A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk -/-mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk -/-HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.

Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel geneedisease and variantedisease associations are expected to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories. (J Mol Diagn 2019, 21: 38e48; https://doi.

Hematopoietic stem and progenitor cell (HSPC) expansion is regulated by intrinsic signaling pathways activated by cytokines. The intracellular kinase JAK2 plays an essential role in cytokine signaling, and activating mutations in JAK2 are found in a number of hematologic malignancies. We previously demonstrated that lymphocyte adaptor protein (Lnk, also known as Sh2b3) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion. Here we show that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced myeloproliferative diseases (MPDs) in mice. Specifically, Lnk deficiency enhanced cytokine-independent JAK/STAT signaling and augmented the ability of oncogenic JAK2 to expand myeloid progenitors in vitro and in vivo. An activated form of JAK2, unable to bind Lnk, caused greater myeloid expansion than activated JAK2 alone and accelerated myelofibrosis, indicating that Lnk directly inhibits oncogenic JAK2 in constraining MPD development. In addition, Lnk deficiency cooperated with the BCR/ABL oncogene, the product of which does not directly interact with or depend on JAK2 or Lnk, in chronic myeloid leukemia (CML) development, suggesting that Lnk also acts through endogenous pathways to constrain HSPCs. Consistent with this idea, aged Lnk -/-mice spontaneously developed a CML-like MPD. Taken together, our data establish Lnk as a bona fide suppressor of MPD in mice and raise the possibility that Lnk dysfunction contributes to the development of hematologic malignancies in humans.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL commonly associated with alterations that affect the tyrosine kinase pathway, tumor suppressors, and lymphoid transcription factors. Loss-of-function mutations in the gene-encoding adaptor protein LNK (also known as SH2B3) are found in Ph-like ALLs; however, it is not clear how LNK regulates normal B cell development or promotes leukemogenesis. Here, we have shown that combined loss of Lnk and tumor suppressors Tp53 or Ink4a/Arf in mice triggers a highly aggressive and transplantable precursor B-ALL. Tp53-/-Lnk-/- B-ALLs displayed similar gene expression profiles to human Ph-like B-ALLs, supporting use of this model for preclinical and molecular studies. Preleukemic Tp53-/-Lnk-/- pro-B progenitors were hypersensitive to IL-7, exhibited marked self-renewal in vitro and in vivo, and were able to initiate B-ALL in transplant recipients. Mechanistically, we demonstrated that LNK regulates pro-B progenitor homeostasis by attenuating IL-7-stimuated JAK/STAT5 signaling via a direct interaction with phosphorylated JAK3. Moreover, JAK inhibitors were effective in prolonging survival of mice transplanted with Lnk-/-Tp53-/- leukemia. Additionally, synergistic administration of PI3K/mTOR and JAK inhibitors further abrogated leukemia development. Hence, our results suggest that LNK suppresses IL-7R/JAK/STAT signaling to restrict pro-/pre-B progenitor expansion and leukemia development, providing a pathogenic mechanism and a potential therapeutic approach for B-ALLs with LNK mutations.

