Genetic predisposition to neuroblastoma results from a regulatory polymorphism that promotes the adrenergic cell state.

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.

Genetic Predisposition to Neuroblastoma Results from a Regulatory Polymorphism that Promotes the Adrenergic Cell State.

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like "mesenchymal" cell state and a more differentiated sympathetic "adrenergic" cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional co-factor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism G/T that affects a G ATA motif in the first intron of LMO1. Here we show that wild-type zebrafish with the G ATA genotype develop adrenergic neuroblastoma, while knock-in of the protective T ATA allele at this locus reduces the penetrance of MYCN-driven tumors, which are restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrates that T ATA/ T ATA tumors also exhibit a mesenchymal cell state and are low risk at diagnosis. Thus, conversion of the regulatory G ATA to a T ATA allele in the first intron of LMO1 reduces the neuroblastoma initiation rate by preventing formation of the adrenergic cell state, a mechanism that is conserved over 400 million years of evolution separating zebrafish and humans.

Disruption of asxl1 results in myeloproliferative neoplasms in zebrafish.

Somatic loss-of-function mutations of the additional sex combs-like transcriptional regulator 1 (ASXL1) gene are common genetic abnormalities in human myeloid malignancies and induce clonal expansion of mutated hematopoietic stem cells (HSCs). To understand how ASXL1 disruption leads to myeloid cell transformation, we generated asxl1 haploinsufficient and null zebrafish lines using genome-editing technology. Here, we show that homozygous loss of asxl1 leads to apoptosis of newly formed HSCs. Apoptosis occurred via the mitochondrial apoptotic pathway mediated by upregulation of bim and bid Half of the asxl1+/- zebrafish had myeloproliferative neoplasms (MPNs) by 5 months of age. Heterozygous loss of asxl1 combined with heterozygous loss of tet2 led to a more penetrant MPN phenotype, while heterozygous loss of asxl1 combined with complete loss of tet2 led to acute myeloid leukemia (AML). These findings support the use of asxl1+/- zebrafish as a strategy to identify small-molecule drugs to suppress the growth of asxl1 mutant but not wild-type HSCs in individuals with somatically acquired inactivating mutations of ASXL1.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis.

Uromodulin-associated kidney disease (UAKD) is caused by mutations in the uromodulin (UMOD) gene that result in a misfolded form of UMOD protein, which is normally secreted by nephrons. In UAKD patients, mutant UMOD is poorly secreted and accumulates in the ER of distal kidney epithelium, but its role in disease progression is largely unknown. Here, we modeled UMOD accumulation in mice by expressing the murine equivalent of the human UMOD p.Cys148Trp point mutation (UmodC147W/+ mice). Like affected humans, these UmodC147W/+ mice developed spontaneous and progressive kidney disease with organ failure over 24 weeks. Analysis of diseased kidneys and purified UMOD-producing cells revealed early activation of the PKR-like ER kinase/activating transcription factor 4 (PERK/ATF4) ER stress pathway, innate immune mediators, and increased apoptotic signaling, including caspase-3 activation. Unexpectedly, we also detected autophagy deficiency. Human cells expressing UMOD p.Cys147Trp recapitulated the findings in UmodC147W/+ mice, and autophagy activation with mTOR inhibitors stimulated the intracellular removal of aggregated mutant UMOD. Human cells producing mutant UMOD were susceptible to TNF-α- and TRAIL-mediated apoptosis due to increased expression of the ER stress mediator tribbles-3. Blocking TNF-α in vivo with the soluble recombinant fusion protein TNFR:Fc slowed disease progression in UmodC147W/+ mice by reducing active caspase-3, thereby preventing tubule cell death and loss of epithelial function. These findings reveal a targetable mechanism for disease processes involved in UAKD.

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage.

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.

IκB kinase ß (IKBKB) mutations in lymphomas that constitutively activate canonical nuclear factor κB (NFκB) signaling.

Somatic mutations altering lysine 171 of the IKBKB gene that encodes (IKKß), the critical activating kinase in canonical (NFκB) signaling, have been described in splenic marginal zone lymphomas and multiple myeloma. Lysine 171 forms part of a cationic pocket that interacts with the activation loop phosphate in the activated wild type kinase. We show here that K171E IKKß and K171T IKKß represent kinases that are constitutively active even in the absence of activation loop phosphorylation. Predictive modeling and biochemical studies establish why mutations in a positively charged residue in the cationic pocket of an activation loop phosphorylation-dependent kinase result in constitutive activation. Transcription activator-like effector nuclease-based knock-in mutagenesis provides evidence from a B lymphoid context that K171E IKKß contributes to lymphomagenesis.

Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins.

Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described-one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

Robust, synergistic regulation of human gene expression using TALE activators.

Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

FLASH assembly of TALENs for high-throughput genome editing.

Engineered transcription activator­like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell­based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.