones, R.L., Lord, C.J., Linardopoulos, S., et al.
(2010). PPM1D gene amplification and overexpression in breast cancer: a qRT-PCR and chromogenic in situ hybridization study. Mod pathol,
PPM1D (protein phosphatase magnesium-dependent 1δ) maps to the 17q23.2 amplicon and is amplified in ∼8% of breast cancers. The PPM1D gene encodes a serine threonine phosphatase, which is involved in the regulation of several tumour suppressor pathways, including the p53 pathway. Along with others, we have recently shown that PPM1D is one of the drivers of the 17q23.2 amplicon and a promising therapeutic target. Here we investigate whether PPM1D is overexpressed when amplified in breast cancers and the correlations between PPM1D overexpression and amplification with clinicopathological features and survival of breast cancer patients from a cohort of 245 patients with invasive breast cancer treated with therapeutic surgery followed by adjuvant anthracycline-based chemotherapy. mRNA was extracted from representative sections of tumours containing >50% of tumour cells and subjected to TaqMan quantitative real-time PCR using primers for PPM1D and for two housekeeping genes. PPM1D overexpression was defined as the top quartile of expression levels. Chromogenic in situ hybridization with in-house-generated probes for PPM1D was performed. Amplification was defined as >50% of cancer cells with >5 signals per nucleus/large gene clusters. PPM1D overexpression and amplification were found in 25 and 6% of breast cancers, respectively. All cases harbouring PPM1D amplification displayed PPM1D overexpression. PPM1D overexpression was inversely correlated with expression of TOP2A, EGFR and cytokeratins 5/6 and 17. PPM1D amplification was significantly associated with HER2 overexpression, and HER2, TOP2A and CCND1 amplification. No association between PPM1D gene amplification and PPM1D mRNA overexpression with survival was observed. In conclusion, PPM1D is consistently overexpressed when amplified; however, PPM1D overexpression is more pervasive than gene amplification. PPM1D overexpression and amplification are associated with tumours displaying luminal or HER2 phenotypes. Co-amplification of PPM1D and HER2/TOP2A and CCND1 are not random events and may suggest the presence of a 'firestorm' genetic profile..
Bouloc, N., Large, J.M., Kosmopoulou, M., Sun, C., Faisal, A., Matteucci, M., Reynisson, J., Brown, N., Atrash, B., Blagg, J., et al.
(2010). Structure-based design of imidazo[1,2-a]pyrazine derivatives as selective inhibitors of Aurora-A kinase in cells. Bioorg med chem lett,
Co-crystallisation of the imidazo[1,2-a]pyrazine derivative 15 (3-chloro-N-(4-morpholinophenyl)-6-(pyridin-3-yl)imidazo[1,2-a]pyrazin-8-amine) with Aurora-A provided an insight into the interactions of this class of compound with Aurora kinases. This led to the design and synthesis of potent Aurora-A inhibitors demonstrating up to 70-fold selectivity in cell-based Aurora kinase pharmacodynamic biomarker assays..
Bavetsias, V., Large, J.M., Sun, C., Bouloc, N., Kosmopoulou, M., Matteucci, M., Wilsher, N.E., Martins, V., Reynisson, J., Atrash, B., et al.
(2010). Imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases: lead optimization studies toward the identification of an orally bioavailable preclinical development candidate. J med chem,
Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss..
Bibby, R.A., Tang, C., Faisal, A., Drosopoulos, K., Lubbe, S., Houlston, R., Bayliss, R. & Linardopoulos, S.
(2009). A cancer-associated aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2. J biol chem,
Mutations in protein kinases can drive cancer through alterations of the kinase activity or by uncoupling kinase activity from regulation. Changes to protein expression in Aurora A, a mitotic Ser/Thr kinase, are associated with the development of several human cancers, but the effects of somatic cancer-associated mutations have not been determined. In this study we show that Aurora A kinase activity is altered in different ways in three somatic cancer-associated mutations located within the catalytic domain; Aurora A(V174M) shows constitutively increased kinase activity, Aurora A(S155R) activity is decreased primarily due to misregulation, and Aurora A(S361*) activity is ablated due to loss of structural integrity. These alterations suggest vastly different mechanisms for the role of these three mutations in human cancer. We have further characterized the Aurora A(S155R) mutant protein, found that its reduced cellular activity and mislocalization are due to loss of interaction with TPX2, and deciphered the structural basis of the disruption at 2.5 A resolution. Previous studies have shown that disruption of the Aurora A/TPX2 interaction results in defective spindles that generate chromosomal abnormalities. In a panel of 40 samples from microsatellite instability-positive colon cancer patients, we found one example in which the tumor contained only Aurora A(S155R), whereas the normal tissue contained only wild-type Aurora A. We propose that the S155R mutation is an example of a somatic mutation associated with this tumor type, albeit at modest frequency, that could promote aneuploidy through the loss of regulated interactions between Aurora A and its protein partners..
