Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

Shared clonal cytogenetic abnormalities in aberrant mast cells and leukemic myeloid blasts detected by single nucleotide polymorphism microarray-based whole-genome scanning.

Systemic mastocytosis (SM) is characterized by a clonal proliferation of aberrant mast cells within extracutaneous sites. In a subset of SM cases, a second associated hematologic non-mast cell disease (AHNMD) is also present, usually of myeloid origin. Polymerase chain reaction and targeted fluorescence in situ hybridization studies have provided evidence that, in at least some cases, the aberrant mast cells are related clonally to the neoplastic cells of the AHNMD. In this work, a single nucleotide polymorphism microarray (SNP-A) was used to characterize the cytogenetics of the aberrant mast cells from a patient with acute myeloid leukemia and concomitant mast cell leukemia associated with a KIT D816A mutation. The results demonstrate the presence of shared cytogenetic abnormalities between the mast cells and myeloid blasts, as well as additional abnormalities within mast cells (copy-neutral loss of heterozygosity) not detectable by routine karyotypic analysis. To our knowledge, this work represents the first application of SNP-A whole-genome scanning to the detection of shared cytogenetic abnormalities between the two components of a case of SM-AHNMD. The findings provide additional evidence of a frequent clonal link between aberrant mast cells and cells of myeloid AHNMDs, and also highlight the importance of direct sequencing for identifying uncommon activating KIT mutations.

Metal-ion rescue revisited: biochemical detection of site-bound metal ions important for RNA folding.

Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage.

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme.

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK(a) = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5' oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2'-O nucleophile by G638 and protonation of the 5'-O leaving group by A756.

Synthesis, properties, and applications of oligonucleotides containing an RNA dinucleotide phosphorothiolate linkage.

RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3'- or 5'-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage. Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3'- or 5'-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3'- or 5'-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3'- and 5'-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities.

Automated solid-phase synthesis of RNA oligonucleotides containing a nonbridging phosphorodithioate linkage via phosphorothioamidites.

This work describes a general method for the synthesis of oligoribonucleotides containing a site-specific nonbridging phosphorodithioate linkage via automated solid-phase synthesis using 5'-O-DMTr-2'-O-TBS-ribonucleoside 3'-N,N-dimethyl-S-(2,4-dichlorobenzyl) phosphorothioamidites (2a-2d). The 3'-phosphorothioamidites (2a-2d) can be conveniently prepared in good yields (86-99%) via a one-pot reaction from the corresponding 5'-O-DMTr-2'-O-TBS-ribonucleosides (1a-1d).

Synthetic antibodies for specific recognition and crystallization of structured RNA.

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 A. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementarity-determining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.

Identification of catalytic metal ion ligands in ribozymes.

Site-bound metal ions participate in the catalytic mechanisms of many ribozymes. Understanding these mechanisms therefore requires knowledge of the specific ligands on both substrate and ribozyme that coordinate these catalytic metal ions. A number of different structural and biochemical strategies have been developed and refined for identifying metal ion binding sites within ribozymes, and for assessing the catalytic contributions of the metal ions bound at those sites. We review these approaches and provide examples of their application, focusing in particular on metal ion rescue experiments and their roles in the construction of the transition state models for the Tetrahymena group I and RNase P ribozymes.

Smoothened stabilizes and protects TRAF6 from degradation: A novel non-canonical role of smoothened with implications in lymphoma biology.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance.

Philadelphia Chromosome-like Mixed-Phenotype Acute Leukemia Demonstrating P2RY8-CRLF2 Fusion and JAK1 Mutation.

OBJECTIVES: Philadelphia chromosome-like (Ph-like) genetic alterations define a subset of B lymphoblastic leukemia/lymphoma (B-ALL), which represents a separate provisional entity in the World Health Organization 2016 updated classification. However, these alterations have not been described outside the context of B-ALL. METHODS: Cytogenomic array and molecular analysis identified a Ph-like signature in a mixed-phenotype acute leukemia (MPAL), B/myeloid, confirmed using conventional immunophenotypic and cytochemical analysis. RESULTS: Flow cytometry identified a blast population demonstrating a B-cell lineage and myeloperoxidase positivity. A P2RY8-CRLF2 fusion and JAK1 mutation were detected, both of which are associated with Ph-like features. CONCLUSIONS: To our knowledge, this is the first report of Ph-like MPAL, which may represent a new diagnostic entity. We emphasize the need for refinement of diagnostic criteria for MPALs and highlight an opportunity for expansion of inclusion criteria in ongoing clinical trials studying the use of tyrosine kinase inhibitor therapy to include cases of Ph-like MPAL.

Systematic review of the effectiveness of fine-needle aspiration and/or core needle biopsy for subclassifying lymphoma.

CONTEXT: The World Health Organization system for lymphoma classification relies on histologic findings from excisional biopsies. In contradistinction to expert guidelines, practitioners increasingly rely on fine-needle aspiration cytology and core needle biopsies rather than excisional biopsies to diagnose lymphomas. OBJECTIVE: To determine a rate at which fine-needle aspiration cytology and core needle biopsies, combined with flow cytometry and/or genetic techniques, can provide a diagnosis sufficient for optimal medical management of lymphoma. DATA SOURCES: The English-language literature on fine-needle aspiration cytology and core needle biopsies for lymphoma was reviewed to identify studies that provided interpretations of all specimens regardless of whether these were deemed diagnostic. CONCLUSIONS: Forty-two studies (1989-2012) specified the lymphoma subtypes for each diagnosis or indicated a rate at which the methods failed to provide a diagnosis. The median rate at which fine-needle aspiration cytology and core needle biopsies yielded a subtype-specific diagnosis of lymphoma was 74%. Strictly adhering to expert guidelines, which state that follicular lymphoma cannot be graded by these techniques, decreased the diagnostic yield further to 66%. Thus, 25% to 35% of fine-needle aspirates and/or core biopsies of nodes must be followed by an excisional lymph node biopsy to fully classify lymphoma.

Separation of RNA phosphorothioate oligonucleotides by HPLC.

Phosphorothioate oligonucleotides are indispensable tools for probing nucleic acid structure and function and for the design of antisense therapeutics. Many applications involving phosphorothioates require site- and stereospecific substitution of individual pro-R(P) or pro-S(P) nonbridging oxygens. However, the traditional approach to phosphorothioate synthesis produces a mixture of R(P) and S(P) diastereomers that must be separated prior to use. High-performance liquid chromatography (HPLC) has proven to be a versatile method for effecting this separation, with both reversed phase (RP) and strong anion exchange (SAX) protocols yielding favorable results. In this chapter, we present several examples of successful separations of RNA phosphorothioate diastereomers by HPLC. We also report the use of complementary DNA oligonucleotides for the separation of poorly resolved phosphorothioate RNAs.

Efficient synthesis of [2'-18O]uridine and its incorporation into oligonucleotides: a new tool for mechanistic study of nucleotidyl transfer reactions by isotope effect analysis.

Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2'-cyclouridine with [(18)O2]benzoic acid/potassium hydride, allowing an efficient "one-pot" synthesis of [2'-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2'-(18)O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases.