Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Abstract

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.

An Autocrine Loop Involving Ret and Glial Cell–Derived Neurotrophic Factor Mediates Retinoic Acid–Induced Neuroblastoma Cell Differentiation

“…First, we asked whether RA-dependent Ret activation depends on the presence of an autocrine loop involving ligand-dependent dimerization or, rather, the receptor itself is transactivated by intracellular mechanisms acting on its kinase domain (20). To this end, we used a 2 ¶-fluoro-RNAbased aptamer, named D4, which is a specific inhibitor of dimerization-dependent Ret activation but has no effects on Ret activation induced by mutations of the intracellular tyrosine kinase domain (19). As shown in Fig.…”

“…All-trans RA (Sigma, St. Louis, MO) was dissolved (10 mmol/L) in DMSO and diluted at the indicated concentration in serum-containing culture medium. Neuroblastoma cells (160,000 per 3.5 cm plate) were treated for 18 hours with 1 mL culture medium containing 10 Amol/L RA plus 50 Ag of the indicated EC-Ret protein or plus 1600 nmol/L of the indicated aptamer after a short denaturation-renaturation step (19). To evaluate the effects of RNA aptamers and EC-Ret proteins on cell differentiation, cells were incubated in 12-well cell plate with 10 Amol/L RA together with 3 Amol/L aptamer or 20 Ag/mL EC-Ret proteins.…”

“…D4 is a 93-base 2 ¶-fluoro-RNA-based aptamer that specifically binds the extracellular domain of Ret thus interfering with ligand-induced stimulation of its intrinsic tyrosine kinase activity (19).…”

“…To minimize the contribution of TrkB, here, we chose to use the human SK-N-BE(2) neuroblastoma cell line (hereafter named SK-N-BE), in which RA-induced differentiation is preceded by a rapid accumulation of Ret but not of Trk family receptors that are not or poorly expressed (14,16). We used two specific interfering molecules to inhibit Ret function: the EC-Ret peptide that blocks Ret stimulation by competing for binding to the ligandGFRa complex (17,18) and the D4 aptamer that inhibits Ret activity by directly binding the receptor (19).…”

Evaluation of systemic targeting of RET oncogene-based MTC with tumor-selective peptide-tagged Ad vectors in clinical mouse models

“…9 Principles of RET inhibition in MTC cells include molecular strategies to interfere with its kinase activity by small molecules [10][11][12] and receptor dimerization using aptamers or soluble RET ectodomain. 13,14 As an alternative, strategies that block RET expression or autophosphorylation are highly promising. We have previously demonstrated that dominant-negative RET proteins that contain mutations in the extreme N-terminal region of the extracellular domain such as HSCR32 or Flag expressed by adenoviral (Ad) vectors inhibit receptor maturation, 15,16 thereby preventing its transport to the membrane.…”