Rayter, S., Elliott, R., Travers, J., Rowlands, M.G., Richardson, T.B., Boxall, K., Jones, K., Linardopoulos, S., Workman, P., Aherne, W., et al.
(2008). A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D. Oncogene,
The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor..
Bavetsias, V., Sun, C., Bouloc, N., Reynisson, J., Workman, P., Linardopoulos, S. & McDonald, E.
(2007). Hit generation and exploration: imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases. Bioorg med chem lett,
A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases with IC(50) values of 0.042, 0.198 and 0.227microM, respectively. Compound 31 inhibits cell proliferation and has good microsomal stability..
Sun, C., Chan, F., Briassouli, P. & Linardopoulos, S.
(2007). Aurora kinase inhibition downregulates NF-kappaB and sensitises tumour cells to chemotherapeutic agents. Biochem biophys res commun,
We have identified that Aurora-A activates NF-kappaB via IkappaBalpha phosphorylation. Here, we analysed different human tumour cell types for their NF-kappaB activity. We found that there is an association between cell resistance to chemotherapeutic agents and NF-kappaB activation. A549 human lung adenocarcinoma cells and SKOV3 human ovarian cancer cells have high levels of NF-kappaB and are resistant to cytotoxic agents such as adriamycin and VP-16 (etoposide). We also found that in A549 and SKOV3 cells treated with a small molecule inhibitor towards Aurora kinases, the NF-kappaB activity was downregulated and the efficacy of cytotoxic drugs was enhanced. In addition, the transcriptional targets Bcl-XL and Bcl-2 were downregulated. This study provides evidence for a potential mechanism of chemoresistance and may be useful for the enhancement of certain chemotherapeutics regimens..
(2007). Aurora-A kinase regulates NF-kappaB activity: lessons from combination studies. J buon,
Vol.12 Suppl 1,
The Aurora-A/STK15 gene encodes a kinase that is frequently amplified in cancer. However, it is not clear what role this plays in the development of cancer since little is known about its biochemical targets. We have showed that Aurora-A induces phosphorylation of IkappaBa, thereby mediating its degradation. Loss of IkappaBa leads to activation of NF-kappaB target gene transcription. We analysed primary human breast cancers and 13.6% of samples showed Aurora-A gene amplification all of which exhibited nuclear localisation of NF-kappaB. We propose that this subgroup of breast cancer patients might benefit from inhibiting Aurora-A. Further analysis of different human tumour cell types for their NF-kappaB activity have showed that there is an association between cell resistance to chemotherapeutic agents and NF-kappaB activation. A549 human lung adenocarcinoma cells and SKOV3 human ovarian cancer cells have high levels of NF-kappaB and are resistant to cytotoxic agents such as adriamycin and VP-16 (etoposide). We also found that in A549 and SKOV3 cells treated with a small molecule inhibitor towards Aurora kinases, the NF-kappaB activity was downregulated and the efficacy of cytotoxic drugs was enhanced. In addition, the transcriptional targets Bcl-XL and Bcl-2 were downregulated. These findings have important implications for cancer chemotherapy. Aurora-A-inhibition enhances the efficacy of chemotherapeutic agents and reverses acquired resistance resulting from the activation of NF-kappaB. Consequently, preventing NF-kappaB activation by inhibition of Aurora-A, may provide a valuable enhancement to specific chemotherapeutic regimens..
Chan, F., Sun, C., Perumal, M., Nguyen, Q.-., Bavetsias, V., McDonald, E., Martins, V., Wilsher, N.E., Raynaud, F.I., Valenti, M., et al.
(2007). Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity. Mol cancer ther,
(12 Pt 1),
The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo..
Whittaker, S.R., Te Poele, R.H., Chan, F., Linardopoulos, S., Walton, M.I., Garrett, M.D. & Workman, P.
(2007). The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis. Cell cycle,
The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials..
Briassouli, P., Chan, F., Savage, K., Reis-Filho, J.S. & Linardopoulos, S.
