Preface

Previously, my laboratory has concentrated on isolation of human DNA repair genes, and in particular checkpoint genes for DNA damage. For this purpose we introduced the approach of isolating human DNA repair genes by expression cloning of cDNA libraries (Teitz et al., 1987; 1988; 1989). This approach has initially resulted in our identification of the regulatory subunit (beta) of human protein kinase CK2 as a suppressor of DNA damage, and our prediction that CK2 acts by enhancing the G2/M checkpoint control (Teitz et al., 1990a). Subsequently, Glover’s group has shown, between the years 1995-1998, that CK2 is essential for cell cycle progression in yeast during G1 and G2/M, while Hartwell’s group demonstrated in 1997 that CK2 is part of the adaptation mechanism to the DNA damage-induced G2/M checkpoint arrest. Using Glover’s conditional yeast CK2 mutants, we have shown that a human CK2 catalytic subunit can substitute the essential cell cycle related functions of the two yeast CK2 catalytic subunits, while the human CK2 regulatory subunit can act as a suppressor of either one of the two conditional yeast catalytic subunits (Dotan et al., 2001). Other works from my group have completed our chromosomal mapping of all three human CK2 subunit genes (Yang-Feng et al., 1991; Yang-Feng et al., 1994), while pointing to the complex regulation of CK2 expression (Dotan et al., 1995).
The second human cDNA suppressor isolated by this approach (Teitz et al., 1990b) turned out to be a novel human gene which we named UV Resistance Associated Gene (UVRAG; Perelman et al., 1997). Surprisingly, the proteolytic cytoplasmic product of this gene confers not only UV but also X-ray resistance, a feature, which is shared by some of the most common DNA damage-responding genes such as p53 and ATR.
As of June 1997, the focus of the research in the laboratory has changed to identification of anticancer drugs as well as new targets for cancer therapy. The experimental approach is based on development of chemical and genetic synthetic lethality screens in cultured, tumor-derived human and mouse cell lines.

Primary and secondary targets for anticancer therapy
In order for chemotherapy to be effective, it must kill tumor cells selectively. Accordingly, a new generation of anticancer drugs is being developed on the basis of understanding the molecular alterations that drive the disease process. Yet, the multitude genetic alterations which occur in tumor cells, make this avenue of drug identification far from trivial. The mechanism-based drug target identification approach has therefore concentrated for the most part on inhibiting the overexpression of key dominant oncogenes such as Ras, the Estrogen Receptor (ER), ErbB-2 (neu), BCR-ABL, ErbB1, etc.
A different approach named, synthetic lethality screening, examines whether known primary tumor specific alterations sensitize the malignant cell to drugs aimed at secondary targets, thus establishing a synergistic lethal linkage between a mutation in a gene of interest and a drug/drug target. In the absence of correct expression of the primary gene (representing either an oncogene or a tumor suppressor gene), secondary targets that become necessary for cell survival are termed synthetically lethal. Crippling of such a target, on the genetic background of the primary mutation, becomes untenable for the tumor cell, incurring lethality. No prior knowledge of the identity of the secondary target is necessary, making this screen unbiased and specifically lethal to cells with the primary alteration. This is particularly relevant in the case of tumor suppressor genes whose loss of function usually eliminates targets/pathways from drug interference (Reviewed in Canaani 2009; 2014).

Projects

Establishment of chemical and genetic synthetic lethality screens in cultured human and mouse cells
In a project initiated in our laboratory in June 1997, we established a chemical synthetic lethality screen in cultured human cells (Simons et al., (2001a)) and Mouse Embryo Fibroblasts (Einav et al., 2003), and demonstrated the feasibility of a genetic synthetic lethality screen in human cells (Simons et al., 2001b), and in mouse embryo fibroblasts (Einav et al., 2005). This was followed by a collaboration with Joerg Hoheisel’s group (DKFZ Heidelberg) in which  we devised a novel method to decode pooled lentiviral shRNA screens via in situ synthesized 25 bp long partially overlapping barcode tiling arrays. We demonstrated how this approach can be used for negative selection screen (shRNAs drop out) while precisely quantifying the abundance of individual shRNAs from a pool (Bottcher et al., 2010). We have also proved the advantage of this barcode tiling arrays over the (barcode-less) half hairpin probes. Noteworthy, in the context of this genetic screening we have succeeded to produce at high yield triple-negative breast cancer cell lines whose ERα-deficiency is complemented by a cDNA expressed from a bicistronic IRES expression vector (Shenfeld et al., 2012). These opened up the possibility of screening isogenic mesenchymal-like breast cancer cell lines for genes synthetic lethal with ERα-deficiency.

Regulation of tumor suppressor genes and metastasis suppressor genes by long noncoding RNAs (lncRNA)

Some time ago, we have initiated another route which may lead to anticancer drug discovery, based on the idea of reactivating transcription of tumor suppressor- or metastasis suppressor-genes, specifically epigenetically silenced in breast cancer. Epigenetic silencing of tumor suppressor gene promoters is a common observation in cancer. Genome-wide promoter analyses, aided by pharmacological activation has uncovered tens of known and putative tumor suppressor genes that are epigenetically silenced in human cancers. Likewise, reduced transcription, rather than mutation, explains a large part of the loss of metastasis suppressor gene expression observed in different tumor types, including breast carcinoma. We started to scan for directional/ bidirectional transcription through promoters of either tumor suppressor or metastasis suppressor- genes known to be epigenetically silenced in vivo in breast carcinomas. Identification of Sense (S) and Anti-Sense (AS) transcripts to promoter regions was performed by RT-PCR, whereby the RT specific primer determines directionality. Surprisingly, we found (Tzadok et al., 2013) that RT-PCR amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT primed by endogenous transcripts originating from promoter or upstream spanning regions.  We have shown that this prevalent “no primer” artifact can be prevented by periodate treatment of the RNA. In severe cases, a combined protocol entailing, both periodate treatment of the RNA preparation, alongside high temperature RT reactions, was required to overcome the endogenous priming ( Tzadok et al., 2013). After having established an amenable protocol for the detection of S and AS noncoding nuclear transcripts, using multiple potential primers, we have identified several genes having undescribed promoter-spanning antisense lncRNAs. Their functional role in the respective suppressor genes, and their physiological in vivo relevance are the focus of our research.

RNA interference mediated transcriptional gene silencing in mammalian cells. It has been known for quite a while that in mammalian cells RNAi directs homologous RNA degradation, causing posttranscriptional gene silencing (PTGS), as well as translational inhibition. But over the  past few years  there were several reports that RNAi/dsRNA also directs sequence-specific transcriptional gene silencing (TGS) in mammalian cells. Presently, together with a group at Bar Ilan University we are examining several model cell systems that we have set up to see whether they exhibit this phenomena, and if so what are the crucial components.