Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of highrisk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.

Over the last decade, anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase (RTK), has been identified as a fusion partner in a diverse variety of translocation events resulting in oncogenic signaling in many different cancer types. In tumors where the full‐length ALK RTK itself is mutated, such as neuroblastoma, the picture regarding the role of ALK as an oncogenic driver is less clear. Neuroblastoma is a complex and heterogeneous tumor that arises from the neural crest derived peripheral nervous system. Although high‐risk neuroblastoma is rare, it often relapses and becomes refractory to treatment. Thus, neuroblastoma accounts for 10–15% of all childhood cancer deaths. Since most cases are in children under the age of 2, understanding the role and regulation of ALK during neural crest development is an important goal in addressing neuroblastoma tumorigenesis. An impressive array of tyrosine kinase inhibitors (TKIs) that act to inhibit ALK have been FDA approved for use in ALK‐driven cancers. ALK TKIs bind differently within the ATP‐binding pocket of the ALK kinase domain and have been associated with different resistance mutations within ALK itself that arise in response to therapeutic use, particularly in ALK‐fusion positive non‐small cell lung cancer (NSCLC). This patient population has highlighted the importance of considering the relevant ALK TKI to be used for a given ALK mutant variant. In this review, we discuss ALK in neuroblastoma, as well as the use of ALK TKIs and other strategies to inhibit tumor growth. Current efforts combining novel approaches and increasing our understanding of the oncogenic role of ALK in neuroblastoma are aimed at improving the efficacy of ALK TKIs as precision medicine options in the clinic.

The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

Activating anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system, we employed CRIPSR/Cas9, incorporating orthologs of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibited enhanced Alk signaling phenotypes, but unexpectedly depended on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains displayed hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We showed that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing, we identified perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of Alk-activating mutations to perturb specification and promote survival in neuronal lineages. This article has an associated First Person interview with the first author of the paper.

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Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.