RNAi screen reveals synthetic lethality between cyclin G-associated kinase and FBXW7 by inducing aberrant mitoses.

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.

Discovery of 2-(3-Benzamidopropanamido)thiazole-5-carboxylate Inhibitors of the Kinesin HSET (KIFC1) and the Development of Cellular Target Engagement Probes.

The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans-cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

A cancer-associated aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2.

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.

Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development.

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 twofold 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.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Quercetin enhances TRAIL-mediated apoptosis in colon cancer cells by inducing the accumulation of death receptors in lipid rafts.

Cytokines such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in colon cancer cells through engagement of death receptors. Nevertheless, evading apoptosis induced by anticancer drugs characterizes many types of cancers. This results in the need for combination therapy. In this study, we have investigated whether the flavonoid quercetin could sensitize human colon adenocarcinoma cell lines to TRAIL-induced apoptosis. We report that quercetin enhanced TRAIL-induced apoptosis by causing the redistribution of DR4 and DR5 into lipid rafts. Nystatin, a cholesterol-sequestering agent, prevented quercetin-induced clustering of death receptors and sensitization to TRAIL-induced apoptosis in colon adenocarcinoma cells. In addition, our experiments show that quercetin, in combination with TRAIL, triggered the mitochondrial-dependent death pathway, as shown by Bid cleavage and the release of cytochrome c to the cytosol. Together, our findings propose that quercetin, through its ability to redistribute death receptors at the cell surface, facilitates death-inducing signaling complex formation and activation of caspases in response to death receptor stimulation. Based on these results, this study provides a challenging approach to enhance the efficiency of TRAIL-based therapies.

Integrated genomics and functional validation identifies malignant cell specific dependencies in triple negative breast cancer.

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.

APC/C is an essential regulator of centrosome clustering.

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.

Integration of RNAi and small molecule screens to identify targets for drug development.

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.

Multifaceted targeting in cancer: the recent cell death players meet the usual oncogene suspects.

Recent complicated advances towards the blueprinting of the altered molecular networks that lie behind cancer development have paved the way for targeted therapy in cancer. This directed a significant part of the research community to the development of specialized targeted agents, many of which are already available or in clinical trials. The prospect of patient-tailored therapeutic strategies, although very close to becoming a reality also raises the level of complexity of the therapeutic approach. This review summarizes the functions, in vivo expression patterns and aberrations of factors presently targeted or representing potential targets by therapeutic agents, focusing on those implicated in death receptor-induced apoptosis. The authors overview the regulation of these factors and death receptor-induced apoptosis by classical oncogenes (e.g., RAS, MYC, HER2) and their effectors/regulators, most of which are also being targeted. In addition, the importance of orthologic systemic approaches in future patient-tailored therapies are discussed.

Microarray analysis of the differential transformation mediated by Kirsten and Harvey Ras oncogenes in a human colorectal adenocarcinoma cell line.

Colorectal cancer arises after a series of mutational events in the colon epithelia and is often used as a model of the multistep progression of tumorigenesis. Mutations in Ki-Ras have been detected in some 50% of cases and are thought to occur at an early stage. Almost never do mutations arise in the loci of other Ras isoforms (Ha- and N-), leading to the assumption that Ki-Ras plays a unique role in tumorigenesis. In order to examine the distinctive function that Ki-Ras plays in cancer development in the colon, we introduced constitutively active mutant Ki- and Ha-Ras genes into an intermediate-stage colon adenoma cell line (Caco-2). We found that mutant active Ha-RasV12 was more efficient at transforming these colon epithelial cells as assessed by anchorage-independent growth, tumor formation in SCID mice and the development of mesenchymal morphology compared to transformation by Ki-RasV12. We conducted microarray analysis in an attempt to reveal the genes whose aberrant expression is a direct result of overexpression of either Ki-RasV12 or Ha-RasV12. We used Clontech's Atlas cancer cDNA (588 genes) and RZPD's Onco Set 1 (1,544 genes) arrays. We identified fewer genes that were commonly regulated than were differentially expressed between Ki- and Ha-RasV12 isoforms. Specifically, we found that Ki-RasV12 regulated genes involved in cytokine signaling, cell adhesion and colon development, whereas Ha-RasV12 mainly regulated genes involved in controlling cell morphology, correlating to an epithelial-mesenchymal transition only observed in these cells. Our results demonstrate how 2 Ras isoforms regulate disparate biologic processes, revealing a number of genes whose deregulated expression may influence colon carcinogenesis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway.

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.