Today I would like to share with you an article published in JACS titled “Telomere Targeting Chimera Enables Targeted Destruction of Telomeric Repeat-Binding Factor Proteins”. This article developed a nucleotide-based PROTAC named TeloTAC, which can degrade telomeric repeat-binding factor 1/2 (TRF1/2), a protein that interacts with the telomeric DNA repeat sequence to regulate telomere length. TeloTAC provides a nucleotide-based degradation approach to shorten telomeres and inhibit tumor cell growth, suggesting a potential therapeutic approach for cancer. The corresponding author of this article is Associate Professor Wen-Yi Wei from Harvard Medical School, whose research group is dedicated to developing novel PROTAC technologies and efficient delivery methods for PROTAC.
Due to the combination of telomere shortening and each cell cycle division, normal cells undergo replicative senescence while cancer cells can obtain unlimited replicative capacity by activating telomerase to extend telomere ends. Therefore, targeting telomerase function is a direct and effective method for delaying cancer progression. In this regard, various therapies have been developed to target telomerase for cancer treatment, but they still face issues with relatively low efficiency and adaptability to drug resistance. Imetelstat is the first nucleotide drug designed to target telomerase and is currently being studied for clinical efficacy. However, further research is needed to fully evaluate its effectiveness and safety due to its side effects. Therefore, better methods are still needed to overcome the problem of untreatable telomerase.
Previous studies have targeted telomeric repeat-binding factor 1/2 (TRF1/2) proteins and developed various therapies targeting telomerase by directly inhibiting its activity or indirectly disrupting its precise localization using small molecules and oligonucleotide inhibitors. However, there remain challenges such as low efficiency and adaptive drug resistance. The author proposes a method to the TeloTAC by attaching the DNA PROTAC to the non-transcription factor protein, TRF1/2. The TRF1/2 protein is the main component of shelterin, whose main function is to form hydrogen bonds to mediate the precise binding between telomere-specific segments and other proteins, including Lys421 (Lys488 TRF2) G4, Asp422 C5′ (antisense variation, Asp489 TRF2), and Arg425 C5′ (Arg492 TRF2). Based on the important role of TRF1/2 in telomerase and telomere binding, the authors designed a series of TeloTACs to induce shelterin deconstruction by using TRF1/2 as a degradation determinant (degron), thereby disrupting the binding of telomerase and telomeres. This method targets the activation of telomerase in tumor cells by degrading the structural protein TRF1/2, rather than directly inhibiting the activity of telomerase.
Schematic 1 shows the role of TRF1/2 in maintaining telomeres. (A) Functional relationship between telomere length and cellular aging. (B) Structural analysis demonstrates the interaction between TRF1 and the telomere repeat sequence (TTAGGG)n. TeloTAC proposes a new strategy to target the necessary telomerase activity in cancer cells by degrading the structural proteins TRF1/2 instead of directly inhibiting telomerase enzyme activity.
Schematic 2. TeloTAC induces TRF2/3 ubiquitination and subsequent degradation by recruitment of E1 enzyme using an E1-linking enzyme. Several prototype TeloTAC molecules were designed by coupling TRF1/2 binding motifs (TTAGGG) with Von Hippel-Lindau (VHL) ligands. Upon entering the cell, the oligonucleotide portion of TeloTAC directly binds to the TRF1/2 protein, while the VHL ligand portion recruits VHL E3 ligase to promote ubiquitination and subsequent degradation of TRF1/2.
Research Results
Result 1: Design of TRF1/2 Ligand TRF-ODN
The author first designed a ligand consisting of three TTAGGG tandem repeat sequences that bind to TRF1/2 proteins, named TRF-ODN. To construct TRF-ODN, the author inserted a single-stranded DNA oligomer (5’-GGGTTAGGGTTAGGGTTATTTTAACCCTAACCC-TAACCC-3’) connected by a “-TTT-linker” to form a stem-loop structure and stabilise the DNA oligomer (Figure 1A). To evaluate the binding affinity of TRF-ODN to TRF1/2 proteins, the author used biotinylated DNA oligomers for streptavidin-biotin pull-down assays (Figure 1B-1C). The results showed that TRF1 and TRF2 competitively bound to the free biotinylated TRF-ODN (oligonucleotide), indicating specific binding (Figure 1D-1E).
Result 2: TeloTAC induces degradation of TRF1/2 in various tumor cell lines.
