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1.  Dual CDK4/CDK6 Inhibition Induces Cell Cycle Arrest and Senescence in Neuroblastoma 
Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes.
Experimental Procedures
We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011, a highly specific CDK4/6 inhibitor.
Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nM in sensitive lines). LEE011 caused cell cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p = 0.01), the identification of additional clinically accessible biomarkers is of high importance.
Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease.
PMCID: PMC3844928  PMID: 24045179
Neuroblastoma; CDK4; CDK6; LEE011; MYCN
2.  Targeting MYCN: A Good BET for Improving Neuroblastoma Therapy? 
Cancer discovery  2013;3(3):255-257.
Direct targeting of oncogenic MYC proteins has been an elusive goal of many cancer drug development efforts. In this issue of Cancer Discovery, Stegmaier and colleagues demonstrate that pharmacologically interfering with the bromodomain and extraterminal (BET) class of proteins potently depletes MYCN in neuroblastoma cells, resulting in cellular cytotoxicity and thus providing a novel approach with a potential impact on a previously undruggable major oncogene.
PMCID: PMC3709873  PMID: 23475876
3.  Common variation at 6q16 within HACE1 and LIN28B influences susceptibility to neuroblastoma 
Nature genetics  2012;44(10):1126-1130.
Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths1. Here we report on a genome-wide association study of 2,817 neuroblastoma cases and 7,473 controls. We identified two new associations at 6q16, the first within HACE1 (rs4336470; combined P = 2.7 × 10−11, odds ratio 1.26, 95% CI: 1.18–1.35) and the second within LIN28B (rs17065417; combined P = 1.2 × 10−8, odds ratio 1.38, 95% CI: 1.23–1.54). Expression of LIN28B and let-7 miRNA correlated with rs17065417 genotype in neuroblastoma cell lines, and we observed significant growth inhibition upon depletion of LIN28B specifically in neuroblastoma cells homozygous for the risk allele. Low HACE1 and high LIN28B expression in diagnostic primary neuroblastomas were associated with worse overall survival (P = 0.008 and 0.014, respectively). Taken together, we show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and that both genes likely play a role in disease progression.
PMCID: PMC3459292  PMID: 22941191
4.  Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform that Influences Neuroblastoma Susceptibility and Oncogenicity 
Cancer Research  2012;72(8):2068-2078.
The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherent, anchorage-independent, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.
PMCID: PMC3328617  PMID: 22350409
genome-wide association; neuroblastoma; BARD1; cancer susceptibility genes; functional genomics; oncogenes; genotype-phenotype correlations
5.  Integrative genomics identifies LMO1 as a neuroblastoma oncogene 
Nature  2010;469(7329):216-220.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
PMCID: PMC3320515  PMID: 21124317
6.  Menin Induces Apoptosis in Murine Embryonic Fibroblasts 
The Journal of biological chemistry  2003;279(11):10685-10691.
Multiple endocrine neoplasia type I (MEN1) is a hereditary tumor syndrome characterized by multiple endocrine and occasionally non-endocrine tumors. The tumor suppressor gene Men1, which is frequently mutated in MEN1 patients, encodes the nuclear protein menin. Although many tumor suppressor genes are involved in the regulation of apoptosis, it is unclear whether menin facilitates apoptosis. Here we show that ectopic overexpression of menin via adenoviruses induces apoptosis in murine embryonic fibroblasts (MEFs). The induction of apoptosis depends on Bax and Bak, two proapoptotic proteins. Moreover, loss of menin expression compromises apoptosis induced by UV irradiation and TNF-α, while complementation of menin-null cells with menin restores sensitivity to UV and TNF-α-induced apoptosis. Interestingly, loss of menin reduces the expression of procaspase 8, a critical protease that is essential for apoptosis induced by death-related receptors, whereas complementation of the menin-null cells upregulates the expression of procaspase 8. Furthermore, complementation of menin-null cells with menin increases the activation of caspase 8 in response to TNF-α treatment. These results suggest a proapoptotic function for menin that may be important in suppressing the development of MEN1.
