Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival.

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.

A somatic mutation in moesin drives progression into acute myeloid leukemia.

Acute myeloid leukemia (AML) arises when leukemia-initiating cells, defined by a primary genetic lesion, acquire subsequent molecular changes whose cumulative effects bypass tumor suppression. The changes that underlie AML pathogenesis not only provide insights into the biology of transformation but also reveal novel therapeutic opportunities. However, backtracking these events in transformed human AML samples is challenging, if at all possible. Here, we approached this question using a murine in vivo model with an MLL-ENL fusion protein as a primary molecular event. Upon clonal transformation, we identified and extensively verified a recurrent codon-changing mutation (Arg295Cys) in the ERM protein moesin that markedly accelerated leukemogenesis. Human cancer-associated moesin mutations at the conserved arginine-295 residue similarly enhanced MLL-ENL-driven leukemogenesis. Mechanistically, the mutation interrupted the stability of moesin and conferred a neomorphic activity to the protein, which converged on enhanced extracellular signal-regulated kinase activity. Thereby, our studies demonstrate a critical role of ERM proteins in AML, with implications also for human cancer.

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm.

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.

In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

Insights into Bioinformatic Applications for Glycosylation: Instigating an Awakening towards Applying Glycoinformatic Resources for Cancer Diagnosis and Therapy.

Glycosylation plays a crucial role in various diseases and their etiology. This has led to a clear understanding on the functions of carbohydrates in cell communication, which eventually will result in novel therapeutic approaches for treatment of various disease. Glycomics has now become one among the top ten technologies that will change the future. The direct implication of glycosylation as a hallmark of cancer and for cancer therapy is well established. As in proteomics, where bioinformatics tools have led to revolutionary achievements, bioinformatics resources for glycosylation have improved its practical implication. Bioinformatics tools, algorithms and databases are a mandatory requirement to manage and successfully analyze large amount of glycobiological data generated from glycosylation studies. This review consolidates all the available tools and their applications in glycosylation research. The achievements made through the use of bioinformatics into glycosylation studies are also presented. The importance of glycosylation in cancer diagnosis and therapy is discussed and the gap in the application of widely available glyco-informatic tools for cancer research is highlighted. This review is expected to bring an awakening amongst glyco-informaticians as well as cancer biologists to bridge this gap, to exploit the available glyco-informatic tools for cancer.

Authenticating apple cider vinegar's home remedy claims: antibacterial, antifungal, antiviral properties and cytotoxicity aspect.

These are the days when one would go online first seeking home remedies before seeing a doctor. Apple cider vinegar (ACV) is one such popular yet scientifically under-validated remedy. Our results prove the unequivocal antimicrobial activity of ACV to be true at full strength concentrations. However, the activity cannot be generalised because although strong antibacterial activity was observed at 25% concentrations, in terms of antifungal activity, yeasts, especially Candida were found to be less susceptible. The antimicrobial/antioxidant properties are ascertained to the total phenolic contents of ACV, as confirmed by our characterisation of the bioactive compounds and antioxidant activity. When checking for its cytotoxicity, ACV exhibited toxicity even at concentrations as low as 0.7%. These results indicate that there is no question of generalising the idea of ACV usage, instead more in vitro and in vivo validations are necessary in order to precisely weigh the pros and cons of ACV.

The unequivocal preponderance of biocomputation in clinical virology.

Bioinformatics and computer based data simulation and modeling are captivating biological research, delivering great results already and promising to deliver more. As biological research is a complex, intricate, diverse field, any available support is gladly taken. With recent outbreaks and epidemics, pathogens are a constant threat to the global economy and security. Virus related plagues are somehow the most difficult to handle. Biocomputation has provided appreciable help in resolving clinical virology related issues. This review, for the first time, surveys the current status of the role of computation in virus related research. Advances made in the fields of clinical virology, antiviral drug design, viral immunology and viral oncology, through input from biocomputation, have been discussed. The amount of progress made and the software platforms available are consolidated in this review. The limitations of computation based methods are presented. Finally, the challenges facing the future of biocomputation in clinical virology are speculated upon.