No abstract

Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.

The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

Genomic aberrations of Cyclin D1 (CCND1), CDK4, and CDK6 in neuroblastoma indicate that dysregulation of the G 1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here, we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive overexpression of Cyclin D1, which correlates with histologic subgroups. Immunohistochemical analysis showed overexpression of Cyclin D1 in neuroblasts and low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G 1 -regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partners CDK4 and CDK6 in several neuroblastoma cell lines. The Cyclin D1 and CDK4 knockdown resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4/CDK6-specific pRb phosphorylation, reduction of E2F transcriptional activity, and a decrease of Cyclin A protein levels. Phenotype analysis showed a significant reduction in cell proliferation, a G 1 -specific cell cycle arrest, and, moreover, an extensive neuronal differentiation. Affymetrix microarray profiling of small interfering RNA-treated cells revealed a shift in expression profile toward a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on overexpression of G 1 -regulating genes to maintain their undifferentiated phenotype. [Cancer Res 2008;68(8):2599-609]

neuroblastoma ͉ synthetic lethal T wo genes are considered to be "synthetically lethal" if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes causes death. This mechanism is best described for loss of function genes but also exits for gain of function genes. For example gene A can become essential for survival if gene B is over-expressed. This mechanism is potentially very attractive for the development of targeted anti cancer compounds that could specifically kill tumor cells while leaving normal cells alive (1-3). However, examples of synthetic lethal oncogenes in human tumors are hardly documented.Neuroblastomas are embryonal tumors that originate from the developing sympathetic nervous system, and although neuroblastomas have a low incidence, they are the second cause of cancer related deaths in children (4, 5). The MYCN gene is amplified in 20-30% of neuroblastoma tumors and amplification strongly correlates with a bad prognosis. MYC genes are potent oncogenes that drive unrestrained cell growth and proliferation. They function as transcription factors that cause up-regulation or repression of genes involved in a variety of oncogenetic pathways. Recently MYC genes were also shown to control protein expression through mRNA translation and to directly regulate DNA replication (6-9). Apart from the function in oncogenesis, MYC genes have also been described to induce apoptosis if over-expressed in non MYC-amplified cells (10). This could indicate that the amplification of the MYC oncogenes requires a specific genetic background and that there could be synthetic lethal relations with other (onco)genes.Cell cycle aberrations occur in all tumors and many targeted compounds inhibiting specific cell cycle kinases have been developed (11,12). These efforts were based on the idea that the targeting of aberrant cell cycle checkpoints in cancer cells could lead to tumor growth inhibition and cell death (13). Several studies have recently shown that most cell cycle kinases are not essential for cell survival in vitro and in vivo (14). Cyclindependent kinase 2 (CDK2) was thought to be a crucial regulator of S-phase progression and was therefore evaluated as an anticancer drug target. Tetsu et al. however showed that CDK2 inhibition in several cancer cell types did not result in cell death, and Santamaria et al. showed that genetic ablation of CDK2 in mice could be compensated for by CDK1 (15,16). These findings severely reduced optimism about CDK2 as therapeutic target.Several cell cycle aberrations involving G1-regulating genes have been identified in neuroblastomas. Cyclin D1 and CDK4 gene amplifications occur at a low frequency, and a CDK6 mutation that inactivates p16-binding has been found (17)(18)(19). Cyclin D1 was found to be extremely over-expressed in about 75% of neuroblastoma tumors. Inhibition of the G1 regulating genes CDK4 or cyclin D1 in neuroblastoma cell lines resulted in restoration of the G1 checkpoint and subsequent neuronal differentiation (20). The ab...

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n ‫؍‬ 18) and older children (noninfants) with ALL (n ‫؍‬ 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P ‫؍‬ .007) and accumulated 2.3-fold more Ara-CTP (P ‫؍‬ .011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P ‫؍‬ .026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P ‫؍‬ .001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r s ‫؍‬ ؊0.58, P ‫؍‬ .006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n ‫؍‬ 14) and germ line MLL cases (n ‫؍‬ 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P ‫؍‬ .001) and a 2.7-fold higher hENT1 (P ‫؍‬ .046) mRNA expression in patients with MLL generearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL generearranged infant ALL.

BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The overexpression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.