Irina Lambertz

Monoallelic but not biallelic loss of Dicer1 promotes tumorigenesis in vivo

Human tumors are characterized by widespread reduction in microRNA (miRNA) expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells. A defect in miRNA processing is one possible mechanism for global downregulation. To explore this possibility in more detail in vivo, we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only a partial decrease in miRNA processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer etiology and cancer therapy.

Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma.

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

Antitumor Activity of the Selective MDM2 Antagonist Nutlin-3 Against Chemoresistant Neuroblastoma With Wild-Type p53

BACKGROUND Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas. METHODS The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided. RESULTS Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX20 cells, as evidenced by increased expression of p53 target genes, G1 cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR10 cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX20 xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm3, difference = 890 mm3, 95% confidence interval = 469 to 1311 mm3, P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR10 xenografts was unaffected by nutlin-3. CONCLUSIONS Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.

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 ALKF1174L or ALKR1275Q 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/ALKF1174L 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 ALKF1174L and ALKR1275Q 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. Clin Cancer Res; 21(14); 3327–39. ©2015 AACR.