A Simple and Scalable Zebrafish Model of Sonic Hedgehog Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ~30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe the first zebrafish model of SHH MB using CRISPR to mutate ptch1, the primary genetic driver in human SHH MB. These tumors rapidly arise adjacent to the valvula cerebelli and resemble human SHH MB by histology and comparative genomics. In addition, ptch1-deficient MB tumors with loss of tp53 have aggressive tumor histology and significantly worse survival outcomes, comparable to human patients. The simplicity and scalability of the ptch1 MB model makes it highly amenable to CRISPR-based genome editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the grk3 kinase as one such target.

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

The scaffolding function of LSD1/KDM1A reinforces a negative feedback loop to repress stem cell gene expression during primitive hematopoiesis.

Lsd1/Kdm1a functions both as a histone demethylase enzyme and as a scaffold for assembling chromatin modifier and transcription factor complexes to regulate gene expression. The relative contributions of Lsd1's demethylase and scaffolding functions during embryogenesis are not known. Here, we analyze two independent zebrafish lsd1/kdm1a mutant lines and show Lsd1 is required to repress primitive hematopoietic stem cell gene expression. Lsd1 rescue constructs containing point mutations that selectively abrogate its demethylase or scaffolding capacity demonstrate the scaffolding function of Lsd1, not its demethylase activity, is required for repression of gene expression in vivo. Lsd1's SNAG-binding domain mediates its scaffolding function and reinforces a negative feedback loop to repress the expression of SNAG-domain-containing genes during embryogenesis, including gfi1 and snai1/2. Our findings reveal a model in which the SNAG-binding and scaffolding function of Lsd1, and its associated negative feedback loop, provide transient and reversible regulation of gene expression during hematopoietic development.

GFI1 Cooperates with IKZF1/IKAROS to Activate Gene Expression in T-cell Acute Lymphoblastic Leukemia.

Growth factor independence-1 (GFI1) is a transcriptional repressor and master regulator of normal and malignant hematopoiesis. Repression by GFI1 is attributable to recruitment of LSD1-containing protein complexes via its SNAG domain. However, the full complement of GFI1 partners in transcriptional control is not known. We show that in T-acute lymphoblastic leukemia (ALL) cells, GFI1 and IKAROS are transcriptional partners that co-occupy regulatory regions of hallmark T-cell development genes. Transcriptional profiling reveals a subset of genes directly transactivated through the GFI1-IKAROS partnership. Among these is NOTCH3, a key factor in T-ALL pathogenesis. Surprisingly, NOTCH3 expression by GFI1 and IKAROS requires the GFI1 SNAG domain but occurs independent of SNAG-LSD1 binding. GFI1 variants deficient in LSD1 binding fail to activate NOTCH3, but conversely, small molecules that disrupt the SNAG-LSD1 interaction while leaving the SNAG primary structure intact stimulate NOTCH3 expression. These results identify a noncanonical transcriptional control mechanism in T-ALL which supports GFI1-mediated transactivation in partnership with IKAROS and suggest competition between LSD1-containing repressive complexes and others favoring transactivation. IMPLICATIONS: Combinatorial diversity and cooperation between DNA binding proteins and complexes assembled by them can direct context-dependent transcriptional outputs to control cell fate and may offer new insights for therapeutic targeting in cancer.

Pediatric Cancer Models in Zebrafish.

Pediatric cancer is a leading cause of death in children and adolescents. Improvements in pediatric cancer treatment that include the alleviation of long-term adverse effects require a deeper understanding of the genetic, epigenetic, and developmental factors driving these cancers. Here, we review how the unique attributes of the zebrafish model system in embryology, imaging, and scalability have been used to identify new mechanisms of tumor initiation, progression, and relapse and for drug discovery. We focus on zebrafish models of leukemias, neural tumors and sarcomas - the most common and difficult childhood cancers to treat.

Growth Factor Independence 1B-Mediated Transcriptional Repression and Lineage Allocation Require Lysine-Specific Demethylase 1-Dependent Recruitment of the BHC Complex.

Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B's obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis.

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.

Zebrafish as a model to study neuroblastoma development.

Neuroblastoma is a pediatric solid tumor arising from embryonic neural crest progenitor cells that normally generate the peripheral sympathetic nervous system. As such, the location of neuroblastoma tumors is correlated with the distribution of major post-ganglionic clusters throughout the sympathetic chain, with the highest incidence in the adrenal medulla or lumbar sympathetic ganglia (~65%). Neuroblastoma is an enigmatic tumor that can spontaneously regress with minimal treatment or become highly metastatic and develop resistance to aggressive treatments, including radiation and high-dose chemotherapy. Age of diagnosis, stage of disease and cellular and genetic features often predict whether the tumor will regress or advance to metastatic disease. Recent efforts using molecular and genomic technologies have allowed more accurate stratification of patients into low-, intermediate- and high-risk categories, thereby allowing for minimal intervention in low-risk patients and providing potential new therapeutic targets, such as the ALK receptor tyrosine kinase, for high-risk or relapsed patients. Despite these advances, the overall survival of high-risk neuroblastoma patients is still less than 50%. Furthermore, next-generation sequencing has revealed that almost two-thirds of neuroblastoma tumors do not contain obvious pathogenic mutations, suggesting that epigenetic mechanisms and/or a perturbed cellular microenvironment may heavily influence neuroblastoma development. Understanding the mechanisms that drive neuroblastoma, therefore, will likely require a combination of genomic, developmental and cancer biology approaches in whole animal systems. In this review, we discuss the contributions of zebrafish research to our understanding of neuroblastoma pathogenesis as well as the potential for this model system to accelerate the identification of more effective therapies for high-risk neuroblastoma patients in the future.

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.