Targeting the Transcription of RET Oncogene in Medullary Thyroid Carcinoma Using Drug-Like Small Molecules

Abstract

Dominant-activating mutations in the RET (Rearranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, was identified as the major cause for the development of medullary thyroid carcinoma (MTC). Hence, the RET protein has been explored as an excellent molecular target for therapeutic intervention to treat progressive and advanced MTC. The main objective of this dissertation is to highlight a clinically relevant therapeutic strategy for the treatment of MTC by selectively suppressing the transcription of mutant RET oncogene. The basal transcriptional activity of the RET gene is mainly regulated by the polypurine/polypyrimidine (pPu/pPy) tract, present within the proximal promoter region of this gene (-51 to -33 relative to transcription start site). The pPu/pPy tract is rich in guanine-repeat sequence containing five runs of consecutive guanine residues that serve as the binding site for transcriptional factors. This stretch of nucleotides in the promoter region is highly dynamic in nature and tend to adopt non-B DNA secondary structures called G-quadruplexes. As we have recently shown, the formation of G-quadruplex structures on the RET promoter region negatively regulates the transcription of this gene through sequestration of the transcription factor binding sites. Hence, we hypothesized that the stabilization of these structures using small molecules could exert transcriptional inhibitory effect on the RET gene. In our study, we validated that the RET promoter G-quadruplex structure is an amenable drug target to silence RET gene expression by demonstrating the anti-RET activity of a well-characterized G-quadruplex stabilizing agent, berberine. This compound inhibits the RET gene transcription in an MTC derived TT cell line by specifically targeting its promoter G-quadruplex structure. Interestingly, the inhibitory effect of berberine on the RET expression is lost in papillary thyroid carcinoma cell line, TPC1, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement. Furthermore, the RET downregulation by berberine results in the inhibition of TT-cell proliferation through cell-cycle arrest and activation of apoptosis. Overall, these data strongly suggest that the formation of G-quadruplex structure and its stabilization by small molecules is an ideal approach for the transcriptional repression of the RET oncogene. In continuation of our study, we next explored for more drug-like small molecules as potential RET G-quadruplex stabilizers that could be used for MTC patients in a clinical setting. We identified the anti-cancer drug, ellipticine, and its derivatives as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that one of the ellipticine analogs, NSC311153 that incorporates a ethylpiperidine group, showed improved binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound possessed selective inhibitory effect on RET transcription in TT cell line over the TPC1 cells. The RET downregulation by NSC311153 selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In this study, we also showed that the systemic administration of a structural analog of NSC311153 in mice, bearing MTC xenografts, results in the inhibition of tumor growth through RET downregulation. Overall, our study supports the rationale for the development of RET G-quadruplex stabilizing-based therapeutic approaches for the treatment of progressive MTC.