Selective inhibition of the p53–MDM2 interaction by nutlin drugs: a new therapeutic perspective for neuroblastoma

Neuroblastoma is one of the most common and most deadly childhood tumors. There is an unmet need to develop new therapeutic modalities for this malignancy that preferentially should be guided by our increasing knowledge of the biology of neuroblastoma. Proliferation and survival of neuroblastoma cells is critically dependent on suppression of the activity of the tumor suppressor protein p53, which is often mediated by increased activity of the MDM2 oncoprotein. Accordingly, small-molecule inhibitors of the interaction between MDM2 and p53 may provide a useful therapeutic option for the treatment of neuroblastoma by restoring the potent antitumor activity of wild-type p53. One of the most promising classes of selective inhibitors of the p53–MDM2 interaction are the nutlins, which have been extensively studied over the last years in several tumor types, including neuroblastoma. We discuss here preclinical data that support the notion that nutlin drugs may offer therapeutic benefit for children with neuroblastoma, on condition that wild-type p53 is present

miRBase Tracker : keeping track of microRNA annotation changes

Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annota- tion. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA se- quences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annota- tion changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, compari- son and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-use on- line database that keeps track of all historical and current miRNA annotation present in the miRBase database. Three basic functionalities allow researchers to keep their miRNA annotation up-to-date, reannotate analytical miRNA platforms and link published results with outdated annotation to the latest miRBase release. We expect miRBase Tracker to increase the transparency and annotation accuracy in the field of miRNA research. Database URL: www.mirbasetracker.or

Dual targeting of MDM2 and BCL2 as a therapeutic strategy in neuroblastoma

Wild-type p53 tumor suppressor activity in neuroblastoma tumors is hampered by increased MDM2 activity, making selective MDM2 antagonists an attractive therapeutic strategy for this childhood malignancy. Since monotherapy in cancer is generally not providing long-lasting clinical responses, we here aimed to identify small molecule drugs that synergize with idasanutlin (RG7388). To this purpose we evaluated 15 targeted drugs in combination with idasanutlin in three p53 wild type neuroblastoma cell lines and identified the BCL2 inhibitor venetoclax (ABT-199) as a promising interaction partner. The venetoclax/idasanutlin combination was consistently found to be highly synergistic in a diverse panel of neuroblastoma cell lines, including cells with high MCL1 expression levels. A more pronounced induction of apoptosis was found to underlie the synergistic interaction, as evidenced by caspase-3/7 and cleaved PARP measurements. Mice carrying orthotopic xenografts of neuroblastoma cells treated with both idasanutlin and venetoclax had drastically lower tumor weights than mice treated with either treatment alone. In conclusion, these data strongly support the further evaluation of dual BCL2/MDM2 targeting as a therapeutic strategy in neuroblastoma

Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples ( serum, plasma) is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5' tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5' tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5' tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs

Genome wide expression profiling of p53 regulated miRNAs in neuroblastoma

Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells. Genome-wide miRNA expression analysis showed that miR-34a-5p, miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p are upregulated following nutlin-3 treatment in a p53 dependent manner. The function of miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p was analyzed by ectopic overexpression of miRNA mimics. We observed that these p53-regulated miRNAs inhibit the proliferation of neuroblastoma cells to varying degrees, with the most profound growth inhibition recorded for miR-182-5p. Overexpression of miR-182-5p promoted apoptosis in some neuroblastoma cell lines and induced neuronal differentiation of NGP cells. Using Chromatin Immunoprecipitation-qPCR (ChIP-qPCR), we did not observe direct binding of p53 to MIR182, MIR203, MIR222, and MIR432 in neuroblastoma cells. Taken together, our findings yield new insights in the network of p53-regulated miRNAs in neuroblastoma

