Telomerase reverse transcriptase (hTERT) is a catalytic subunit of the enzyme telomerase which is frequently overexpressed in human tumors. It has recently been shown that the hTERT promoter is commonly mutated in a malignant melanoma and glioblastoma (>75% of tumors). These mutations occur at four specific sites in a G-rich region which has been shown to form quadruplex DNA and to downregulate hTERT expression. We have shown that these mutations destabilize quadruplex-formation resulting in increased hTERT expression and cellular proliferation. Stabilization of the quadruplex structure with TMPYP4 reverses these changes. Treatment of cells in which the hTERT promoter is mutated with strand-invading oligonucleotides downregulates hTERT expression and dramatically inhibits the growth of transformed cells. This application will test the hypothesis tht stabilization of the mutated hTERT promoter quadruplex structure by sequence specific oligonucleotides will result in downregulation of hTERT and inhibition of growth by cells with the mutated sequence(s).
The Specific Aims of this application are: 1. To characterize the gene and sequence specificity of oligonucleotides targeted to the mutated or wild type hTERT promoter sequences. 2. To characterize the potential synergy of strand invading hTERT promoter targeted oligonucleotides with targeted agents and standard chemotherapy drugs. 3. To characterize the therapeutic potential of hTERT strand invading oligonucleotides in animal models, alone and in conjunction with targeted therapies. The results of the proposed studies will have important clinical implications as inhibition of hTERT gene expression is relevant to a wide variety of tumor types. In addition, the proposed experiments will provide important new information about the role of quadruplex-formation in the regulation of hTERT gene expression.
Telomerase is overexpressed in over 90% of human tumors. It represents a very attractive target for anticancer therapy. The recent discovery of 'common mutations' in the hTERT promoter has suggested that quadruplex formation may play an important physiologic role in hTERT silencing. We have shown that the mutations destabilize quadruplex and that these changes can be reversed by sequence-specific strand-invading oligonucleotides. This approach allows the stabilization of mutated hTERT promoter sequences and the reversal of hTERT dysregulation. We believe that these observations suggest that inhibition of hTERT expression is a reasonable goal with considerable clinical potential.
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