Palbociclib releases the latent differentiation capacity of neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

The proneural transcription factor ASCL1 regulates cell proliferation and primes for differentiation in neuroblastoma.

Neuroblastoma is believed to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme, but instead become locked in a pro-proliferative developmental state. Achaete-scute homolog 1 (ASCL1) is a proneural master regulator of transcription which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells during development, while its expression has been implicated in the maintenance of an oncogenic programme in MYCN-amplified neuroblastoma. However, the role of ASCL1 expression in neuroblastoma is not clear, especially as its levels vary considerably in different neuroblastoma cell lines. Here, we have investigated the role of ASCL1 in maintaining proliferation and controlling differentiation in both MYCN amplified and Anaplastic Lymphoma Kinase (ALK)-driven neuroblastoma cells. Using CRISPR deletion, we generated neuroblastoma cell lines lacking ASCL1 expression, and these grew more slowly than parental cells, indicating that ASCL1 contributes to rapid proliferation of MYCN amplified and non-amplified neuroblastoma cells. Genome-wide analysis after ASCL1 deletion revealed reduced expression of genes associated with neuronal differentiation, while chromatin accessibility at regulatory regions associated with differentiation genes was also attenuated by ASCL1 knock-out. In neuroblastoma, ASCL1 has been described as part of a core regulatory circuit of developmental regulators whose high expression is maintained by mutual cross-activation of a network of super enhancers and is further augmented by the activity of MYC/MYCN. Surprisingly, ASCL1 deletion had little effect on the transcription of CRC gene transcripts in these neuroblastoma cell lines, but the ability of MYC/MYCN and CRC component proteins, PHOX2B and GATA3, to bind to chromatin was compromised. Taken together, our results demonstrate several roles for endogenous ASCL1 in neuroblastoma cells: maintaining a highly proliferative phenotype, regulating DNA binding of the core regulatory circuit genes to chromatin, while also controlling accessibility and transcription of differentiation targets. Thus, we propose a model where ASCL1, a key developmental regulator of sympathetic neurogenesis, plays a pivotal role in maintaining proliferation while simultaneously priming cells for differentiation in neuroblastoma.

Tumoral heterogeneity in neuroblastoma.

Neuroblastoma is a solid, neuroendocrine tumor with divergent clinical behavior ranging from asymptomatic to fatal. The diverse clinical presentations of neuroblastoma are directly linked to the high intra- and inter-tumoral heterogeneity it presents. This heterogeneity is strongly associated with therapeutic resistance and continuous relapses, often leading to fatal outcomes. The development of successful risk assessment and tailored treatment strategies lies in evaluating the extent of heterogeneity via the accurate genetic and epigenetic profiling of distinct cell subpopulations present in the tumor. Recent studies have focused on understanding the molecular mechanisms that drive tumoral heterogeneity in pursuing better therapeutic and diagnostic approaches. This review describes the cellular, genetic, and epigenetic aspects of neuroblastoma heterogeneity. In addition, we summarize the recent findings on three crucial factors that can lead to heterogeneity in solid tumors: the inherent diversity of the progenitor cells, the presence of cancer stem cells, and the influence of the tumor microenvironment.

Super-enhancer associated core regulatory circuits mediate susceptibility to retinoic acid in neuroblastoma cells.

Neuroblastoma is a pediatric tumour that accounts for more than 15% of cancer-related deaths in children. High-risk tumours are often difficult to treat, and patients' survival chances are less than 50%. Retinoic acid treatment is part of the maintenance therapy given to neuroblastoma patients; however, not all tumours differentiate in response to retinoic acid. Within neuroblastoma tumors, two phenotypically distinct cell types have been identified based on their super-enhancer landscape and transcriptional core regulatory circuitries: adrenergic (ADRN) and mesenchymal (MES). We hypothesized that the distinct super-enhancers in these different tumour cells mediate differential response to retinoic acid. To this end, three different neuroblastoma cell lines, ADRN (MYCN amplified and non-amplified) and MES cells, were treated with retinoic acid, and changes in the super-enhancer landscape upon treatment and after subsequent removal of retinoic acid was studied. Using ChIP-seq for the active histone mark H3K27ac, paired with RNA-seq, we compared the super-enhancer landscape in cells that undergo neuronal differentiation in response to retinoic acid versus those that fail to differentiate and identified unique super-enhancers associated with neuronal differentiation. Among the ADRN cells that respond to treatment, MYCN-amplified cells remain differentiated upon removal of retinoic acid, whereas MYCN non-amplified cells revert to an undifferentiated state, allowing for the identification of super-enhancers responsible for maintaining differentiation. This study identifies key super-enhancers that are crucial for retinoic acid-mediated differentiation.

Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation.

BACKGROUND: The pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an "on-target" pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme. RESULTS: Increasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles. CONCLUSIONS: Our findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.

Dietary Patterns and Associated Microbiome Changes that Promote Oncogenesis.

