Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus.

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus.

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

Inhibition of human cytomegalovirus immediate-early gene expression by cyclin A2-dependent kinase activity.

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

The latency-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (TNF-alpha) signaling by upregulating TNF-alpha receptor 1 cell surface expression.

Many viruses antagonize tumor necrosis factor alpha (TNF-α) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-α receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-α has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb' region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb'-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb' gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-α responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-α on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-α-mediated reactivation of HCMV.

Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle.

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

CD11b is a therapy resistance- and minimal residual disease-specific marker in precursor B-cell acute lymphoblastic leukemia.

A consistently increased mRNA expression of the adhesion receptor CD11b is a hallmark of the reported genomewide gene expression changes in precursor B-cell acute lymphoblastic leukemia (PBC-ALL) after 1 week of induction therapy. To investigate its clinical relevance, CD11b protein expression in leukemic blasts has been prospectively measured at diagnosis (159 patients) and during therapy (53 patients). The initially heterogeneous expression of CD11b inversely correlated with cytoreduction rates measured at clinically significant time points of induction therapy in the ALL-Berlin-Frankfurt-Münster 2000 protocol. CD11b positivity conferred a 5-fold increased risk of minimal residual disease (MRD) after induction therapy (day 33) and of high-risk group assignment after consolidation therapy (day 78). In the multivariate analysis CD11b expression was an independent prognostic factor compared with other clinically relevant parameters at diagnosis. During therapy, CD11b expression increased early in most ALL cases and remained consistently increased during induction/consolidation therapy. In more than 30% of MRD-positive cases, the CD11b expression on blast cells exceeded that of mature memory B cells and improved the discrimination of residual leukemic cells from regenerating bone marrow. Taken together, CD11b expression has considerable implications for prognosis, treatment response monitoring, and MRD detection in childhood PBC-ALL.

Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing.

Eraserhead: Stem cells seem to erase epigenetic information by decarboxylation of the newly discovered epigenetic base 5-carboxycytosine (caC; see picture). This reaction is likely to involve a nucleophilic attack of the C5-C6 double bond.

General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism.

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36-38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis.

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells.

The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G(0)/G(1) phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G(1) DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G(1). Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G(2). Moreover, MCMV can also replicate in G(2) cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G(1) or G(2). Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.

A role for E2F6 in the restriction of male-germ-cell-specific gene expression.

E2F transcription factors play a pivotal role in the regulation of cellular proliferation and can be subdivided into activating and repressing family members [1]. Like other E2Fs, E2F6 binds to E2F consensus sites, but in contrast to E2F1-5, it lacks an Rb binding domain and functions as an Rb-independent transcriptional repressor [2, 3, 4 and 5]. Instead, E2F6 has been shown to complex with Polycomb (PcG) group proteins [6 and 7], which have a well-established role in gene silencing. Here, we show that E2F6 plays an unexpected and essential role in the tissue specificity of gene expression. E2F6-deficient mice ubiquitously express the alpha-tubulin 3 and 7 genes, which are expressed strictly testis-specifically in control mice. Like an additional E2F6 target gene, Tex12, that we identified, tubulin 3 and 7 are normally expressed in male germ cells only. The promoters of the alpha-tubulin and Tex12 genes share a perfectly conserved E2F site, which E2F6 binds to. Mechanistically, E2F6-mediated repression involves CpG hypermethylation locking target promoters in an inactive state. Thus, E2F6 is essential for the long-term somatic silencing of certain male-germ-cell-specific genes, but it is dispensable for cell-cycle regulation.

Combined effect of proteasome and calpain inhibition on cisplatin-resistant human melanoma cells.

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Expression of late cell cycle genes and an increased proliferative capacity characterize very early relapse of childhood acute lymphoblastic leukemia.

PURPOSE: In childhood acute lymphoblastic leukemia (ALL), approximately 25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood. EXPERIMENTAL DESIGN: Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group. RESULTS: We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G(2)-M phase cells and this correlated well with the expression level of cell cycle genes. CONCLUSIONS: Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.

HuSiDa--the human siRNA database: an open-access database for published functional siRNA sequences and technical details of efficient transfer into recipient cells.

Small interfering RNAs (siRNAs) have become a standard tool in functional genomics. Once incorporated into the RNA-induced silencing complex (RISC), siRNAs mediate the specific recognition of corresponding target mRNAs and their cleavage. However, only a small fraction of randomly chosen siRNA sequences is able to induce efficient gene silencing. In common laboratory practice, successful RNA interference experiments typically require both, the labour and cost-intensive identification of an active siRNA sequence and the optimization of target cell line-specific procedures for optimal siRNA delivery. To optimize the design and performance of siRNA experiments, we have established the human siRNA database (HuSiDa). The database provides sequences of published functional siRNA molecules targeting human genes and important technical details of the corresponding gene silencing experiments, including the mode of siRNA generation, recipient cell lines, transfection reagents and procedures and direct links to published references (PubMed). The database can be accessed at http://www.human-siRNA-database.net. We used the siRNA sequence information stored in the database for scrutinizing published sequence selection parameters for efficient gene silencing.

A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma.

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/ß-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.