Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2008; 105(28):9751-6. DOI: 10.1073/pnas.0710413105
Source: PubMed


The discovery of new small molecules and their testing in rational combination poses an ongoing problem for rare diseases, in particular, for pediatric cancers such as neuroblastoma. Despite maximal cytotoxic therapy with double autologous stem cell transplantation, outcome remains poor for children with high-stage disease. Because differentiation is aberrant in this malignancy, compounds that modulate transcription, such as histone deacetylase (HDAC) inhibitors, are of particular interest. However, as single agents, HDAC inhibitors have had limited efficacy. In the present study, we use an HDAC inhibitor as an enhancer to screen a small-molecule library for compounds inducing neuroblastoma maturation. To quantify differentiation, we use an enabling gene expression-based screening strategy. The top hit identified in the screen was all-trans-retinoic acid. Secondary assays confirmed greater neuroblastoma differentiation with the combination of an HDAC inhibitor and a retinoid versus either alone. Furthermore, effects of combination therapy were synergistic with respect to inhibition of cellular viability and induction of apoptosis. In a xenograft model of neuroblastoma, animals treated with combination therapy had the longest survival. This work suggests that testing of an HDAC inhibitor and retinoid in combination is warranted for children with neuroblastoma and demonstrates the success of a signature-based screening approach to prioritize compound combinations for testing in rare diseases.

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Available from: Renee D. Wright-Michaud, Oct 10, 2015
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    • "Inducing NB cells to differentiate is an important therapeutic approach that seems to be particularly promising for NBs [44]. Administration of VPA to NB cells such as Neuro2a, SHSY-5Y and BE (2)-C in vitro leads to proliferative arrest and neuronal differentiation [20], [45], [46]. Very recently, a whole-genome sequence analysis of 87 neuroblastomas reveals novel molecular defects in neurite genesis genes including a series of regulators of the Rac/Rho pathway, which frequently occur in high-risk tumours [47]. "
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    ABSTRACT: The inverse F-BAR (IF-BAR) domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA)-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1R542A and srGAP2R527A exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.
    PLoS ONE 03/2013; 8(3). DOI:10.1371/journal.pone.0057865 · 3.23 Impact Factor
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    • "These drug combinations are pharmaco-dynamically synergistic, additive or antagonistic as their effects are greater than, equal to, or less than the summed effects of individual drugs, respectively [8]. These models have garnered interest in the possibility of effective combinatorial drug discovery based on systems-oriented approaches [9]–[13]. "
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    ABSTRACT: Anticancer therapies that target single signal transduction pathways often fail to prevent proliferation of cancer cells because of overlapping functions and cross-talk between different signaling pathways. Recent research has identified that balanced multi-component therapies might be more efficacious than highly specific single component therapies in certain cases. Ideally, synergistic combinations can provide 1) increased efficacy of the therapeutic effect 2) reduced toxicity as a result of decreased dosage providing equivalent or increased efficacy 3) the avoidance or delayed onset of drug resistance. Therefore, the interest in combinatorial drug discovery based on systems-oriented approaches has been increasing steadily in recent years. Here we describe the development of Combinatorial Drug Assembler (CDA), a genomics and bioinformatics system, whereby using gene expression profiling, multiple signaling pathways are targeted for combinatorial drug discovery. CDA performs expression pattern matching of signaling pathway components to compare genes expressed in an input cell line (or patient sample data), with expression patterns in cell lines treated with different small molecules. Then it detects best pattern matching combinatorial drug pairs across the input gene set-related signaling pathways to detect where gene expression patterns overlap and those predicted drug pairs could likely be applied as combination therapy. We carried out in vitro validations on non-small cell lung cancer cells and triple-negative breast cancer (TNBC) cells. We found two combinatorial drug pairs that showed synergistic effect on lung cancer cells. Furthermore, we also observed that halofantrine and vinblastine were synergistic on TNBC cells. CDA provides a new way for rational drug combination. Together with phExplorer, CDA also provides functional insights into combinatorial drugs. CDA is freely available at
    PLoS ONE 08/2012; 7(8):e42573. DOI:10.1371/journal.pone.0042573 · 3.23 Impact Factor
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    • "BE(2)-C cells treated with RA display morphologic changes characteristic of neuronal differentiation with extensive outgrowth of neurites [25], [26], [27]. At the molecular level, RA-induced neuronal differentiation is characterized by downregulation of progenitor cell markers, for example, paired-like homeobox 2B (PHOX2B) [25], [35], and upregulation of neuronal differentiation markers, such as neurofilament medium polypeptide (NEFM) [25], [36]. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 in BE(2)-C cells also led to morphologic changes of neuronal differentiation with marked neurite outgrowth (Figure 4 A). "
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    ABSTRACT: Retinoic acid (RA) can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13) that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2)-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1) or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13) on BE(2)-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.
    PLoS ONE 08/2012; 7(8):e40728. DOI:10.1371/journal.pone.0040728 · 3.23 Impact Factor
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