פרופ' דן [דני] כנעני

בדימוס בביוכימיה וביולוגיה מולקולרית
ביוכימיה וביולוגיה מולקולרית בדימוס
ניווט מהיר:
פרופ' דן [דני] כנעני
טלפון פנימי: 03-6408985
פקס: 09-7422298
משרד: שרמן, 603

Research Interests




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).


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.




Selected Publications


 Boettcher, M., Fredebohm J., Gholami A.M., Hachmo, Y., Dotan, I., Canaani, D. Hoheisel, J.,   (2010) Decoding pooled RNAi screens by means of barcode tiling arrays.  BMC Genomics  11 :7

 Canaani, D. (2009) Methodological approaches in application of synthetic lethality  screening      towards anticancer therapy.   Br. J. Cancer 100, 1213-1218.

Canaani D. (2014) Application of the Concept Synthetic Lethality Toward Anticancer Therapy: A Promise Fulfilled? Cancer Letters 352 (1) 59-65.


Dotan, I., Kopatz, I., Naiman, T., Perelman, B., Dafni, N., and Canaani, D. (1995) Establishment of an autocatalytic conditional mammalian system for expression of stringently regulated genes. Nucleic Acids Res.  23 : 307-309.


Dotan, I., Ziv, E., Dafni, N., Beckman, J.S., McCann, R.D.,  Glover, C.V.C., and Canaani, D. (2001) Functional conservation between the human, nematode and yeast CK2  cell cycle genes. Biochem. Biophys. Res. Commun.  288 : 603-609.  


Einav, Y., Shistik, Y., Shenfeld, M., Simons, A.H., Melton, D.W., and Canaani, D. (2003) Replication and episomal maintenance of EBV-based vectors in mouse embryo fibroblasts enable synthetic lethality screens. Mol. Cancer Ther., 2, 1121-1128.


Einav, Y., Agami, R., and Canaani, D. (2005) shRNA-mediated RNA interference as a tool for genetic synthetic lethality screening in mouse embryo fibroblasts. FEBS Lett. 579, 199-2002.


Kopatz, I., Naiman, T., Eli, D., and Canaani, D. (1990) The nucleotide sequence of the mouse cDNA encoding the beta subunit of casein kinase II. Nucleic Acids Res.  18 : 3639.


Liang Chengyu, Feng Pinghui, Ku Bonsu, Dotan Iris, Canaani Dan, Oh Byung-Ha, and Jung U. Jae (2006) Autophagic and  tumor suppressor activity of a novel Beclin1-binding protein UVRAG. Nature Cell Biology,  8 , 688-699.


Naiman, T., and Canaani, D. (1989) A hypodiploid karyotype, found in immortal human cells, is selected from a wide spectrum of posttransformation chromosomal complements. Cancer Genet. and Cytogenet.  40 :65-71.


Perelman, B., Dafni, N., Naiman, T., Eli, D., Yaakov, M., Yang-Feng, T.-L., Sinha, S., Weber, G., Khodaei, S., Sancar, A., Dotan, I., and Canaani, D. (1997)  Molecular cloning of a novel human gene encoding a 63-kDa protein and its sublocalization within the 11q13 locus. Genomics  41 : 397-405.


Shenfeld,  M., Hachmo, Y.,  Dafni, N Boettcher M., Hoheisel J, .,  Dotan, I., and Canaani, D. (2012) ER a   cDNA expressed as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones. PLoS One. 7 (2) e31977


Simons, A., Dafni, N., Dotan, I., Oron, Y., and Canaani, D. (2001a) Establishment of a chemical synthetic lethality screen in cultured human cells. Genome Research.  11 : 266-273.


Simons, A.H., Dafni, N., Dotan, I., Oron, Y., and Canaani, D. (2001b) Genetic synthetic lethality screen at the single gene level in cultured human cells. Nucleic Acids Res.  29:  e100 (7 pages).


Stark, M., Naiman, T., and Canaani, D. (1989) Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient. Biochem. Biophys. Res. Commun.  162 :1351-1356.


Teitz, T., Naiman, T., Avissar, S.S., Bar, S., Okayama, H., and Canaani, D. (1987) Complementation of the UV-sensitive phenotype of xeroderma pigmentosum human cell line by transfection with a cDNA clone library. Proc. Natl. Acad. Sci. USA  84 : 8801-8804.


 Teitz, T., Naiman, T., Eli, D., Bakhanashvili, M., and Canaani, D. (1988) Stable correction of excision-repair deficiency in a xeroderma pigmentosum human cell line. In: "Mechanisms and Consequences of DNA Damage Processing", UCLA Symposia on Molecular and Cellular Biology, new series, vol. 83, pp. 313-317, ed. E. Friedberg and P. Hanawalt, Alan R. Liss Inc., New York, N.Y.


Teitz, T., Naiman, T., Eli, D., Bakhanashvili, M., and Canaani, D. (1989) Complementation of excision-repair deficiency in a human cell: advantage in the use of a cDNA clone library for gene transfer. In "Gene Transfer and Gene Therapy", UCLA Symposia on Molecular and Cellular Biology, new series, vol. 87, pp. 215-223, eds. A.L. Beaudet, R. Mulligan and I.M. Verma, Alan R. Liss Inc., New York, NY.


Teitz, T., Eli, D., Penner, M., Bakhanashvili, M., Naiman, T., Timme, T.L., Wood, C.M., Moses, R.E., and Canaani, D. (1990a) Expression of the cDNA for the beta subunit of human casein kinase II confers partial UV resistance on xeroderma pigmentosum cells. Mutat. Res.  236 : 85-97.


Teitz, T., Penner, M., Eli, D., Stark, M., Bakhanashvili, M., Naiman, T., and Canaani, D. (1990b) Isolation by polymerase chain reaction of a cDNA whose product partially complements the UV sensitivity of xeroderma pigmentosum group C cells. Gene  87 : 295-298.  


Tzadok S., Caspin Y., Hachmo Y., Canaani D., and Dotan I. (2013) Directionality of noncoding human RNAs: how to avoid artifacts. Anal. Biochem. 439: 23-29.


Yang-Feng, T.L., Naiman, T., Kopatz, I., Eli, D., Dafni, N., and Canaani, D. (1994) Assignment of the human casein kinase II  a '-subunit gene to 16p13.2-p13.3. Genomics  19 : 173.


Yang-Feng, T.L., Zheng, K., Kopatz, I., Naiman, T., and Canaani, D. (1991) Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the  a - subunit. Nucleic Acids Res.  19 : 7125-7129.


Yang-Feng, T.L., Teitz, T., Cheung, M.C., Kan, Y.W., and Canaani, D. (1990) Assignment of the human casein kinase II beta-subunit gene to 6p12-p21. Genomics  8 : 741-742.




1995  U.S. patent 5,882,880 on the human UVRAG gene and its usages - approved 1999.

2000 PCT patent 00956768.6 on Genetic Screening Methods: Chemical and genetic synthetic lethality screens- approved 2005.

1999 U.S. patent 6,569,231 B1 on Genetic Screening Methods: Genetic Synthetic Lethality Screening in Human and Mouse Cells-issued May 2003.

2001 U.S. patent 6,861,220 on Genetic Screening Methods: Chemical Synthetic Lethality Screening in Human and Mouse Cells-issued March 2005.



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