MYC regulates metabolism through vesicular transfer of glycolytic kinases.

Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs.

ATR is a MYB regulated gene and potential therapeutic target in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a rare cancer that preferentially occurs in the head and neck, breast, as well as in other sites. It is an aggressive cancer with high rates of recurrence and distant metastasis. Patients with advanced disease are generally incurable due to the lack of effective systemic therapies. Activation of the master transcriptional regulator MYB is the genomic hallmark of ACC. MYB activation occurs through chromosomal translocation, copy number gain or enhancer hijacking, and is the key driving event in the pathogenesis of ACC. However, the functional consequences of alternative mechanisms of MYB activation are still uncertain. Here, we show that overexpression of MYB or MYB-NFIB fusions leads to transformation of human glandular epithelial cells in vitro and results in analogous cellular and molecular consequences. MYB and MYB-NFIB expression led to increased cell proliferation and upregulation of genes involved in cell cycle control, DNA replication, and DNA repair. Notably, we identified the DNA-damage sensor kinase ATR, as a MYB downstream therapeutic target that is overexpressed in primary ACCs and ACC patient-derived xenografts (PDXs). Treatment with the clinical ATR kinase inhibitor VX-970 induced apoptosis in MYB-positive ACC cells and growth inhibition in ACC PDXs. To our knowledge, ATR is the first example of an actionable target downstream of MYB that could be further exploited for therapeutic opportunities in ACC patients. Our findings may also have implications for other types of neoplasms with activation of the MYB oncogene.

A Promyelocytic Leukemia Protein-Thrombospondin-2 Axis and the Risk of Relapse in Neuroblastoma.

PURPOSE: Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations. EXPERIMENTAL DESIGN: Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays. RESULTS: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas. CONCLUSIONS: Our work reveals a novel PML-I-TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398-409. ©2016 AACR.

Identification and pharmacological inactivation of the MYCN gene network as a therapeutic strategy for neuroblastic tumor cells.

The MYC family of transcription factors consists of three well characterized members, c-MYC, L-MYC, and MYCN, deregulated in the majority of human cancers. In neuronal tumors such as neuroblastoma, MYCN is frequently activated by gene amplification, and reducing its expression by RNA interference has been shown to promote growth arrest and apoptosis of tumor cells. From a clinical perspective, RNA interference is not yet a viable option, and small molecule inhibitors of transcription factors are difficult to develop. We therefore planned to identify, at the global level, the genes interacting functionally with MYCN required to promote fitness of tumor cells facing oncogenic stress. To find genes whose inactivation is synthetically lethal to MYCN, we implemented a genome-wide approach in which we carried out a drop-out shRNA screen using a whole genome library that was delivered into isogenic neuroblastoma cell lines expressing or not expressing MYCN. After the screen, we selected for in-depth analysis four shRNAs targeting AHCY, BLM, PKMYT1, and CKS1B. These genes were chosen because they are directly regulated by MYC proteins, associated with poor prognosis of neuroblastoma patients, and inhibited by small molecule compounds. Mechanistically, we found that BLM and PKMYT1 are required to limit oncogenic stress and promote stabilization of the MYCN protein. Cocktails of small molecule inhibitors of CKS1B, AHCY, BLM, and PKMYT1 profoundly affected the growth of all neuroblastoma cell lines but selectively caused death of MYCN-amplified cells. Our findings suggest that drugging the MYCN network is a promising avenue for the treatment of high risk, neuroblastic cancers.

Physical interaction between MYCN oncogene and polycomb repressive complex 2 (PRC2) in neuroblastoma: functional and therapeutic implications.

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5'-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.

Polyphenon [corrected] E enhances the antitumor immune response in neuroblastoma by inactivating myeloid suppressor cells.

PURPOSE: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. EXPERIMENTAL DESIGN: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E-drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. RESULTS: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. CONCLUSIONS: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.

Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas.

Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the non-canonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.

Addiction of MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop.

MYCN is a member of the MYC family of oncoproteins frequently amplified or overexpressed in aggressive, paediatric tumours of the nervous system. In this study we have identified the gene B-MYB, encoding the transcription factor also known as MYBL2, as a downstream target of MYCN. Using multiple in silico databases we show that expression of B-MYB significantly correlates with that of MYCN in neuroblastoma patients. MYCN binds to and activates the B-MYB gene in vivo and in vitro. Blunting B-MYB expression by RNA interference causes reduced proliferation of MYCN amplified, but not MYCN-non amplified, neuroblastoma cell lines, indicating that tumour cells are addicted to B-MYB in a MYCN dependent manner. Notably, B-MYB binds in vivo to the MYCN amplicon and is required for its expression. We conclude that MYCN and B-MYB are engaged in a reciprocal regulatory loop whose pharmacological targeting could be beneficial to patients with the aggressive forms of cancer in which MYCN is amplified.