(2007). Aurora-A regulation of nuclear factor-kappaB signaling by phosphorylation of IkappaBalpha. Cancer res,
The Aurora-A/STK15 gene encodes a kinase that is frequently amplified in cancer. Overexpression of Aurora-A in mammalian cells leads to centrosome amplification, genetic instability, and transformation. In this study, we show that Aurora-A activates nuclear factor-kappaB (NF-kappaB) via IkappaBalpha phosphorylation. Inhibition of endogenous Aurora-A reduces tumor necrosis factor alpha (TNFalpha)-induced IkappaBalpha degradation. We analyzed primary human breast cancers, and 13.6% of samples showed Aurora-A gene amplification, all of which exhibited nuclear localization of NF-kappaB. We propose that this subgroup of patients with breast cancer might benefit from inhibiting Aurora-A. We also show that down-regulation of NF-kappaB via Aurora-A depletion can enhance cisplatin-dependent apoptosis. These data define a new role for Aurora-A in regulating IkappaBalpha that is critical for the activation of NF-kappaB-directed gene expression and may be partially responsible for the oncogenic effect of Aurora-A when the gene is amplified and overexpressed in human tumors..
Briassouli, P., Chan, F. & Linardopoulos, S.
(2006). The N-terminal domain of the Aurora-A Phe-31 variant encodes an E3 ubiquitin ligase and mediates ubiquitination of IkappaBalpha. Hum mol genet,
Aurora-A is an important regulator of mitosis and is frequently amplified in human cancer. Ectopic expression of Aurora-A in mammalian cells induces centrosome amplification, genomic instability and transformation. A common genetic variant in Aurora-A (F31I) is preferentially amplified and is associated with the occurrence and the status of colon, oesophageal and breast cancers. Here we demonstrate that the N-terminal domain of Aurora-A Phe-31 variant exhibits an intrinsic ubiquitin ligase activity. Mutation of cysteines 8, 33 and 49 of Aurora-A abolishes the ubiquitin ligase activity of the protein. Aurora-A in a complex with UBE2N/MMS2 catalyses polyubiquitination of IkappaBalpha in vitro and in vivo..
Chieffi, P., Cozzolino, L., Kisslinger, A., Libertini, S., Staibano, S., Mansueto, G., De Rosa, G., Villacci, A., Vitale, M., Linardopoulos, S., et al.
(2006). Aurora B expression directly correlates with prostate cancer malignancy and influence prostate cell proliferation. Prostate,
BACKGROUND: Chromosomal instability is one of the most common features of prostate cancer (PC), especially in advanced stages. Recent studies suggest that defects in mitotic checkpoints play a role in carcinogenesis. Lack of mitotic regulation induces aneuploidy in cancer cells acting thereafter as a driving force for malignant progression. Serine/threonine protein kinases of the Aurora genes family play an important throughout the entire cell cycle. In that Aurora B regulates chromosome segregation by ensuring the orientation of sister chromatids. As a consequence, the overexpression of Aurora B in diploid human cells NHDF induces the appearance of multinucleate cells. METHODS: Archive samples of normal and neoplastic prostate tissue, and prostate derived cell lines were screened for the expression of Aurora B. RESULTS: Immunohistochemical analysis showed increased nuclear expression of Aurora-B in high Gleason grade PCs respect to low and intermediate grade cases and in all cancers in respect to hyperplastic and normal glands. Furthermore, in the high Gleason grade anaplastic cancer tissues Aurora B expression was accompanied by the phosphorylation of the histone H3. In analogy to the in vivo situation, Aurora B was vigorously expressed in the androgen independent PC cell lines PC3 and DU145, while a very modest expression of the kinase was observed in the androgen sensitive LnCap cells and in the EPN cells, a line of epithelial cells derived from normal prostate tissue. In addition, in PC3 cells Aurora B expression is accompanied the by the phosphorylation of the histone H3. The block of Aurora B expression induced by an inhibitor of Aurora kinase activity significantly reduced the growth of prostate carcinoma cells, but not that of non-transformed EPN cells. CONCLUSIONS: Our data are the first demonstration of a role of Aurora B in PC progression. In addition, the observation that Aurora B specific inhibitors interfere with PC cell proliferation but not with that of non-transformed prostate epithelial cells suggest that Aurora B is a potential therapeutic target for PC..
Sorrentino, R., Libertini, S., Pallante, P.L., Troncone, G., Palombini, L., Bavetsias, V., Spalletti-Cernia, D., Laccetti, P., Linardopoulos, S., Chieffi, P., et al.
