Radiolabeled Oligonucleotides Targeting the RNA Subunit of Telomerase Inhibit Telomerase and Induce DNA Damage in Telomerase-Positive Cancer Cells

Abstract

Telomerase is expressed in the majority (>85%) of tumors, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterization of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (Match) and noncomplementary (Scramble or Mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111 In). Match oligonucleotides inhibited telomerase activity with high potency, which was not observed with Scramble or Mismatch oligonucleotides. DTPA-conjugation and 111 In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled Match oligonucleotides had no effect on survival, however, 111 In-labeled Match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker gH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111 In-Match oligonucleotides. Match oligonucleotides localized in close proximity to nuclear Cajal bodies in telomerasepositive cells. In comparison with Match oligonucleotides, 111 In-Scramble or 111 In-Mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells and paves the way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers. Significance: These findings present a novel radiolabeled oligonucleotide for targeting telomerase-positive cancer cells that exhibits dual activity by simultaneously inhibiting telomerase and promoting radiation-induced genomic DNA damage.

Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies

“…In myeloma, ILF2 antisense oligonucleotide (Interleukin Enhancer Binding Factor, a key modulator of the DNA repair pathway) is a synthetic lethal target with DNA2 (DNA replication helicase/nuclease 2) [284]. A radiolabeled oligonucleotide that targets the RNA-associated telomerase promotes radiation-induced genomic DNA damage in telomerase-positive cancer cells [285]. A short, double-stranded oligonucleotide linked to a cholesterol molecule, AsiDNA™, acts as a decoy, mimics DSBs, and triggers a false DNA break signal to activate and attract DNA repair proteins, preventing their recruitment to the site of genomic DNA damage.…”

Oligonucleotide-Functionalized Gold Nanoparticles for Synchronous Telomerase Inhibition, Radiosensitization, and Delivery of Theranostic Radionuclides

“…Match, but not Scramble, oligonucleotides inhibited telomerase in telomerase-expressing MDA-MB-435 (log IC 50 −6.93 ± 0.072) ( Figure 3 A), consistent with previous results. 21 The effect of ON–AuNPs on telomerase activity was also tested using TRAP assays ( Figure 3 B). Match–AuNP inhibited the telomerase activity in a dose-dependent manner (log IC 50 −7.49 ± 0.04, Hill slope −1.46 ± 0.19).…”

“… 14 − 20 To exploit this radiosensitizing effect, an oligonucleotide hTR inhibitor was conjugated to indium-111 ( 111 In) for concomitant telomerase inhibition and targeted radionuclide therapy (TRT). 21 111 In emits Auger electrons that cause dense ionizations over a short range (nm to μm), thus allowing for irradiation of individual cells and sparing of non-targeted tissue. 22 , 23 This 111 In-labeled hTR-targeted oligonucleotide construct causes sequence- and telomerase-dependent DNA damage and cell-killing effects in cancer cells.…”

“… 22 , 23 This 111 In-labeled hTR-targeted oligonucleotide construct causes sequence- and telomerase-dependent DNA damage and cell-killing effects in cancer cells. 21 However, the use of oligonucleotides as anticancer agents is hampered by poor cellular uptake and unfavorable pharmacokinetics. There is therefore an impetus in the field of DNA therapeutics to devise new oligonucleotide delivery methods that can overcome this barrier to the clinic.…”

An 111In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells

“…4 With sufficient uptake into the cell nucleus, AEs are able to induce radiotoxic DNA damage in the form of single-strand and double-strand breaks (SSBs and DSBs), with DSBs exhibiting greater cytotoxicity. 5 Due to the short path-length of AEs in biological media, it follows that AEs directed to specic regions within the genome will efficiently provide cell-specic radiotoxicity [6][7][8] and that nonspecic radiotoxicity to neighbouring cells can be limited. 9 Furthermore, many Auger electron emitting radionuclides also emit gamma ray radiation, making them compatible with whole-body SPECT imaging.…”