Regulation of CLU gene expression by oncogenes and epigenetic factors implications for tumorigenesis.

In no other field has the function of clusterin (CLU) been more controversial than in cancer genetics. After more than 20 years of research, there is still uncertainty with regard to the role of CLU in human cancers. Some investigators believe CLU to be an oncogene, others-an inhibitor of tumorigenesis. However, owing to the recent efforts of several laboratories, the role of CLU in important cellular processes like proliferation, apoptosis, differentiation, and transformation is beginning to emerge. The "enigmatic" CLU is becoming less so. In this chapter, we will review the work of research teams interested in understanding how CLU is regulated by oncogenic signaling. We will discuss how and under what circumstances oncogenes and epigenetic factors modify CLU expression, with important consequences for mammalian tumorigenesis.

Myb-induced chromatin remodeling at a dual enhancer/promoter element involves non-coding rna transcription and is disrupted by oncogenic mutations of v-myb.

The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor (v-Myb) that transforms myelomonocytic cells by deregulating the expression of specific target genes. v-myb has acquired its oncogenic potential by truncation as well as by a number of point mutations of its cellular progenitor c-myb. As a result of these changes, the target gene spectrum v-Myb differs from that of c-Myb. We recently showed that the chicken mim-1 gene, a c-Myb target gene that is not activated by v-Myb harbors a powerful cell type-specific and Myb-inducible enhancer in addition to a Myb-responsive promoter. We now show that Myb-dependent activation of the mim-1 gene is accompanied by extensive remodeling of the nucleosomal architecture at the enhancer. We found that the mim-1 enhancer region also harbors a promoter whose activity is stimulated by Myb and which directs the transcription of an apparently non-coding RNA. Furthermore, our data show that the oncogenic mutations of AMV have disrupted the ability of v-Myb to induce remodeling of chromatin structure at the mim-1 enhancer. Together, our results demonstrate for the first time directly that Myb induces alterations of the nucleosomal organization at a relevant target site and provide new insight into the functional consequences of the oncogenic amino acid substitutions.

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.

BACKGROUND: Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. METHODS: We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription-polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. RESULTS: Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). CONCLUSIONS: We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.

C/EBPbeta induces chromatin opening at a cell-type-specific enhancer.

We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBPbeta cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBPbeta activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBPbeta triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBPbeta in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region.

A dual activation mechanism for Myb-responsive genes in myelomonocytic cells.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. Here we have used DNaseI hypersensitive site mapping and reporter gene assays to study the activation of three Myb target genes--mim-1, the lysozyme gene and the C/EBPbeta gene--all of which are activated by Myb in myelomonocytic cells but not in other hematopoietic lineages. We have found that these genes are activated by Myb via more than one cis-regulatory region. Our data suggest that all three genes are activated by Myb by dual mechanisms involving the promoters as well as enhancers. Using a cell line that expresses an estrogen-inducible v-Myb/estrogen receptor fusion protein we have also determined the effect of Myb on the expression of the C/EBPalpha gene. Our results show that C/EBPalpha expression is down-regulated by v-Myb. Thus, v-Myb affects the expression of two C/EBP family members in opposite directions.

v-Myb mediates cooperation of a cell-specific enhancer with the mim-1 promoter.

The oncogenic transcription factor v-Myb disrupts myelomonocytic differentiation and transforms myelomonocytic cells by deregulating the expression of specific target genes. One of these genes, the chicken mim-1 gene, is activated by Myb exclusively in myelomonocytic cells and, therefore, has been an interesting model system to study how Myb activates a target in a lineage-specific manner. Previous work has suggested that Myb activates mim-1 by cooperating with CCAAT box/enhancer binding protein beta (C/EBPbeta) or other C/EBP transcription factors at the mim-1 promoter. We have now identified and characterized a powerful Myb-dependent enhancer located 2 kb upstream of the mim-1 promoter. The enhancer is preferentially active in myelomonocytic cells, confers Myb responsiveness onto a heterologous promoter, and dramatically increases Myb responsiveness of the mim-1 promoter. Chromatin immunoprecipitation demonstrates that v-Myb and C/EBPbeta are bound to the enhancer in v-Myb-transformed cells; furthermore, cooperation of the enhancer with the mim-1 promoter is greatly stimulated by C/EBPbeta and p300. Taken together, our results show that the regulation of mim-1 expression by v-Myb is more complex than previously assumed and involves two distinct regions of the mim-1 gene. A major function of v-Myb (in addition to its role at the mim-1 promoter) apparently is to activate the mim-1 enhancer and, together with C/EBPbeta and p300, facilitate its cooperation with the promoter. Interestingly, our work also shows that the v-Myb protein encoded by avian myeloblastosis virus is defective in this function, suggesting an explanation for why primary avian myeloblastosis virus-transformed myeloblasts do not express the mim-1 gene.