(2005). Aurora B overexpression associates with the thyroid carcinoma undifferentiated phenotype and is required for thyroid carcinoma cell proliferation. J clin endocrinol metab,
Alterations in chromosome number (aneuploidy) are common in human neoplasias. Loss of mitotic regulation is believed to induce aneuploidy in cancer cells and act as a driving force during the malignant progression. The serine/theronine protein kinases of aurora family genes play a critical role in the regulation of key cell cycle processes. Aurora B mediates chromosome segregation by ensuring orientation of sister chromatids and overexpression of Aurora B in diploid human cells NHDF (normal human diploid fibroblast) induces multinuclearity. We analyzed Aurora B expression in human thyroid carcinomas. Cell lines originating from different histotypes showed an increase in Aurora B expression. Immunohistochemical analysis of archive samples showed a high expression of Aurora B in anaplastic thyroid carcinomas; conversely, Aurora B expression was not detectable in normal thyroid tissue. Real-time PCR analysis confirmed a strong expression of Aurora B in anaplastic thyroid carcinomas. The block of Aurora B expression induced by RNA interference or by using an inhibitor of Aurora kinase activity significantly reduced the growth of thyroid anaplastic carcinoma cells..
Chieffi, P., Troncone, G., Caleo, A., Libertini, S., Linardopoulos, S., Tramontano, D. & Portella, G.
(2004). Aurora B expression in normal testis and seminomas. J endocrinol,
Aurora/Ipl1-related kinases are a conserved family of proteins that have multiple functions during mitotic progression. High levels of Aurora kinases are characteristic of rapidly dividing cells and tumours. Aurora B encodes a protein that associates with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. In this study the expression and the localisation of Aurora B throughout germinal epithelial progression in normal testis and its neoplastic counterpart were analysed. Immunocytochemistry and RT-PCR analysis of mouse germinal epithelium cells showed the presence of Aurora B in spermatogonia and occasionally in spermatocytes. Western blot analysis revealed the typical Aurora B isoform ( approximately 41 kDa) in the same cellular types. A similar distribution was observed in human testis by immunohistochemistry. Moreover, the distribution and the expression of Aurora B were investigated in neoplasms derived from germ cells. Surgical samples of seminomas were analysed, and a high percentage of Aurora B positive cells (51%) was detected; the expression of Aurora B was significantly related to the MIB-1 proliferation marker (R=0.816). The data presented here demonstrate that Aurora B expression occurs in spermatogonial division. Furthermore, our results indicate that the expression of Aurora B is a consistent feature of human seminomas..
Sun, C., Newbatt, Y., Douglas, L., Workman, P., Aherne, W. & Linardopoulos, S.
(2004). High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase. J biomol screen,
STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity..
Ewart-Toland, A., Briassouli, P., de Koning, J.P., Mao, J.-., Yuan, J., Chan, F., MacCarthy-Morrogh, L., Ponder, B.A., Nagase, H., Burn, J., et al.
(2003). Identification of Stk6/STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and human. Nat genet,
Linkage analysis and haplotype mapping in interspecific mouse crosses (Mus musculus x Mus spretus) identified the gene encoding Aurora2 (Stk6 in mouse and STK15 in human) as a candidate skin tumor susceptibility gene. The Stk6 allele inherited from the susceptible M. musculus parent was overexpressed in normal cells and preferentially amplified in tumor cells from F(1) hybrid mice. We identified a common genetic variant in STK15 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors. The Ile31 variant transforms rat1 cells more potently than the more common Phe31 variant. The E2 ubiquitin-conjugating enzyme UBE2N was a preferential binding partner of the 'weak' STK15 Phe31 variant form in yeast two-hybrid screens and in human cells. This interaction results in colocalization of UBE2N with STK15 at the centrosomes during mitosis. These results are consistent with an important role for the Ile31 variant of STK15 in human cancer susceptibility..
Linardopoulos, S., Silva, S., Klein, G. & Balmain, A.
(2000). Allele-specific loss or imbalance of chromosomes 9, 15, and 16 in B-cell tumors from interspecific F1 hybrid mice carrying Emu-c-myc or N-myc transgenes. Int j cancer,
Mice carrying an immunoglobulin enhancer (Emu-) linked c- or N-myc transgene develop fatal monoclonal or oligoclonal pre-B or B-cell lymphomas. This indicates that, beside the Emu-activated myc gene, additional genetic changes are required for tumor development. To trace these additional changes, we carried out a genome-wide search for loss of heterozygosity (LOH) and allelic imbalance (AI). This was done at 53 microsatellite markers in a panel of 34 lymphomas and four plasmacytomas from c- or N-myc transgene carrying (BALB/c x Mus spretus)F1 hybrids. An additional 43 lymphomas and three plasmacytomas from non-transgenic F1 mice were also investigated. Losses of one or more spretus-derived chromosome 9 markers were detected in 19 of 23 (83%) of the lymphomas, but in none of the four plasmacytomas that developed in N-myc F1 mice. No LOH-9 was found in any of the 11 lymphomas from Emu-c-myc F1 mice and only in 1 of 46 (2%) tumors derived from non-transgenic (BALB/c x spretus)F1 hybrid controls. These results suggest that a gene on spretus chromosome 9 confers resistance to the development of N-myc but not c-myc-induced lymphomas. AI of chromosome 15 markers (AI-15) was detected in 57 of 77 (74%) lymphomas and in 5 of 7 (72%) plasmacytomas, independently of the transgenic status and the mode of induction. All of the lymphomas and plasmacytomas with AI-15 revealed a relative gain of the spretus-derived D15Mit6 allele (located at 13.7 cM from the centromere), together with a gain of the BALB/c allele of the more distal (29.6 cM) D15Mit64 marker, suggesting somatic recombination. LOH in the region close to c-myc was detected in a proportion of tumors with AI-15. The observation of complex genetic alterations includes somatic recombination, AI and LOH involving chromosome 15 in tumors induced by a myc transgene. This indicates that at least two genes in addition to c-myc on this chromosome can be involved in lymphoma development..
