Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs

MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology

Identification and characterization of new lymphoma-associated geneticaberrations involving protein tyrosine kinase loci

Despite major advances made over the last decades in the treatment of cancer, it is today still a leading cause of mortality worldwide. Cancer is the generic term for a group of diseases that are characterized by uncontrolled cell division, invasion and sometimes metastasis. Multiple cellular processes, including apoptosis, differentiation, cell cycle regulation and proliferation, may be disturbed in malignant neoplasms. Protein tyrosine kinases (PTKs) are important regulators of these processes. PTKs are signaling proteins that activate intracellular signal transduction pathways by phosphorylation of substrate proteins. Their deregulationis a key event in the transformation and progression of many human malignancies. The resulting constitutive kinase activity may be caused by several mechanisms such as gene amplification, the formation of fusion genes due to chromosomal rearrangements, and activating mutations. Although PTK aberrations have been identified and studied in several hematological and non-hematological malignancies, the precise mechanisms leading to defective tyrosine phosphorylation still need to be identified in a variety of cancers, including lymphoid neoplasms. In thisstudy, we focused on the characterization of previously unidentified PTK abnormalities in lymphoid malignancies. We discovered two lymphoma-associated fusion genes involving the PTKs ALK and JAK2, SEC31A-ALK and SEC31A-JAK2 , in ALK-expressing large B-cell lymphoma and classical Hodgkin lymphoma, respectively. Both genes result in fusion proteins important in malignant cell transformation and responsive to ALK- and JAK-specific tyrosine kinase inhibitors in vitro . We established a murine bone marrow transplant model for SEC31A-JAK2 and could demonstrate that this fusion protein induces either a T-lymphoblastic lymphoma or a myeloid hyperplasia in vivo . We further confirmed previous findings of amplification of the JAK2 /9p24 region in classical Hodgkin lymphoma and demonstrated the presence of JAK2 /9p24 amplicons in approximately 40% of analyzed cases. Whether this amplification preferentially targets JAK2 or activates other co-amplified genes, such as the programmed death ligand genes PDL1 ( CD274 ) and PDL2 ( PDCD1LG2 ), remains to be elucidated. In B-cell non-Hodgkin lymphomas, we identified a 9p24 breakpoint cluster in close vicinity, but outside the JAK2 gene. Our results indicate that, at least in a proportion of cases with 9p24 rearrangements, these aberrations may target PDL1 and/or PDL2 , leading to overexpression of these gene products. The mechanisms underlying thisoverexpression are diverse and possibly include the formation of a fusion gene, promoter exchange and juxtaposition with immunoglobulin enhancer elements. Further studies are required to precisely determine the roleof PDL1 and/or PDL2 overexpression in lymphomagenesis. A variety of B-cell non-Hodgkin lymphomas contained deletions of 9p24 sequences. We identified a commonly deleted region of 2Mb covering, among others, the tyrosine kinase gene JAK2 and two significantly downregulated genes, the transcription factor RFX3 and the microRNA hsa-mir-101-2. MicroRNAs are a recently identified class of non-coding RNAs that negatively regulate gene expression and that are often deregulated in cancer. Hsa-mir-101 is a microRNA with a known role in tumorigenesis. However, within the scope of this work, we were not able to identify consistent upregulation of any of the known targets of microRNA-101. The significance of the downregulation of microRNA-101 as well as RFX3 needs to be further explored. Given that cancer patients with aberrant expression of PTKs may benefit from targeted therapies with tyrosine kinase inhibitors, the identification and characterization of novel PTK abnormalities is clinically important and should continue to be a focus in cancer research.nrpages: 117status: publishe

PDS5A, a novel translocation partner of MLL in acute myeloid leukemia

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ALK-positive large B-cell lymphomas with cryptic SEC31A-ALK and NPM1-ALK fusions

We report 2 ALK-positive large B-cell lymphoma cases showing granular cytoplasmic and cytoplasmic/nuclear ALK immunostaining in which cryptic ALK rearrangements were identified by fluorescent in situ hybridization and molecular analysis. In the first case, the ALK-involving t(2;3)(p23;q27) masked the cryptic SEC31A-ALK fusion generated by an insertion of the 5′ end of SEC31A (4q21) upstream of the 3′ end of ALK. This rearrangement was associated with loss of the 5′ end of ALK and duplication of SEC31A-ALK on der(20). In the second case with complex rearrangements of both chromosomes 2, a submicroscopic NPM1-ALK fusion created by insertion of the 3′ end of ALK into the NPM1 locus was evidenced. Further studies of SEC31A-ALK showed that this variant fusion transforms IL3-dependent Ba/F3 cells to growth factor independence, and that the ALK inhibitor TAE-684 reduces cell proliferation and kinase activity of SEC31A-ALK and its downstream effectors ERK1/2, AKT, STAT3 and STAT5

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19

The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies