Prevention of metastasis with 5-aza-2′-deoxycytidine

Details Prevention of metastasis with 5-aza-2′-deoxycytidine Prevention of metastasis with 5-aza-2′-deoxycytidine

1999-07-26

2001-07-03

Schwartzman, Robert A. (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S005000, C435S006120, C435S007100

Reexamination Certificate

active

06255293

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for screening cancer patients for susceptibility to a particular treatment protocol and to a method of treatment of those patients found positive in the screen, and more particularly to such screens and methods based on screening for DAP-kinase methylation and treatment with 5-aza-2′-deoxycytidine.
BACKGROUND OF THE INVENTION
Loss or inactivation of tumor suppressor genes is among the genetic defects which participate in the multi-step process which causes cancer. Some of the tumor suppressor genes are part of the genetic machinery which controls programmed cell death, also named apoptosis, and therefore their deregulation severely disrupts the normal tissue homeostasis terminating in tumor growth. Various types of genetic and epigenetic deregulations can inactivate tumor suppressor genes including DNA deletions, mutations, translocations, chromosomal losses and DNA methylations.
DAP-kinase is a positive mediator of apoptosis, recently identified as a tumor suppressor gene (Deiss et al., 1995; Cohen et al., 1997; Kissil et al., 1997; Inbal et al., 1997; Cohen et al., 1999). This calcium/calmodulin-dependent serine/threonine kinase mediates cell death triggered by various signals including different cytokines, detachment from extracellular matrix and oncogenes. Several independent research lines suggested that DAP-kinase is a potential tumor suppressor gene:
1. DAP-kinase gene expression was found to be lost at high frequency in human tumor cell lines. Both the mRNA and protein expression levels were found below detection limits in 70% of B-cell lymphoma and leukemia cell lines and in 30% of cell lines derived from bladder carcinomas, breast carcinomas, and renal cell carcinomas (Kissil et al., 1997). This stood in sharp contrast to the finding that DAP-kinase mRNA was widely expressed in all the tested human and murine tissues, as well as in many immortalized cell lines established from normal cells. In two bladder carcinoma and in one of the B cell lymphoma cell lines, DAP-kinase expression could be restored by treatment of cells with 5-aza-2′-deoxycytidine, a drug which inhibits DNA methylation (Kissil, et al., 1997). This suggested that loss of expression in these particular cases was due to DNA methylation, as previously reported for other tumor suppressor genes, such as p16, VHL, and pRB. Yet, it was found, from the tumor cell lines screen, that demethylation is not an exclusive mechanism for suppressing DAP-kinase expression, in accordance with the well established paradigm that tumor suppressor genes may be lost or inactivated by multiple genetic or epigenetic alterations. These experiments provided the first hint that DAP-kinase inactivation may possibly be a causative factor in the formation of tumors, as was further tested in experimental animal model systems.
2. In parallel, a second approach was carried out that directly tested in animal models whether the DAP-kinase gene has tumor suppressor functions. In these experiments, the normal expression levels of DAP-kinase were restored into tumor cells that had lost it, and the impact of this genetic manipulation on the tumorigenic properties of the cells was assayed. It was found that high-metastatic lung carcinoma clones, originating from two independent murine lung tumors, lacked DAP-kinase expression, in contrast to their low-metastatic counterparts which expressed normal levels. Wild-type DAP-kinase was introduced into the high-metastatic Lewis carcinoma cells and stable transfected clones in which DAP-kinase expression was restored to physiological levels were isolated and assayed for their tumorigenic and metastatic activity in syngeneic mice. Strikingly, restoration of physiological levels of DAP-kinase into the high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injections into mice. The in vivo effects were proportional to the levels of the ectopically expressed DAP-kinase. The transgene also delayed local tumor growth in a foreign micro environment, yet this feature was less sensitive to DAP-kinase ectopic expression than the metastatic activity. Once tumors appeared at the late time points, they were capable of generating spontaneous metastases in the lungs. Examination of these secondary lung lesions indicated that the tumor cells have lost expression from the DAP-kinase transgene. The loss was due to specific DNA methylation which suppressed expression, since treatment of the cells with 5-aza-2′-deoxycytidine in vitro, after their removal from the lung lesions, restored DAP-kinase expression to high levels (Inbal et al., 1997).
3. By selecting in vivo rare lung metastases, after injections of the original low-metastatic cells into irradiated syngeneic mice, it was found that the development of these metastatic lesions correlated with loss of the endogenous DAP kinase expression. Moreover, treatment of cells recovered from one of these DAP-kinase negative lung lesions with 5-aza-2′-deoxycytidine, restored protein expression to the normal levels (Inbal et al., 1997). DNA methylation was therefore also responsible for silencing the endogenous DAP-kinase gene in some of the in vivo selected lung lesions. Altogether, these experiments suggested that loss of DAP-kinase expression provides a positive selective advantage during the formation of lung metastases.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to overcome the deficiencies of the prior art.
It is another object of the present invention to provide a screening procedure for determining whether a given cancer patient is most likely to be susceptible to treatment for the prevention of metastases using a DNA demethylating agent.
It is a further object of the present invention to provide such a screening method which first involves determining whether DAP-kinase protein is produced by the patient's tumor cells and, if not, determining if such cells contain DAP-kinase genes which are methylated.
It is still a further object of the present invention to provide a method of treatment to decrease the chance that tumor cells will metastasize.
It is still another object of the present invention to provide such a method of treatment which involves administering a DNA demethylating agent if the screening procedure shows that the patient is likely to be susceptible to such treatment.
These and other objects of the present invention are accomplished by means of the embodiments disclosed in the present specification and equivalent procedures. A specific embodiment of a screen which is in accordance with the present invention is a screening procedure to determine whether primary tumor cells or metastatic tumor cells lack DAP gene expression, and particularly DAP kinase expression. More particularly, such a screening procedure is to determine whether such cells which do not have DAP, and particularly DAP-kinase activity, display a methylated form of the DAP gene, and particularly of the DAP-kinase gene. Cells which are positive for such a screen are particularly good candidates for treatment by means of demethylation agents, such as 5-aza-2′-deoxycytidine, in order to demethylate the gene and restore DAP function, and particularly DAP-kinase function.
Further in accordance with the present invention is the therapeutic method of preventing or limiting the spread of metastases by treatment of a patient with a therapeutically effective amount of a demethylating agent, such as 5-aza-2′-deoxycytidine, so as to prevent methylation of the DAP genes, and particularly the DAP-kinase gene, or to demethylate genes which have lost their ability to express the DAP product due to methylation of the gene, thereby restoring the biological metastasis-preventing activity of the endogenous DAP genes, and particularly the DAP-kinase gene. This method of therapeutic treatment is particularly useful for the treatment of patients whose tumor cells have been shown to have a methylated DAP-kinase gene. It is

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