Zoumpourlis, V., Papassava, P., Linardopoulos, S., Gillespie, D., Balmain, A. & Pintzas, A.
(2000). High levels of phosphorylated c-Jun, Fra-1, Fra-2 and ATF-2 proteins correlate with malignant phenotypes in the multistage mouse skin carcinogenesis model. Oncogene,
Analysis of the functions of AP-1 transcription factor in cellular systems has shown its key role as a mediator of oncogenic signals. The employment of suitable animal model systems greatly facilitates the study of changes in the composition and activity of the AP-1 complex. Here, we have analysed the quantitative and qualitative changes of AP-1 at different stages of carcinogenesis in mouse skin cell lines, derived from tumours induced by chemical mutagens. The findings of this study suggest that elevated AP-1 DNA binding and transactivation activity characterize the carcinoma cell lines, most notably the highly malignant spindle carcinomas. In addition, increased amounts and post-translational modifications of c-Jun, Fra-1, Fra-2 and ATF-2 proteins account for a high percentage of the increased AP-1 activity. Remarkably, high levels of phosphorylated ATF-2 protein were detected in malignant cell lines, indicating a novel role of ATF-2 in tumour progression. c-Jun and ATF-2 proteins are phosphorylated by highly active JNK kinases present in tumour cells. Finally, our results indicate distinct functions for different AP-1 components in the promotion and progression of mouse skin tumours. Oncogene (2000) 19, 4011 - 4021..
Frame, S., Crombie, R., Liddell, J., Stuart, D., Linardopoulos, S., Nagase, H., Portella, G., Brown, K., Street, A., Akhurst, R., et al.
(1998). Epithelial carcinogenesis in the mouse: correlating the genetics and the biology. Philos trans r soc lond b biol sci,
Tumour formation relies on a complex combination of genetic and environmental factors. In particular, the contributions from inherited predisposition genes as well as carcinogens, for example from cigarettes or in the diet, are amongst the major contributors to tumorigenesis. Since the study of such processes in particularly difficult in human cancers, the availability of a well-defined model system is of obvious benefit. The mouse skin model of multistage carcinogenesis offers an excellent tool for the study of the target cells, the target genes and the biological events associated with neoplasia. In this system, tumorigenesis occurs in a series of defined stages, each of which is characterized by specific and reproducible alterations in genes such as H-ras, cyclin D1, p53 and p16INK4A. Additional changes occur in the production of, or response to, factors such as transforming growth factor beta (TGF beta). These genetic and biological alterations are mirrored in human tumours of epithelial origin. Hence, research into the general principles of tumour initiation, promotion and progression in the context of the mouse skin model is likely to prove valuable in the continual search for new methods for the diagnosis, prevention, and therapeutic treatment of human cancers..
Tsopanomichalou, M., Ergazaki, M., Linardopoulos, S., Kouroumalis, E. & Spandidos, D.A.
(1997). Detection of hepatitis C virus in sera and genotyping according to the 5' non-coding region. Oncol rep,
A reverse transcription (RT)-polymerase chain reaction (PCR) method was used for detection of the RNA of hepatitis C virus (HCV) in 120 samples of sera from Crete, which were positive for HCV-specific antibodies, by ELISA and Western blot analyses. A segment of 255 bp, located in the most conserved region of the HCV genome (the 5' untranslated region, 5' UTR), was amplified. For the identification of sequence variation from the HCV-1 strain, twenty of these samples were sequenced and compared to prototype strain (HCV-1) according to current genotypic classification. We were able to identify fourteen of the twenty as type 1a (i.e. similar to the prototype), two as type 1b, two as type 3a and two as type 4a. These findings generally agree with the geographic distribution of the already identified genotypes, though 3a type has not been reported previously in Crete (Greece)..
Karachristos, A., Linardopoulos, S., Ergazaki, M. & Spandidos, D.A.