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis

The human CD14+ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16+ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by theCD16+CD163+MerTK+pSTAT3+ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16+CD163+MerTK+pSTAT3+ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16+CD163+MerTK+pSTAT3+ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy,and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoringof treatment efficacy.Fil: Lastrucci, Claire. Centre National de la Recherche Scientifique; FranciaFil: Bénard, Alan. Centre National de la Recherche Scientifique; FranciaFil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pingris, Karine. Centre National de la Recherche Scientifique; FranciaFil: Souriant, Shanti. Centre National de la Recherche Scientifique; FranciaFil: Poincloux, Renaud. Centre National de la Recherche Scientifique; FranciaFil: Al Saati, Talal. Inserm; FranciaFil: Rasolofo, Voahangy. Pasteur Institute in Antananarivo; MadagascarFil: González Montaner, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Inwentarz, Sandra. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Moraña, Eduardo José. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Kondova, Ivanela. Biomedical Primate Research Centre; Países BajosFil: Verreck, Franck A. W.. Biomedical Primate Research Centre; Países BajosFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; FranciaFil: Maridonneau Parini, Isabel. Centre National de la Recherche Scientifique; FranciaFil: Lugo Villarino, Geanncarlo. Centre National de la Recherche Scientifique; FranciaFil: Cougoule, Celine. Centre National de la Recherche Scientifique; Franci

Pharmacological restoration of tumour suppressor protein p53 in neuroblastoma : seeking synergy and non-invasive biomarkers

Neuroblastoma is a rare developmental tumour of young children accounting for a disproportionally high proportion of paediatric cancer deaths. Increasing insights into neuroblastoma genetics and biology led to the identification of the MDM2 antagonist idasanutlin as a promising therapeutic strategy. In this thesis, we investigated two key aspects that could facilitate the clinical implementation of idasanutlin for the treatment of neuroblastoma patients. First, we identified the BCL2 inhibitor venetoclax as a synergistic drug partner of idasanutlin using both in vitro and in vivo model systems of neuroblastoma. Second, we discovered circulating miRNAs that dynamically respond to p53 activation in mice carrying human neuroblastoma xenografts; opening up possibilities for non-invasive treatment monitoring

MicroRNA expression analysis using small RNA sequencing discovery and RT-qPCR-based validation

miRNAs are small noncoding RNA molecules that function as regulators of gene expression. Deregulated miRNA expression has been reported in various diseases including cancer. Due to their small size and high degree of homology, accurate quantification of miRNA expression is technically challenging. In this chapter, we present two different technologies for miRNA quantification: small RNA sequencing and RT-qPCR

Evaluation of targeted drugs in combination with the MDM2 antagonist RG7388 in neuroblastoma

Background: Neuroblastoma is the second most common solid malignant tumor in children and accounts for approximately 15% of all pediatric cancer-related deaths. As in most pediatric cancers, TP53 mutations are relatively rare in neuroblastoma and the p53 tumor suppressor protein is mainly kept inactive by aberrations in the p14ARF/MDM2/TP53 axis resulting in increased activity of MDM2. These biological conditions make neuroblastoma specifically suitable for treatment with targeted inhibitors of the TP53–MDM2 interaction. As well-matched combination therapy is considered decisive for targeted therapeutics by increasing therapeutic efficacy and preventing the development of treatment resistance, the aim of this study was to identify appropriate drug combination partners for the small-molecule MDM2 antagonist RG7388 in neuroblastoma. Methods: Using an in vitro cell viability screen, 15 targeted drugs were evaluated in combination with RG7388 in a panel of four neuroblastoma cell lines, three of which with wild-type TP53 (NGP, IMR-32 and SH-SY5Y) and one with mutated TP53 (SK-N-BE(2c)). Results: Several compounds displayed synergism in combination with RG7388 in at least one of the cell lines studied. The highest degree of synergism was observed when RG7388 was combined with the BCL2 inhibitor ABT-199. The lowering of the apoptotic threshold by ABT-199 complemented with the potent induction of pro-apoptotic TP53 target genes by RG7388 resulted in enhanced induction of apoptosis and is proposed to account for the observed synergism. Conclusion: Dual targeting of MDM2 and BCL2 is highly synergistic in neuroblastoma cells trough enhanced induction of apoptosis