The recent increases in cancer incidences have been linked to lifestyle changes that result in obesity and metabolic syndrome. It is now evident that these trends are associated with the profound changes that occur in the intestinal microbiome, producing altered microbial population signatures that interact, directly or indirectly, with potentially pro-carcinogenic molecular pathways of transcription, proliferation, and inflammation. The effects of the entire gut microbial population on overall health are complex, but individual bacteria are known to play important and definable roles. Recent detailed examinations of a large number of subjects show a tight correlation between habitual diets, fecal microbiome signatures, and markers of metabolic health. Diets that score higher in healthfulness or diversity such as plant-based diets, have altered ratios of specific bacteria, including an increase in short-chain fatty acid producers, which in turn have been linked to improved metabolic markers and lowered cancer risk. Contrarily, numerous studies have implicated less healthy, lower-scoring diets such as the Western diet with reduced intestinal epithelial defenses and promotion of specific bacteria that affect carcinogenic pathways. In this review, we will describe how different dietary patterns affect microbial populations in the gut and illustrate the subsequent impact of bacterial products and metabolites on molecular pathways of cancer development, both locally in the gut and systemically in distant organs.

Dephosphorylation of the Proneural Transcription Factor ASCL1 Re-Engages a Latent Post-Mitotic Differentiation Program in Neuroblastoma.

Pediatric cancers often resemble trapped developmental intermediate states that fail to engage the normal differentiation program, typified by high-risk neuroblastoma arising from the developing sympathetic nervous system. Neuroblastoma cells resemble arrested neuroblasts trapped by a stable but aberrant epigenetic program controlled by sustained expression of a core transcriptional circuit of developmental regulators in conjunction with elevated MYCN or MYC (MYC). The transcription factor ASCL1 is a key master regulator in neuroblastoma and has oncogenic and tumor-suppressive activities in several other tumor types. Using functional mutational approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells drives coordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating an enduring gene program driving mitotic exit and neuronal differentiation. IMPLICATIONS: These findings indicate that targeting phosphorylation of ASCL1 may offer a new approach to development of differentiation therapies in neuroblastoma. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/12/1759/F1.large.jpg.

Cell cycle regulation of proliferation versus differentiation in the central nervous system.

Formation of the central nervous system requires a period of extensive progenitor cell proliferation, accompanied or closely followed by differentiation; the balance between these two processes in various regions of the central nervous system gives rise to differential growth and cellular diversity. The correlation between cell cycle lengthening and differentiation has been reported across several types of cell lineage and from diverse model organisms, both in vivo and in vitro. Furthermore, different cell fates might be determined during different phases of the preceding cell cycle, indicating direct cell cycle influences on both early lineage commitment and terminal cell fate decisions. Significant advances have been made in the last decade and have revealed multi-directional interactions between the molecular machinery regulating the processes of cell proliferation and neuronal differentiation. Here, we first introduce the modes of proliferation in neural progenitor cells and summarise evidence linking cell cycle length and neuronal differentiation. Second, we describe the manner in which components of the cell cycle machinery can have additional and, sometimes, cell-cycle-independent roles in directly regulating neurogenesis. Finally, we discuss the way that differentiation factors, such as proneural bHLH proteins, can promote either progenitor maintenance or differentiation according to the cellular environment. These intricate connections contribute to precise coordination and the ultimate division versus differentiation decision.

The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.

Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.

The SLC6A4 VNTR genotype determines transcription factor binding and epigenetic variation of this gene in response to cocaine in vitro.

We demonstrated that the genotype of the variable number tandem repeats (VNTRs) in the linked polymorphic region (LPR) of the 5' promoter and in the intron 2 (Stin2) transcriptional regulatory domains of the serotonin transporter SLC6A4 gene determined its promoter interactions with transcription factors and co-activators in response to cocaine in the JAr cell line. The LPR variants contain 14 (short, s) or 16 (long, l) copies of a 22-23 bp repeat element, whereas the Stin2 VNTR exists as three variants containing 9, 10 or 12 copies of a 16-17 bp repeat. We observed a differential effect of cocaine on the association of the promoter with the transcription factor CTCF, which bound to both LPR alleles prior to cocaine exposure but only to the l-allele following exposure. Significantly, this differential effect of cocaine was correlated with the binding of the transcriptional regulator MeCP2 specifically to the s-allele and recruiting the histone deacetylase complex (HDAC). Concurrently, cocaine increased the association of positive histone marks over the SLC6A4 gene locus. At the Stin2 domain, we lost binding of the transcription factor YB-1, while CTCF remained bound. Our biochemical data are consistent with differential reporter gene activity directed by the individual or dual domains in response to cocaine in an Epstein-Barr virus-based episome model of stable transfections. These observations suggest that exposure of JAr cells to cocaine may result in differential binding of transcription factors and activators based on a specific genotype that might alter epigenetic parameters affecting gene expression after the initial challenge.

Assessing the impact of genetic variation on transcriptional regulation in vitro.

Two alleles of a gene that contain polymorphic cis-regulatory regions can contribute differently to expression levels. Evolutionary changes in such cis-regulatory domains are believed to have participated in the cognitive evolution of H. sapiens as well as phenotypic diversity. There have been many studies that associate genetic variations to individual's susceptibility to behavioural and affective disorders. Cis-acting regulatory polymorphisms can effect gene expression at many levels, such as transcription, mRNA processing efficiency, pre-mRNA splicing, and mRNA stability. Trans-acting modulators (such as transcription factors) also play a major role in determining mRNA concentration of a specific allele. Several studies have demonstrated that VNTRs within various genes can support differential gene expression based on copy number and that the function of the VNTR as a transcriptional regulator can be modulated, in part, by transcription factors.A better understanding of the pathways regulating expression mediated by the VNTRs would complement clinical studies, demonstrating how these domains may be mechanistically involved in the progression of the disorder and may supply more defined targets for pharmaceutical intervention.

Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF.

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene (SLC6A4) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5'-promoter exists as short (s) and long (l) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.