Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis. Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2^{Kip2} targeting

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27$^{Kip1}$ and p57$^{Kip2}$, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS

Zebrafish as a Model for Cancer Self-Renewal

Self-renewal is the process by which normal stem cells and cancer cells make more of themselves. In cancer, this process is ultimately responsible for the infinite replicative potential of malignant cells and is likely found in residual cell populations that evade conventional therapy. Two intrinsically opposing hypotheses have emerged to explain how self-renewal occurs in cancer. The cancer stem cell hypothesis states that self-renewal is confined to a discrete subpopulation of malignant cells, whereas the stochastic model suggests that all tumor cells have the potential to self-renew. Presently, the gold standard for measuring cancer self-renewal is limiting dilution cell transplantation into immune-matched or immune-deficient animals. From these experiments, tumor-initiating frequency can be calculated based on the number of animals that engraft disease following transplantation of various doses of tumor cells. Here, we describe how self-renewal assays are performed, summarize the current experimental models that support the cancer stem cell and stochastic models of cancer self-renewal, and enumerate how the zebrafish can be used to uncover important pathways in cancer self-renewal

Distinct Functional and Temporal Requirements for Zebrafish <i>Hdac1</i> during Neural Crest-Derived Craniofacial and Peripheral Neuron Development

<div><p>The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In <i>hdac1^b382</i> mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer <i>hoxb3a, dlx2</i> and <i>dlx3-</i>expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in <i>hdac1^b382</i> mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in <i>hdac1^b382</i> mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce <i>hdac1^b382</i> defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish <i>hdac1</i> during neural crest-derived craniofacial and peripheral neuron development.</p></div

Craniofacial progenitor differentiation defects in the mandibular and hyoid arches of <i>hdac1^b382</i>mutants.

<p>A, C, E, G wild-type, B, D, F, H <i>hdac1^b382</i> mutants; A, B Lateral views of the head with <i>dlx2</i> expression labeling developing jaw elements in 48 hpf embryos. C, D, Lateral views of the head with <i>dlx3</i> expression labeling developing jaw elements in 48 hpf embryos. E, F ventral views of the head in 68 hpf embryos expressing <i>col2a1</i> in different jaw structures, <i>col2a1</i>. G, H ventral views of the head of 68 hpf embryos expressing <i>sox9a</i> in different jaw elements. M, mandibular; H, hyoid; BA, branchial arches.</p

Temporal requirements of HDAC function during craniofacial development.

<p>Embryos were assigned the following groups based on the severity of craniofacial malformations when compared to wild-type embryos;+++ wild-type, +++/− Mild malformation,++ Moderate malformation,+ Severe malformation, - Jaw elements absent or not stained.</p><p>Percentages reflect proportion of embryos with alcian blue stained cartilage elements to the total number of treated embryos expressed as a percentage.</p

Craniofacial defects in <i>hdac1^b382</i> mutants.

<p>A–E alcian blue stained jaw elements in 5 dpf wild type A, C, E and <i>hdac1^b382</i> mutants B, D, F; A, B Ventral view of dissected craniofacial cartilages of wild-type <i>and hdac1^b382</i> mutant; C, D lateral view of head region in wild-type and <i>hdac1^b382</i> mutant; E,F, High magnification of the mandibular chondrocytes (arrows) in wild-type and <i>hdac1^b382</i> mutant; m, meckels; pq, platoquadrate; M, mandibular; ch, ceratohyal; hs, hyosymplectic; H, hyoid; cb1-5, ceratobrachials 1-5; BA, branchial arches.</p

Effect of 800 nM of TSA on sympathetic neuron <i>th</i> expression.

<p>Percentages reflect the proportion of embryos with <i>th</i>-expression to the total number of treated embryos expressed as a percentage.</p

Treatment with the HDAC inhibitor TSA can reproduce the <i>hdac1^b382</i> mutant phenotype.

<p>A–d Lateral views of wild-type embryos treated with DMSO, 400 nM, 600 nM and 800 nM TSA from 16–24 hpf after which embryos were fixed and stained for <i>dlx2</i> expression. A–D and A’–D’ Alcian blue stained 3.5 dpf wild-type embryos under different TSA treatment conditions, all embryos were treated between 16 hpf and 3.5 dpf after which embryos were fixed and then stained with alcian blue; A–A’ DMSO controls, B–B’ 400 nM TSA, C–C’ 600 nM TSA, D–D’ 800 nM TSA. A–D lateral view; A’–D’ ventral views. A’’–D’’ schematic with summary of craniofacial defects at different TSA treatment conditions. M, mandibular; H, hyoid; cb1-5, cerato-branchials 1-5; BA, branchial arches,+++ wild-type,++ reduced in size compared to wild-type,+severely reduced compared to wild-type, +/− severely reduced or absent altogether.</p