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.

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.

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.

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.

ASCL1 phosphorylation and ID2 upregulation are roadblocks to glioblastoma stem cell differentiation.

The growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine-Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.

Tracing oncogene-driven remodelling of the intestinal stem cell niche.

Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1-3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model-the Red2Onco system-that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones.

Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids.

Emerging three-dimensional (3D) cultures of glioblastoma are becoming powerful models to study glioblastoma stem cell behavior and the impact of cell-cell and cell-microenvironment interactions on tumor growth and invasion. Here we describe a method for culturing human glioblastoma stem cells (GSCs) in 3D by co-culturing them with pluripotent stem cell-derived brain organoids. This requires multiple coordinated steps, including the generation of cerebral organoids, and the growth and fluorescence tagging of GSCs. We highlight how to recognize optimal organoid generation and how to efficiently mark GSCs, before describing optimized co-culture conditions. We show that GSCs can efficiently integrate into brain organoids and maintain a significant degree of cell fate heterogeneity, paving the way for the analysis of GSC fate behavior and lineage progression. These results establish the 3D culture system as a viable and versatile GBM model for investigating tumor cell biology and GSC heterogeneity.This article has an associated First Person interview with the first author of the paper.

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.

Accelerating drug development for neuroblastoma: Summary of the Second Neuroblastoma Drug Development Strategy forum from Innovative Therapies for Children with Cancer and International Society of Paediatric Oncology Europe Neuroblastoma.

Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain. This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials. Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial. Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration. In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy.

Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells.

The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.

Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems.

Xenopus extract systems have been used to study ubiquitylation of proteins, and to uncover some of the fundamental processes of the ubiquitylation pathway itself. They provide a simple, quick, and robust method for studying ubiquitylation. In this protocol, methods are provided for studying protein ubiquitylation using Xenopus egg or embryo extracts and in vitro radiolabeled proteins. These methods also enable examination of whether proteins undergo noncanonical ubiquitylation, through modification of the protein by covalent linkage to ubiquitin through residues other than lysine, such as cysteine, serine, and threonine.

Xenopus Models of Cancer: Expanding the Oncologist's Toolbox.

The use of the Xenopus model system has provided diverse contributions to cancer research, not least because of the striking parallels between tumour pathogenesis and early embryo development. Cell cycle regulation, signalling pathways, and cell behaviours such as migration are frequently perturbed in cancers; all have been investigated using Xenopus, and these developmental events can additionally act as an assay for drug development studies. In this mini-review, we focus our discussion primarily on whole embryo Xenopus models informing cancer biology; the contributions to date and future potential. Insights into tumour immunity, oncogene function, and visualisation of vascular responses during tumour formation have all been achieved with naturally occurring tumours and induced-tumour-like-structures in Xenopus. Finally, as we are now entering the era of genetically modified Xenopus models, we can harness genome editing techniques to recapitulate human disease through creating embryos with analogous genetic abnormalities. With the speed, versatility and accessibility that epitomise the Xenopus system, this new range of pre-clinical Xenopus models has great potential to advance our mechanistic understanding of oncogenesis and provide an early in vivo model for chemotherapeutic development.

Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration.

The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism of, and requirement for, progenitor plasticity in mediating pathological response remain unknown. Here we show that phosphorylation of the transcription factor Atoh1 is required for both the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. As confirmed by lineage tracing, Atoh1+ cells (Atoh1(WT)CreERT2 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant Atoh1(9S/T-A)CreERT2 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis, Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenicity that affects overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium, and the balance between stem and progenitor fate is directly coordinated by ATOH1 multisite phosphorylation.

The developmental origin of brain tumours: a cellular and molecular framework.

The development of the nervous system relies on the coordinated regulation of stem cell self-renewal and differentiation. The discovery that brain tumours contain a subpopulation of cells with stem/progenitor characteristics that are capable of sustaining tumour growth has emphasized the importance of understanding the cellular dynamics and the molecular pathways regulating neural stem cell behaviour. By focusing on recent work on glioma and medulloblastoma, we review how lineage tracing contributed to dissecting the embryonic origin of brain tumours and how lineage-specific mechanisms that regulate stem cell behaviour in the embryo may be subverted in cancer to achieve uncontrolled proliferation and suppression of differentiation.

