Adam Cheuk

The MYCN oncogene is a direct target of miR-34a.

Loss of 1p36 heterozygosity commonly occurs with MYCN amplification in neuroblastoma tumors, and both are associated with an aggressive phenotype. Database searches identified five microRNAs that map to the commonly deleted region of 1p36 and we hypothesized that the loss of one or more of these microRNAs contributes to the malignant phenotype of MYCN-amplified tumors. By bioinformatic analysis, we identified that three out of the five microRNAs target MYCN and of these miR-34a caused the most significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines with MYCN amplification. Quantitative RT–PCR showed that neuroblastoma tumors with 1p36 loss expressed lower level of miR-34a than those with normal copies of 1p36. Furthermore, we demonstrated that MYCN is a direct target of miR-34a. Finally, using a series of mRNA expression profiling experiments, we identified other potential direct targets of miR-34a, and pathway analysis demonstrated that miR-34a suppresses cell-cycle genes and induces several neural-related genes. This study demonstrates one important regulatory role of miR-34a in cell growth and MYCN suppression in neuroblastoma.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

microRNA Profiling Identifies Cancer-Specific and Prognostic Signatures in Pediatric Malignancies

Purpose: microRNAs have been shown to be involved in different human cancers. We therefore have performed expression profiles on a panel of pediatric tumors to identify cancer-specific microRNAs. We also investigated if microRNAs are coregulated with their host gene. Experimental Design: We performed parallel microRNAs and mRNA expression profiling on 57 tumor xenografts and cell lines representing 10 different pediatric solid tumors using microarrays. For those microRNAs that map to their host mRNA, we calculated correlations between them. Results: We found that the majority of cancer types clustered together based on their global microRNA expression profiles by unsupervised hierarchical clustering. Fourteen microRNAs were significantly differentially expressed between rhabdomyosarcoma and neuroblastoma, and 8 of them were validated in independent patient tumor samples. Exploration of the expression of microRNAs in relationship with their host genes showed that the expression for 43 of 68 (63%) microRNAs located inside known coding genes was significantly correlated with that of their host genes. Among these 43 microRNAs, 5 of 7 microRNAs in the OncomiR-1 cluster correlated significantly with their host gene MIRHG1 (P < 0.01). In addition, high expression of MIRHG1 was significantly associated with high stage and MYCN amplification in neuroblastoma tumors, and the expression level of MIRHG1 could predict the outcome of neuroblastoma patients independently from the current neuroblastoma risk-stratification in two independent patient cohorts. Conclusion: Pediatric cancers express cancer-specific microRNAs. The high expression of the OncomiR-1 host gene MIRHG1 correlates with poor outcome for patients with neuroblastoma, indicating important oncogenic functions of this microRNA cluster in neuroblastoma biology. (Clin Cancer Res 2009;15(17):5560–8)

Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534).

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

CLPTM1L Promotes Growth and Enhances Aneuploidy in Pancreatic Cancer Cells

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33.

Human neuroblastoma cells rapidly enter cell cycle arrest and apoptosis following exposure to C-28 derivatives of the synthetic triterpenoid CDDO

Synthetic triterpenoids, such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives, are an extremely potent class of new anti-cancer therapeutic agents, characterized by high anti-tumor potency and low toxicity to normal tissues. This report is the first to investigate the effects of C-28 derivatives of CDDO on 22 pediatric solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, osteosarcoma, and Ewings sarcoma. We determined IC50s in the range of 5 – 170 nM for inhibition of colony formation and DNA synthesis, and 110 – 630 nM for metabolic cell death and decrease in cell number, using the C-28 CDDO analogs, CDDO methyl ester (CDDO-Me), CDDO imidazolide (CDDO-Im), CDDO ethyl amide (CDDO-EA), CDDO trifluoroethyl amide (CDDO-TFEA), and CDDO diethylamide (CDDO-DE). After treatment of human neuroblastoma cells with CDDO-Me, cell cycle studies show depletion of the S-phase, while apoptosis studies show conformational activation and mitochondrial translocation of Bax protein, as well as activation of caspases -3 and -8. These data demonstrate the potential utility of CDDO analogs as promising novel therapeutic agents for high-risk pediatric solid tumors.

screening a panel of drugs with diverse mechanisms of action yields potential therapeutic agents against neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at ≤ 10 μM concentration. More interestingly, 17 compounds are active at ≤ 1 μM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, 11 compounds are not currently used to treat NB. Among them, 9 are FDA approved compounds, and 3 agents are undergoing clinical trials for various malignancies. Furthermore, we identified 4 agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.