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. The adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs (APPL) 5 was first identified as an AKT2-interacting protein by yeast two-hybrid screening (1). APPL1, also dubbed DIP13␣ (DCC-interacting protein 13␣) (2), comprises three domains, i.e. Bar, PH, and PTB motifs. Whereas all of these domains can bind to lipids, each has unique binding preferences, which theoretically enables APPL to bind to various signaling proteins. The membrane binding-bending feature of the APPL protein permits it to be trafficked among many subcellular compartments (3). Moreover, the APPL Bar domain interacts with its own PH domain to form a unique Bar-PH structure that distinguishes APPL from other Bar domain-containing molecules (4). We previously showed that APPL1 can bind to AKT2 through its PTB domain (1), and subsequent co-immunoprecipitation studies showed that APPL1 also binds to AKT1, AKT3, and the p110 catalytic subunit of phosphatidylinositol 3-kinase. APPL1 does not bind to phosphorylated (active) AKT, and our initial report did not ascertain whether binding to APPL1 affects AKT activity (1). Subsequent work identified a second APPL protein, APPL2, which shares a high homology with APPL1 as well as some of the same binding partners (5).The function of APPL proteins was first demonstrated in HeLa cells, where APPL1 and APPL2 were found to represent key signaling links from the endosome to the nucleus by switching and activating binding partners from the small GTPase Rab5 on endosomes to the nucleosome remodeling and histone deacetylase multiprotein complex NuRD-MeCP1. Furthermore, knock down of APPL protein inhibited DNA synthesis and resulted in cell cycle arrest (6). Structural analysis revealed that APPL binds to Rab5 mainly through its PH domain, but the Bar domain at the other side of the dimer also binds to Rab5 (7). A broader role for APPL in signal transduction was soon discovered when various groups found that APPL proteins are implicated in nerve growth factor and follicle stimulating hormone signaling, as well as in lipid and glucose metabolism. Two groups independently reported that APPL1 tethers GIPC1 to * This work was supported, in whole or in part, by National Institutes of Health Grants CA77429 (to J. R. T.) and CA06927 (to Fox Chase Cancer Center) from the NCI and an appropriation from the Commonwealth of Pennsylvania. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedur...

Dysregulated lipid metabolism represents an important metabolic alteration in cancer. Fatty acids, cholesterol, and phospholipid are the three most prevalent lipids that act as energy producers, signaling molecules, and source material for the biogenesis of cell membranes. The enhanced synthesis, storage, and uptake of lipids contribute to cancer progression. The rewiring of lipid metabolism in cancer has been linked to the activation of oncogenic signaling pathways and cross talk with the tumor microenvironment. The resulting activity favors the survival and proliferation of tumor cells in the harsh conditions within the tumor. Lipid metabolism also plays a vital role in tumor immunogenicity via effects on the function of the noncancer cells within the tumor microenvironment, especially immune‐associated cells. Targeting altered lipid metabolism pathways has shown potential as a promising anticancer therapy. Here, we review recent evidence implicating the contribution of lipid metabolic reprogramming in cancer to cancer progression, and discuss the molecular mechanisms underlying lipid metabolism rewiring in cancer, and potential therapeutic strategies directed toward lipid metabolism in cancer. This review sheds new light to fully understanding of the role of lipid metabolic reprogramming in the context of cancer and provides valuable clues on therapeutic strategies targeting lipid metabolism in cancer.

Abstract:Chromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization.We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb. One Sentence Summary:Super-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.. CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/374058 doi: bioRxiv preprint first posted online Jul. 28, 2018; 4 Main text:In terms of organization, chromosomal DNA is among the most challenging of biological molecules to study. It is massive, differs in sequence and structure from one region to the next, and often comes in pairs of homologs that defy easy distinction. Nevertheless, much progress has been made via genetic and epigenetic analyses, including DNA-DNA proximity ligation assays, such as Hi-C and other Chromosome Conformation Capture technologies (1-8), as well as three-dimensional (3D) modeling (reviewed by (9, 10)). Recently, researchers have walked along chromosomes using widefield microscopy and fluorescent in situ hybridization (FISH) probes in rounds of hybridization (11), wherein each round targeted the central portion of a Hi-C defined self-interacting contact domain, also known as a topologically associating domain, or TAD (3-7). This strategy revealed the overall path of chromosomes, which was then used as a guideline for Hi-C contact-based modeling (bioRxiv, https://doi.org/10.1101/318493 May 2018).Other studies, using super-resolution microscopy, have explored a multitude of genomic features (12-18), some lending support for a physical basis for contact domains (15,18). What remains relatively unexplored, is the physical nature of compartmental intervals, that is, the contiguous chromosomal regions whose loci share long-range contact patterns and collectively, generate the plaid "compartment" pattern often seen extending above and below the Hi-C diagonal (3).Here, we present a method for visualizing chromosomal regions at super-resolution and then integrating the images with Hi-C interaction matrices via a data-driven computational protocol to produce 3D models of chromosome organization at the level...