Kinome profiling of non-Hodgkin lymphoma identifies Tyro3 as a therapeutic target in primary effusion lymphoma.

Non-Hodgkin lymphomas (NHLs) make up the majority of lymphoma diagnoses and represent a very diverse set of malignancies. We sought to identify kinases uniquely up-regulated in different NHL subtypes. Using multiplexed inhibitor bead-mass spectrometry (MIB/MS), we found Tyro3 was uniquely up-regulated and important for cell survival in primary effusion lymphoma (PEL), which is a viral lymphoma infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Tyro3 was also highly expressed in PEL cell lines as well as in primary PEL exudates. Based on this discovery, we developed an inhibitor against Tyro3 named UNC3810A, which hindered cell growth in PEL, but not in other NHL subtypes where Tyro3 was not highly expressed. UNC3810A also significantly inhibited tumor progression in a PEL xenograft mouse model that was not seen in a non-PEL NHL model. Taken together, our data suggest Tyro3 is a therapeutic target for PEL.

YaxAB, a Yersinia enterocolitica pore-forming toxin regulated by RovA.

The transcriptional regulator RovA positively regulates transcription of the Yersinia enterocolitica virulence gene inv. Invasin, encoded by inv, is important for establishment of Y. enterocolitica infection. However, a rovA mutant is more attenuated for virulence than an inv mutant, implying that RovA regulates additional virulence genes. When the Y. enterocolitica RovA regulon was defined by microarray analysis, YE1984 and YE1985 were among the genes identified as being upregulated by RovA. Since these genes are homologous to Xenorhabdus nematophila cytotoxin genes xaxA and xaxB, we named them yaxA and yaxB, respectively. In this work, we demonstrate the effects of YaxAB on the course of infection in the murine model. While a yaxAB mutant (ΔyaxAB) is capable of colonizing mice at the same level as the wild type, it slightly delays the course of infection and results in differing pathology in the spleen. Further, we found that yaxAB encode a probable cytotoxin capable of lysing mammalian cells, that both YaxA and YaxB are required for cytotoxic activity, and that the two proteins associate. YaxAB-mediated cell death occurs via osmotic lysis through the formation of distinct membrane pores. In silico tertiary structural analysis identified predicted structural homology between YaxA and proteins in pore-forming toxin complexes from Bacillus cereus (HBL-B) and Escherichia coli (HlyE). Thus, it appears that YaxAB function as virulence factors by inducing cell lysis through the formation of pores in the host cell membrane. This characterization of YaxAB supports the hypothesis that RovA regulates expression of multiple virulence factors in Y. enterocolitica.

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

Inhibition of Aurora A Kinase in Combination with Chemotherapy Induces Synthetic Lethality and Overcomes Chemoresistance in Myc-Overexpressing Lymphoma.

BACKGROUND: Aberrant Myc expression plays a critical role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-positive lymphoma is clinically aggressive, more resistant to chemotherapy, and associated with high mortality. OBJECTIVE: The current study aimed to show inhibition of aurora A kinase (AURKA) may overcome resistance to chemotherapy and improve outcomes in Myc-overexpressing lymphoma. METHODS: Myc-overexpressing lymphoma cell lines were evaluated by trypan blue, annexin V/propidium iodide staining, and western blotting for cytotoxicity, cell cycle, apoptosis, and Myc-associated protein expression, respectively, in the presence of cyclophosphamide with or without MLN8237, an AURKA inhibitor. Immunofluorescence for apoptosis-inducing factor (AIF) and acridine orange staining were used to analyze levels of autophagy. EµMyc genetically modified mouse model and xenograft models bearing Myc-overexpressing lymphoma cells were used to determine the efficacy of cyclophosphamide, MLN8237, or the combination in chemosensitive and chemoresistant tumors. RESULTS: In our in vitro experiments using chemoresistant lymphoma cells, MLN8237 and cyclophosphamide showed synergistic effects. Mice bearing lymphoma xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, the combination of cyclophosphamide and MLN8237 induced complete tumor regression in all mice, which led to improvement in survival compared with the single agent control (p = 0.022). Kinome analysis of tumors treated with MLN8237 showed global suppression of various kinases. CONCLUSION: Our data demonstrate that AURKA inhibition induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing lymphoma. The combination of MLN8237 and conventional chemotherapy showed promising safety and anti-tumor activities in preclinical models of Myc-positive NHL.

Bespoke Pretargeted Nanoradioimmunotherapy for the Treatment of Non-Hodgkin Lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common types of hematologic malignancies. Pretargeted radioimmunotherapy (PRIT), the sequential administration of a bispecific antibody-based primary tumor-targeting component followed by a radionucleotide-labeled treatment effector, has been developed to improve the treatment efficacy and to reduce the side effects of conventional RIT. Despite the preclinical success of PRIT, clinical trials revealed that the immunogenicity of the bispecific antibody as well as the presence of competing endogenous effector molecules often compromised the treatment. One strategy to improve PRIT is to utilize bio-orthogonal ligation reactions to minimize immunogenicity and improve targeting. Herein, we report a translatable pretargeted nanoradioimmunotherapy strategy for the treatment of NHL. This pretargeting system is composed of a dibenzylcyclooctyne (DBCO)-functionalized anti-CD20 antibody (α-CD20) tumor-targeting component and an azide- and yttrium-90-(90Y) dual-functionalized dendrimer. The physicochemical properties of both pretargeting components have been extensively studied. We demonstrated that an optimized dual-functionalized dendrimer can undergo rapid strain-promoted azide-alkyne cycloaddition with the DBCO-functionalized α-CD20 at the physiological conditions. The treatment effector in our pretargeting system can not only selectively deliver radionucleotides to the target tumor cells but also increase the complement-dependent cytotoxicity of α-CD20 and thus enhance the antitumor effects, as justified by comprehensive in vitro and in vivo studies in mouse NHL xenograft and disseminated models.

Differential IgM expression distinguishes two types of pediatric Burkitt lymphoma in mouse and human.

Endemic Burkitt lymphoma (eBL) is primarily a childhood cancer in parts of Africa and Brazil. Classic studies describe eBL as a homogeneous entity based on t(8;14) IgH-Myc translocation and clinical response to cytotoxic therapy. By contrast, sporadic BL (sBL) in Western countries is considered more heterogeneous, and affects both children and adults. It is overrepresented in AIDS patients. Unlike diffuse large B cell lymphoma (DLBCL), molecular subtypes within BL have not been well defined. We find that differential IgM positivity can be used to describe two subtypes of pediatric Burkitt lymphoma both in a high incidence region (Brazil), as well as in a sporadic region (US), suggesting the phenotype is not necessarily geographically isolated. Moreover, we find that IgM positivity also distinguishes between early and late onset BL in the standard Eµ-Myc mouse model of BL. This suggests that the t(8;14) translocation not only can take place before, but also after isotype switch recombination, and that IgM-negative, t(8;14) positive lymphomas in children should nevertheless be considered BL.

Discovery and Characterization of piRNAs in the Human Fetal Ovary.

Piwi-interacting RNAs (piRNAs), a class of 26- to 32-nt non-coding RNAs (ncRNAs), function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis-developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc.

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.

Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.

Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.

A mammalian microRNA expression atlas based on small RNA library sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.