PMCID: PMC2858560  PMID: 14688275
menin; MEN I; apoptosis; caspase 8; Bax/Bak; MEFs
7.  Menin-mediated caspase 8 expression in suppressing multiple endocrine neoplasia type 1 
The Journal of biological chemistry  2007;282(43):31332-31340.
Multiple endocrine neoplasia type 1 (MEN1) is a familial tumor syndrome linked to mutation of the MEN1 gene, which encodes a tumor suppressor, menin. We previously reported that menin upregulates the caspase 8 expression and promotes TNF-α-induced apoptosis. However, it remains unclear how menin upregulates caspase 8 expression and whether menin-mediated caspase 8 expression plays a role in repressing MEN1 development. Here we show that menin binds the 5’-untranslated region (5’-UTR) of the Caspase 8 locus in vivo and activates transcription of a reporter gene through the 5’-UTR. Menin directly binds the 5’-UTR in a sequence-independent manner in vitro. Moreover, Men1 ablation in cells reduces acetylation of histones H3 and H4 at the 5’-UTR of the caspase 8 locus bound by menin in vivo. Notably, the MEN1-derived menin point mutants lose their ability to bind the caspase 8 locus and fail to induce caspase 8 expression and TNF-α-mediated apoptosis. Consistent with these observations, the expression level of caspase 8 is markedly reduced in insulinomas from Men1+/− mice. Together, our results indicate that menin enhances the caspase 8 expression by binding the caspase 8 locus, and these results also suggest that menin suppresses MEN1 tumorigenesis, at least in part, by upregulating caspase 8 expression.
PMCID: PMC2858561  PMID: 17766243
8.  Tumor Suppressor Menin Regulates Expression of Insulin-Like Growth Factor Binding Protein 2 (IGFBP-2) 
Endocrinology  2004;145(7):3443-3450.
Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that meinin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the Dnase I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.
PMCID: PMC2858565  PMID: 15044367
menin; MEN1; IGFBP-2; transcription
9.  Direct Binding of DNA by Tumor Suppressor Menin 
The Journal of biological chemistry  2004;279(47):49045-49054.
Menin is a tumor suppressor that is mutated in patients with multiple endocrine neoplasia type I (MEN1), an inherited tumor-prone syndrome. Since there is no obvious conserved structural domain in menin that suggests a function, little is known as to how menin suppresses tumorigenisis. Although menin interacts with a variety of nuclear proteins including transcription factors, it is unknown whether menin itself can directly bind DNA. Here we show that menin directly binds to double stranded DNA in a sequence-independent manner. It also binds a variety of DNA structures, including Y-structures, branched structures, and 4-way junction structures. The C-terminus of menin mediates binding to DNA, but MEN1 disease-derived mutations in the C-terminus abolish the ability of menin to bind DNA. Importantly, these MEN1 disease-related menin mutants also fail to repress cell proliferation as well as cell cycle progression at G2/M phase. Furthermore, detailed mutagenesis studies indicate that positively charged residues in two nuclear localization signals mediate direct DNA binding as well as repression of cell proliferation. Collectively, these results demonstrate, for the first time, a novel biochemical activity of menin, binding to DNA, and link its DNA binding to the regulation of cell proliferation.
PMCID: PMC2858586  PMID: 15331604
menin; MEN1; DNA binding; cell proliferation; inhibition
10.  Mutation of Tumor Suppressor Gene Men1 Acutely Enhances Proliferation of Pancreatic Islet Cells 
Cancer research  2006;66(11):5707-5715.
Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEFs) accelerates G0/G1 to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18Ink4c and p27Kip1. Moreover, Men1 excision results in decreased expression of p18Ink4c and p27Kip1 in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as seven days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets fourteen days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repressing G0/G1 to S transition.
PMCID: PMC2839933  PMID: 16740708
menin; tumor suppressor; MEN1; cell proliferation; islet cells; cell cycle

Results 1-10 (10)