(1995). Detection and analysis of hepatitis C virus by a combined RT-PCR method: variation in the 5' non-coding region of the viral genome. J med microbiol,
A combined reverse transcription-polymerase chain reaction (RT-PCR) method was employed for the detection of hepatitis C virus (HCV) RNA in serum from patients with chronic active hepatitis, with primers corresponding to the 5' non-coding region. The diagnosis was based on serological and biochemical methods and on liver biopsy. HCV-RNA was detected in 27 (90%) of 30 sera examined. The nucleotide sequence of PCR-amplified HCV cDNAs (256 bp) was determined from five specimens and heterogeneity varying between 0.58% and 2.89% among the clinical samples and the prototype HCV-1 was found..
Linardopoulos, S., Street, A.J., Quelle, D.E., Parry, D., Peters, G., Sherr, C.J. & Balmain, A.
(1995). Deletion and altered regulation of p16INK4a and p15INK4b in undifferentiated mouse skin tumors. Cancer res,
p16INK4a and p15INK4b are cell cycle regulators that specifically bind to and inhibit the cyclin D-dependent kinases, cdk4 and cdk6. Because these genes undergo frequent deletions and/or mutations in various human cancers, we examined the status and expression of the cognate mouse cdk inhibitors in a panel of 29 cell lines, as well as in 12 primary tumors, representing different stages of mouse skin carcinogenesis. Deletion of p16INK4a and/or p15INK4b was seen in 8 of 10 cell lines derived from spindle carcinomas, the most advanced stage of skin carcinogenesis. Five showed deletion of both genes, and three had independent deletions of p16INK4a or p15INK4b, but in those retaining p16INK4a, expression of the protein was not detected. By contrast, none of 19 more differentiated squamous cell lines exhibited such deletions. In several cases, primary tumor DNA was available, and two spindle tumors showed the same deletion pattern as observed in the corresponding cell lines. In apparent contrast, comparison of two clonally related squamous and spindle cell lines derived from a single carcinoma showed unusually high levels of p16INK4a and p15INK4b only in the invasive spindle cells. Therefore, deletion or altered regulation of p16INK4a and p15INK4b occur concomitantly with the loss of differentiation associated with the late spindle stage of tumor progression in mouse skin..
LINARDOPOULOS, S. & SPANDIDOS, D.A.
(1994). REGULATION OF THE RB GENE BY NORMAL AND MUTATED RAS, TPA AND EGF. Oncol rep,
Complete inactivation of the human retinoblastoma gene is believed to be an essential step in tumorigenesis of several different cancers. Using the plasmid pRbCAT2 that contains the Rb promoter region was tested for its ability to promote transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene in a transient expression assay. This plasmid was co-transfected in a short term transfections with the plasmids pHO6T1 and pHO6N1 that contains the mutant and normal H-ras gene respectively, into the human cell line HeLa, by the calcium phosphate technique. It was found that the mutant H-ras gene enhances the activity of the Rb gene promoter in contrast to the normal H-ras gene that inhibits it. The expression of the CAT gene in stable clones of HeLa cells carrying the promoter of Rb gene after treatment with TPA and EGF respectively, was also investigated, whereas TPA enhanced, EGF had no effect on the activity of the Rb gene promoter..
Linardopoulos, S., Papadakis, E., Delakas, D., Theodosiou, V., Cranidis, A. & Spandidos, D.A.
(1993). Human lung and bladder carcinoma tumors as compared to their adjacent normal tissue have elevated AP-1 activity associated with the retinoblastoma gene promoter. Anticancer res,
Examination of the nucleotide sequence of the retinoblastoma (Rb) promoter revealed the presence of a DNA region highly homologous to the recognition site for the cellular transcription factor AP-1. A pair of complementary oligonucleotides containing the AP-1 site was synthesized and used in gel retardation assays to determine the role of the AP-1 protein in the regulation of the Rb gene expression. Using nuclear extracts from Hela cells as well as from lung and bladder tumors, we found specific binding of the AP-1 protein to this oligonucleotide. This binding is elevated in Hela cells, in 10/13 lung and 3/8 bladder tumors as compared to adjacent normal tissue. These results suggest that AP-1 could be implicated in Rb gene transcriptional regulation through its interaction with the AP-1 binding site of the Rb gene promoter..
Linardopoulos, S., Gonos, E.S. & Spandidos, D.A.