Subcellular localisation modulates ubiquitylation and degradation of Ascl1.

The proneural transcription factor Ascl1 is a master regulator of neurogenesis, coordinating proliferation and differentiation in the central nervous system. While its expression is well characterised, post-translational regulation is much less well understood. Here we demonstrate that a population of chromatin-bound Ascl1 can be found associated with short chains of ubiquitin while cytoplasmic Ascl1 harbours much longer ubiquitin chains. Only cytoplasmic ubiquitylation targets Ascl1 for destruction, which occurs by conjugation of ubiquitin to lysines in the basic helix-loop-helix domain of Ascl1 and requires the E3 ligase Huwe1. In contrast, chromatin-bound Ascl1 associated with short ubiquitin-chains, which can occur on lysines within the N-terminal region or the bHLH domain and is not mediated by Huwe1, is not targeted for ubiquitin-mediated destruction. We therefore offer further insights into post-translational regulation of Ascl1, highlighting complex regulation of ubiquitylation and degradation in the cytoplasm and on chromatin.

Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma.

Neuroblastoma (NB), although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs) and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1 in its multiply phosphorylated state might prevent terminal differentiation in NB, which could offer new approaches for differentiation therapy in NB.

An oncologist׳s friend: How Xenopus contributes to cancer research.

One of the most striking features of the Xenopus system is the versatility in providing a unique range of both in vitro and in vivo models that are rapid, accessible and easily manipulated. Here we present an overview of the diverse contribution that Xenopus has made to advance our understanding of tumour biology and behaviour; a contribution that goes beyond the traditional view of Xenopus as a developmental model organism. From the utility of the egg and oocyte extract system to the use of whole embryos as developmental or induced tumour models, the Xenopus system has been fundamental to investigation of cell cycle mechanisms, cell metabolism, cell signalling and cell behaviour, and has allowed an increasing appreciation of the parallels between early development and the pathogenesis of tumour progression and metastasis. Although not the prototypical oncological model system, we propose that Xenopus is an adaptable and multifunctional tool in the oncologist׳s arsenal.

Non-canonical ubiquitylation: mechanisms and consequences.

Post-translational protein modifications initiate, regulate, propagate and terminate a wide variety of processes in cells, and in particular, ubiquitylation targets substrate proteins for degradation, subcellular translocation, cell signaling and multiple other cellular events. Modification of substrate proteins is widely observed to occur via covalent linkages of ubiquitin to the amine groups of lysine side-chains. However, in recent years several new modes of ubiquitin chain attachment have emerged. For instance, covalent modification of non-lysine sites in substrate proteins is theoretically possible according to basic chemical principles underlying the ubiquitylation process, and evidence is building that sites such as the N-terminal amine group of a protein, the hydroxyl group of serine and threonine residues and even the thiol groups of cysteine residues are all employed as sites of ubiquitylation. However, the potential importance of this "non-canonical ubiquitylation" of substrate proteins on sites other than lysine residues has been largely overlooked. This review aims to highlight the unusual features of the process of non-canonical ubiquitylation and the consequences of these events on the activity and fate of a protein.

The cell cycle and pluripotency.

PSCs (pluripotent stem cells) possess two key properties that have made them the focus of global research efforts in regenerative medicine: they have unlimited expansion potential under conditions which favour their preservation as PSCs and they have the ability to generate all somatic cell types upon differentiation (pluripotency). Conditions have been defined in vitro in which pluripotency is maintained, or else differentiation is favoured and is directed towards specific somatic cell types. However, an unanswered question is whether or not the core cell cycle machinery directly regulates the pluripotency and differentiation properties of PSCs. If so, then manipulation of the cell cycle may represent an additional tool by which in vitro maintenance or differentiation of PSCs may be controlled in regenerative medicine. The present review aims to summarize our current understanding of links between the core cell cycle machinery and the maintenance of pluripotency in ESCs (embryonic stem cells) and iPSCs (induced PSCs).