Longitudinal evaluation of serum microRNAs as biomarkers for neuroblastoma burden and therapeutic p53 reactivation.

Accurate assessment of treatment response and residual disease is indispensable for the evaluation of cancer treatment efficacy. However, performing tissue biopsies for longitudinal follow-up poses a major challenge in the management of solid tumours like neuroblastoma. In the present study, we evaluated whether circulating miRNAs are suitable to monitor neuroblastoma tumour burden and whether treatment-induced changes of miRNA abundance in the tumour are detectable in serum. We performed small RNA sequencing on longitudinally collected serum samples from mice carrying orthotopic neuroblastoma xenografts that were exposed to treatment with idasanutlin or temsirolimus. We identified 57 serum miRNAs to be differentially expressed upon xenograft tumour manifestation, out of which 21 were also found specifically expressed in the serum of human high-risk neuroblastoma patients. The murine serum levels of these 57 miRNAs correlated with tumour tissue expression and tumour volume, suggesting potential utility for monitoring tumour burden. In addition, we describe serum miRNAs that dynamically respond to p53 activation following treatment of engrafted mice with idasanutlin. We identified idasanutlin-induced serum miRNA expression changes upon one day and 11 days of treatment. By limiting to miRNAs with a tumour-related induction, we put forward hsa-miR-34a-5p as a potential pharmacodynamic biomarker of p53 activation in serum.

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients.

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of noninvasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in 2 independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis, and overall survival revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential abundance analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume. These findings were validated in longitudinal serum samples from metastatic neuroblastoma patients, where the 9 miRNAs were associated with disease burden and treatment response.

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.

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.

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment.

PURPOSE: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. EXPERIMENTAL DESIGN: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK(F1174L) or ALK(R1275Q) hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK(F1174L) double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. RESULTS: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK(F1174L) and ALK(R1275Q) regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. CONCLUSIONS: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.

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.

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 annotation. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA sequences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annotation 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, comparison 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 online 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.org.

Pharmacologic activation of wild-type p53 by nutlin therapy in childhood cancer.

A peculiar feature of several types of childhood cancer is that loss-of-function mutations of the TP53 (p53) tumor suppressor gene are uncommon, in contrast to many adult tumors. As p53 needs to be inactivated in order for tumor cells to survive and thrive, pediatric tumors typically make use of other mechanisms to keep p53 in check. One of the critical negative regulators of p53 is the MDM2 oncoprotein. Many anticancer drug development efforts in the past decade have therefore been devoted to the discovery and optimization of small molecules that selectively disrupt the interaction between MDM2 and p53, which could provide, in principle, a potent means to restore p53 function in tumor cells with wild-type p53. The nutlins are the class of selective inhibitors of the p53-MDM2 interaction that are currently most advanced in their clinical development. We review here the preclinical data that support the potential therapeutic use of nutlin drugs in the treatment of various pediatric tumors, including neuroblastoma, retinoblastoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma, medulloblastoma, and childhood acute lymphoblastic leukemia.