Synthetic Lethal Screen Identifies NF-κB as a Target for Combination Therapy with Topotecan for patients with Neuroblastoma

BackgroundDespite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma.MethodsWe took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies.ResultsWe found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments.ConclusionsSynthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

Abstract 2556: Functional characterization of the pancreatic cancer TERT-CLPTM1L risk locus on chr5p15.33.

Pancreatic cancer is a highly lethal cancer with few well established risk factors. PanScan, a genome wide association study (GWAS) of pancreatic cancer has identified four pancreatic cancer susceptibility loci in populations of European ancestry. One of these is in a multi cancer locus on chr5p15.33 where the most significant SNP from the GWAS (rs401681, P=3.7x10-7, ORAllele=1.19) lies in a region containing two genes, TERT and CLPTM1L. The TERT gene encodes the catalytic subunit of telomerase, well known for its essential role in maintaining telomere ends. The function of CLPTM1L is not as clear, although it has been proposed to play a role in apoptosis. It is predicted to encode a protein with 6 transmembrane (TM) domains and two large hydrophilic domains: a loop of 253 aa between the first and second TM domains, and a C-terminal tail of 89 aa. We have performed imputation to fine-map the signal to a SNP three orders of magnitude more significant than the GWAS SNP (PImputed=1.4x10-10, ORAllele=1.30). As this SNP is located in the CLPTM1L gene we have performed a series of experiments to investigate the function of the CLPTM1L gene and its encoded protein. Immunofluorescence analysis in pancreatic cancer cells (PANC-1) indicates that it localizes to the endoplasmic reticulum. Affinity purification and mass spectrometry (HEK-293T, hTERT-HPNE and PANC-1 cells) identified MYH9, a non-muscle heavy chain myosin, as a potential interacting protein. The interaction has been validated by co-immunoprecipitation and co-localization experiments. To examine if CLPTM1L plays a role in growth control, we created stable PANC-1 cell lines overexpressing the full length CLPTM1L gene as well as two deletions, a C-terminal deletion and a loop deletion, and assayed growth in vitro and in vivo. Cell lines overexpressing full length CLPTM1L grow faster in vitro and in vivo as compared to cells containing empty vector. Interestingly, the two CLPTM1L mutants abolish this effect. Furthermore, we have shown by RNA-seq that the CLPTM1L gene is overexpressed in pancreatic tumors as compared to normal pancreatic tissues. Our results indicate that CLPTM1L may play a role in the control of cell growth and oncogenesis in the pancreas. Our current efforts aim at further characterizing the function of CLPTM1L and to correlate pancreatic cancer risk variants on 5p15.33 to molecular phenotypes to attempt to explain the underlying biology of the risk. Citation Format: Jinping Jia, Irene Collins, Marta Dzyadyk, Abbey Thompson, Adam Cheuk, Hemang Parikh, Zhaoming Wang, Chris Westlake, Allen Bosley, Gloria Petersen, Thorkell Andresson, Laufey Amundadottir. Functional characterization of the pancreatic cancer TERT-CLPTM1L risk locus on chr5p15.33. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2556. doi:10.1158/1538-7445.AM2013-2556 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2619: Whole genome and transcriptome sequencing identifies an activating mutation of LPAR1 in a patient with metastatic neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumor of childhood, and is one of the most genomically heterogeneous malignances studied to date. Despite advance of multimodal therapies, the current overall survival rate for patient with metastatic neuroblastoma is Citation Format: Jun S. Wei, Peter Johansson, Li Chen, Young K. Song, Catherine Tolman, Samuel Li, Laura Hurd, Rajesh Patidar, Xinyu Wen, Thomas C. Badgett, Adam Cheuk, Jean-Claude Marshall, Patricia Steeg, Jose Pedro Vaque Diez, J Silvio Gutkind, Javed Khan. Whole genome and transcriptome sequencing identifies an activating mutation of LPAR1 in a patient with metastatic neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2619. doi:10.1158/1538-7445.AM2013-2619