A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we ...identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.
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•LIN28B and regional gain of chromosome 12q24 mediate RAN oncogene overexpression•RAN promotes cell proliferation in neuroblastoma•LIN28B promotes RAN levels by binding RAN mRNA and via RAN binding protein 2•LIN28B promotes Aurora kinase A expression in a let-7-dependent manner
Extending from prior identification of LIN28B as an oncogenic driver in high-risk neuroblastoma, Schnepp et al. show that LIN28B regulates the RAN level directly by mRNA binding and indirectly via let-7-regulated RANBP2. LIN28B and RAN signaling converge on Aurora kinase A, suggesting therapeutic potential.
Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths. Here, we report 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% confidence interval (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 that were 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, these data show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and indicate that both genes likely have a role in disease progression.
Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), ...a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated
, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance
. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation
and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance
. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in
-hyperactivated neuroblastomas.
Mutations in the MEN1 gene are associated with the multiple endocrine neoplasia syndrome type 1 (MEN1), which is characterized by parathyroid hyperplasia and tumors of the pituitary and pancreatic ...islets. The mechanism by which MEN1 acts as a tumor suppressor is unclear. We have recently shown that menin, the MEN1 protein product, interacts with mixed lineage leukemia (MLL) family proteins in a histone methyltransferase complex including Ash2, Rbbp5, and WDR5. Here, we show that menin directly regulates expression of the cyclin-dependent kinase inhibitors p27Kip1and p18Ink4c. Menin activates transcription by means of a mechanism involving recruitment of MLL to the p27Kip1and p18Ink4cpromoters and coding regions. Loss of function of either MLL or menin results in down-regulation of p27Kip1and p18Ink4cexpression and deregulated cell growth. These findings suggest that regulation of cyclin-dependent kinase inhibitor transcription by cooperative interaction between menin and MLL plays a central role in menin's activity as a tumor suppressor.
Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel ...therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, decreases self-renewal and migration. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7i and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.
Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available ...targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-3-(3-methyl-1,2,4-triazolo4,3-bpyridazin-6-yl)phenylacetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.
Background
The treatment of high‐risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors ...can inhibit MYCN expression and suppress MYCN‐amplified neuroblastoma in vivo. Furthermore, alterations within RAS‐MAPK (mitogen‐activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen‐activated extracellular signal‐regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS‐MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I‐BET726 or I‐BET762 and trametinib in high‐risk neuroblastoma.
Procedure
Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models.
Results
Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non‐MYCN–amplified, NRAS‐mutated neuroblastoma xenograft model, but had no efficacy in an MYCN‐amplified model harboring a loss‐of‐function mutation in NF1.
Conclusions
Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high‐risk neuroblastoma.
Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses ...fusion‐positive rhabdomyosarcoma, which expresses either PAX3‐FOXO1 or PAX7‐FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3‐FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient‐derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC‐209 and PTC‐028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas.
Treatment of alveolar rhabdomyosarcoma (ARMS) remains a major therapeutic challenge in pediatric oncology. Here, we demonstrate that genetic and pharmacologic inhibition of BMI1 reduces ARMS viability. We show that BMI1 inhibits the tumor suppressive Hippo pathway and, conversely, that BMI1 disruption upregulates Hippo signaling. Collectively, these findings provide an initial framework for targeting BMI1 in ARMS and additional sarcomas.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. 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.
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