(1993). Abnormalities of retinoblastoma gene structure in human lung tumors. Cancer lett,
The retinoblastoma (Rb) gene is associated with the pathogenesis of several types of human cancer, including retinoblastoma, osteosarcoma, soft tissue sarcomas, and lung, breast and bladder carcinomas. Loss of heterozygosity is a common mode in allelic inactivation of Rb and other tumor-suppressor genes. We investigated DNA from 15 human lung tumors for loss of heterozygosity of the Rb locus using a polymerase chain reaction (PCR) based restriction fragment length polymorphism assay. Of informative cases we found loss of heterozygosity in 2 out of 3 squamous cell carcinomas and 1 out of 2 adenocarcinomas of the lung. We also found structural rearrangements in two out of fourteen Hind III digested lung tumors examined at the 5' region of the human Rb gene using Southern blot hybridization analysis. Since these two tumors were classified as stage III it is possible that the alteration of Rb gene is involved in the progression of this type of cancer. Using specific primers for exons 15, 16, 21 and 22 of the Rb gene, we carried out amplification of these exons by polymerase chain reaction. None of these tumors showed a deletion of exons 15, 16, 21 and 22..
Spandidos, D.A., Karaiossifidi, H., Malliri, A., Linardopoulos, S., Vassilaros, S., Tsikkinis, A. & Field, J.K.
(1992). Expression of ras Rb1 and p53 proteins in human breast cancer. Anticancer res,
The ras, Rb and p53 genes have been implicated in the development of human breast cancer. Qualitative or quantitative changes in the expression of the ras p21 may lead to cell transformation, and this has been previously demonstrated in breast cancer. Both the retinoblastoma protein (Rb1) and the p53 gene product appear to function as negative regulators of cell division. We have investigated the expression of ras p21, Rb1 and p53 proteins in human breast cancer patients immunohistochemically, and correlated the results with a range of clinical and pathological parameters. Ras p21 expression was elevated in 65 per cent and p53 in 23 per cent of cases. Rb1 was expressed in 58 per cent of breast cancer tissues and in 75 per cent of normal tissue. Only four patients were found to have loss of Rb1 expression and also overexpression of both p53 and ras gene products. No correlations were found between the expression of these three genes and menopausal status, histological types or tumour grade. However, a correlation was found between Rb1 loss of expression and tumour diameter (greater than 2 cms), and no lymph node metastasis. Also, a significantly higher number of p53 staining specimens were found to be overexpressing the ras gene. These results suggest that all three oncogenes are most likely involved in the development of breast cancer but that their role is complex..
PAPADAKIS, E., MALLIRI, A., LINARDOPOULOS, S., KARAIOSSIFIDI, H., FIELD, J.K. & SPANDIDOS, D.A.
(1992). RAS AND P53 EXPRESSION IN NON-SMALL-CELL LUNG-CANCER PATIENTS - P53 OVER-EXPRESSION CORRELATES WITH A POOR PROGNOSIS. Int j oncol,
Expression of the tumor suppressor gene p53 and the ras oncogene were examined in 46 tumor and nodal specimens of non-small cell lung cancer (NSCLC) using the antibodies p53 pAb 240 and ras Y13-259 respectively. p53 expression was elevated in 46% and ras p21 was over-expressed in 85% of the tumor specimens analyzed. Fifteen cases of benign lessions were also assessed for both ras p21 and p53 expression; all were found to have negative staining. p53 over-expression was found to correlate with a poor prognosis in both the tumor specimens (p<0.05) and in the nodal tissues (p<0.005). Ras p21 over-expression was found to be associated with survival (p<0.1) in both the tumor and the nodal specimens. Stage of the disease correlated with survival; similarly both p53 and ras p21 over-expression correlated with stage. No correlations were found with the pathological grade of the tumors nor with a history of smoking or duration of smoking. No K-ras mutations at codon 12 were observed in a further 15 NSCLC specimens analyzed. These results indicate that the p53 gene in particular plays a role in the stages of NSCLC..
Linardopoulos, S., Corrigall, V. & Panayi, G.S.
(1992). Activation of HLA-DR and interleukin-6 gene transcription in resting T cells via the CD2 molecule: relevance to chronic immune-mediated inflammation. Scand j immunol,
Only a minority of T cells at cell-mediated immune lesions are antigen specific. In the lesions of human autoimmune disease, such as the synovial membrane in rheumatoid arthritis, the T cells are activated as shown by a variety of phenotypic and functional changes including the expression of HLA-DR and the production of interleukin-6 (IL-6). The stimulatory pathway involved is unknown but does not seem to involve the T-cell receptor. Alternative pathways of activation which may be involved include the CD2 molecule. It is shown that the formation of sheep red blood cell (SRBC) rosettes with resting T cells from human peripheral blood, which is equivalent to CD2/LFA-3 binding, leads to the de novo transcription of the HLA-DR and IL-6 genes and the expression of HLA-DR on the surface of the T cells. There was no transcription of the interleukin-2 (IL-2) or the interleukin-2 receptor (IL-2R) genes and Tac expression was not seen. The rosetted T cells did not proliferate. These are all characteristics of T cells at chronic inflammatory sites. It is concluded that receptor-ligand interactions between CD2/LFA-3, which are expressed in increased amounts in the rheumatoid joint, may be one pathway by which antigen non-specific T cells are recruited as effector cells in lesions of human autoimmune disease..
