Sergey Nemzer

Widespread occurrence of antisense transcription in the human genome

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that ≥60% of this data set, or ∼1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.

Systematic identification of abundant A-to-I editing sites in the human transcriptome

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

Is abundant A-to-I RNA editing primate-specific?

A-to-I RNA editing is common in all eukaryotes, and is associated with various neurological functions. Recently, A-to-I editing was found to occur frequently in the human transcriptome. In this article, we show that the frequency of A-to-I editing in humans is at least an order of magnitude higher than in the mouse, rat, chicken or fly genomes. The extraordinary frequency of RNA editing in human is explained by the dominance of the primate-specific Alu element in the human transcriptome, which increases the number of double-stranded RNA substrates.

Systematic antigenic profiling of hematopoietic antigens on ovarian carcinoma cells identifies membrane proteins for targeted therapy development

OBJECTIVE We studied ovarian cancers for the expression of membrane markers of hematopoietic origin. STUDY DESIGN We used flow cytometry to systematically characterize the expression of more than 30 hematologic antigens on ovarian carcinoma cell lines and to assess their stability under estrogen exposure. The expression of the antigens was validated by a bioinformatics survey and immunohistochemical staining of ovarian cancer specimens. RESULTS Several antigens were expressed by the majority of the cells, such as CD15, CD71, and CD138, whereas others were found on small and distinct cellular subpopulations. The expression patterns of the different markers were unaffected by estrogen exposure, indicating their stability. CONCLUSION The antigens described in our work may serve as potential targets for new and existing targeted drugs.

Abstract B291: Identification of novel immune checkpoints and their implementation as mAb targets for cancer immunotherapy.

Immune checkpoints, such as CTLA4 and PD-1, have emerged as promising drug targets for cancer immunotherapy. We hypothesize that additional novel members of the B7/CD28 family play a role in T cell regulation and thus may serve as targets for therapeutic mAbs. However, the discovery of novel family members is challenging since proteins of the immune system, including proteins of the B7 protein In order to identify novel members of the B7/CD28 protein family, Compugen has developed a discovery approach integrating gene and protein information with extensive expression data, and has identified nine novel membrane proteins that possess characteristics of the B7/CD28 protein family members and are therefore predicted to play a role in T cell co-stimulation. In order to validate our predictive discovery findings, we evaluated the effect of our proteins on immune cells, particularly T cells. For that goal, we expressed the proteins on the cell surface upon ectopic expression, and also produced fusion proteins consisting of the extracellular domain of the predicted proteins, fused to an IgG Fc domain. Here we present results obtained for two of our novel proteins, CGEN-15001T and CGEN-15022. Both display robust inhibition of T cell activation. Interestingly, CGEN-15001, one of the Fc-fused proteins, leads to increased levels of anti-inflammatory cytokines such as IL-4 and IL5, while reducing pro-inflammatory cytokines such as IFN-γ and IL-17. In addition, CGEN-15001 was found to enhance iTregs differentiation. Furthermore, these molecules showed therapeutic efficacy in mouse models of multiple sclerosis and rheumatoid arthritis. To investigate the potential of these membrane proteins as drug targets for treatment of cancer we have performed extensive IHC studies, on a variety of healthy and malignant tissues. Both CGEN-15001T and CGEN-15022 were found to be expressed in numerous types of cancers, each showing a unique pattern of expression. CGEN-15001T, in addition to its expression on tumor cells, was found to be expressed on tumor infiltrating immune cells, especially on Macrophages and Mast cells. Based on the immunomodulatory activities and the expression pattern on malignant and immune cells, CGEN15001T and CGEN15022 may serve as mAb targets for cancer immunotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B291. Citation Format: Gady Cojocaru, Ofer Levy, Amir Toporik, Liat Dassa, Iris Hecht, Ilan Vaknin, Sergey Nemzer, Tania Pergan, Amit Novik, Shirley Sameah-Greenwald, Anat Oren, Zohar Tiran, Peter Steinberger, Joseph Podojil, Nora Tarcic, Eyal Neria, Galit Rotman, Zurit Levine. Identification of novel immune checkpoints and their implementation as mAb targets for cancer immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B291.