Symonds, R.P., Habeshaw, T., Paul, J., Kerr, D.J., Darling, A., Burnett, R.A., Sotsiou, F., Linardopoulos, S. & Spandidos, D.A.
(1992). No correlation between ras, c-myc and c-jun proto-oncogene expression and prognosis in advanced carcinoma of cervix. Eur j cancer,
55 patients suffering from stage III or IV carcinoma of cervix were treated with two pulses of neo-adjuvant chemotherapy prior to radical radiotherapy. 51% (26/51) had a partial response. The initial response to chemotherapy is associated with significantly better long-term survival. The 3-year survival of chemotherapy responders is 62% against 21% for non-responders (P = 0.009 log-rank test). To detect possible differences in oncogene expression in biopsy specimens taken from responding and non-responding patients, paraffin-fixed material was immunocytochemically stained for the expression of the protein products of ras, c-myc and c-jun proto-oncogenes. The frequency of oncogene expression was ras 80.4%, c-myc 45.1% and c-jun 39.2%. There was no statistically significant association between oncogene expression, time to local recurrence or development of metastases or survival..
Tosca, A., Linardopoulos, S., Malliri, A., Hatziolou, E., Nicolaidou, A. & Spandidos, D.A.
(1991). Implication of the ras and myc oncoproteins in the pathogenesis of mycosis fungoides. Anticancer res,
In the present work, we studied the expression of the c-myc oncoprotein p-62 and the ras oncoprotein p-21 in the dermal cellular infiltrate of paraffin embedded skin specimens, obtained from patients suffering from Mycosis Fungoides and Sezary syndrome. Nineteen specimens from early stage Mycosis Fungoides, nineteen from advanced stage Mycosis Fungoides and four from Sezary syndrome were included in the study. The oncoprotein detection was achieved immunohistochemically, using the mouse monoclonal antibody myc 1-9E10 and the rat monoclonal antibody Y13-259 for p-62 and p-21 respectively. Increased detection of both p-62 and p-21 in atypic lymphoid cells was shown in advanced stages of Mycosis Fungoides (third stage plaques and tumors) as compared to early stages (premycotic erythema, second stage plaques). In advanced stages, however, the percentage of P-62+ atypic cells proved to be higher than that of p-21+ atypic lymphoid cells. The implication of increased p-62 and p-21 oncoprotein expression in the process of lymphomagenesis in cutaneous T-cell lymphomas is discussed..
Arvanitis, D., Malliri, A., Antoniou, D., Linardopoulos, S., Field, J.K. & Spandidos, D.A.
(1991). Ras p21 expression in brain tumors: elevated expression in malignant astrocytomas and glioblastomas multiforme. In vivo,
We have employed an immunohistochemical analysis to study the ras p21 oncoprotein in a total of 41 brain tumors and 1 reactive gliosis. The tumors included 33 astrocytomas, 1 oligodendroglioma, 2 ependymomas, 1 neurilemmoma, 1 malignant meningioma, 1 neuroblastoma and 2 medulloblastomas. Our results indicate that elevated ras p21 expression is a common feature in a range of brain tumors. In particular, elevated ras p21 expression has been found in 18 out of 24 high grade astrocytomas (malignant astrocytomas and glioblastomas multiforme) compared to 3 out of 9 low grade (well differentiated astrocytomas) (P less than 0.05). These results suggest that ras p21 expression may be an important molecular marker of the malignant astrocytomas and glioblastomas multiforme..
Linardopoulos, S., Malliri, A., Pintzas, A., Vassilaros, S., Tsikkinis, A. & Spandidos, D.A.
(1990). Elevated expression of AP-1 activity in human breast tumors as compared to normal adjacent tissue. Anticancer res,
The levels of AP-1 activity in human breast lesions and in adjacent normal tissue were studied by a gel retardation assay. A thirty nucleotide long consensus oligonucleotide to the adenovirus E3 gene AP-1 sequence (E3AP-1) was end labelled and reacted with nuclear extracts from breast lesions and adjacent normal tissue. A total of 20 tissue extracts (8 pairs of tumor and normal tissue from the same patient and 4 tumors) were examined. All 12 tumor tissues showed elevated levels of AP-1 as compared to the 8 normal tissues. These results suggest that the AP-1 transcription factor may play a role in breast neoplasia..