Using TRF-ODN as a ligand, the authors coupled it to VHL E3 ligand through SPAAC reaction (1353016-70-2, diphenylcyclooctyne-carboxylic acid for copper-catalyzed azide-alkyne cycloaddition without copper ion). They then constructed PROTACs for TRF1/2, which were referred to as TeloTAC degraders (Figure 2A). The type and length of the linker determine the correct positioning of the E3 ligase and the substrate structure of the PROTAC. Therefore, the authors synthesized a series of VHL ligands, namely compounds 1-18a. After connecting these ligands to TRF-DON at room temperature for 16 hours, their degradation efficiency was verified using PAGE gel. The results showed that the degradation efficiency of TeloTAC degraders in all groups except 7a-10a was above 80% (Figure 2A-2B). Treatment of HeLa cells with compounds 15b-17b (5μg/mL, 24h) induced significant degradation (Figure 2C), while the effects of compounds 1b, 2b, and 6b-10b were relatively weak. The weak effects of 1b and 2b may be attributed to their shorter linker length, while the increased hydrophobicity of the longer linkers in 6b-10b may also contribute to the weaker effect. These results suggest that optimizing the length and affinity of the linker is the key to improving degradation efficiency. This also explains why compounds 3b-5b have lower efficiency compared to other alkane degraders due to their shorter length and higher hydrophobicity. The lower efficiency of compounds 11b-13b may be due to the conformation of the linker, which causes displacement of VHL E3 ligase and TRF localization.
Except for TRF2, the 18 TeloTACs showed almost identical degradation efficiency towards TRF1 (Figure 2C). In addition, the degradation of 15b (5μg/mL) was blocked by free TRF-ODN (5μg/mL), indicating that TRF was degraded in a TRF-ODN-dependent manner (Figure 2D). Moreover, the VHL degradation mediated by TeloTAC 15b was blocked by the proteasome inhibitor MG132 (2.5μM) or the VHL ligand VH-032 (5μM), suggesting that the TRF degradation mediated by TeloTAC involved TRF-ODN-, VHL-, and proteasome-associated pathways (Figure 2D). In addition to HeLa cells, TeloTACs 6b, 14b, and 15b were also effective in degrading TRF1 and TRF2 in A431 and MDA-MB-231 cells (Figure 2E and S6), as well as other tumor cell lines of different species, as shown in Figure S7. TeloTACs 6b, 14b, and 15b (5μg/mL, 24h) significantly inhibited the proliferation of A431 cells (Figure 2F), indicating that these TeloTAC compounds have good anti-tumor effects. Moreover, the authors investigated whether the degradation of TRF1 and TRF2 would affect the physiological function of telomerase. The authors used qPCR to detect telomere length. The results showed that in MDA-MB-231, A431, and HeLa cells, telomere length was significantly shortened after treatment with 14b and 15b (Figure S8).
Result 3: TeloTAC-mediated degradation of TRF1/2 has selective anti-tumor activity.
To further study the anti-proliferative activity of TeloTAC, the authors conducted cell growth curve experiments, colony formation experiments, and MTT assays in various tumor cell lines including A431 and HeLa cells. The results showed that 5μg/mL TRF-ODN, 14b, and 15b could inhibit the proliferation of MDA-MB-231, A431, and HeLa cells (Figure 3A). In addition, the authors treated these cell lines with 2μM telomerase inhibitors RHPS429-31 and BIBR1532. The results showed that the inhibitory effect of TeloTACs 14b and 15b was stronger than that of TRF-ODN and the two inhibitors (Figure 3A). Cell viability assays (Figure 3A) and colony formation experiments (Figure 3B) showed that TeloTAC 14b and 15b significantly inhibited the proliferative ability of cells, while the effect of TRF-ODN was relatively weak. These results suggest that TRF-ODN may function as a DNA decoy to inhibit the function of TRF, while TeloTAC causes the destruction of TRF protein, making it more effective than TRF-ODN.
Due to the different degrees of dependence on telomerase function between tumor cells and non-cancerous normal cells, the authors further studied the effects of TeloTACs 14b and 15b on two other normal cell lines LF1 and MCF10A. The results showed that TeloTACs 14b and 15b could effectively degrade TRF1/2 in both cell types (Figures S10 and S12), but had no significant effect on cell proliferation (Figures S9 and S11). Moreover, the IC50 values of TeloTAC 15b in MDA-MB-231, A431, and HeLa cells were approximately 0.2, 0.3, and 0.39 μM, respectively, while the IC50 value of 15b in non-cancerous normal cells was about 10 times that in tumor cell lines (Figures 3C and S13). In addition, the IC50 values of telomerase inhibitors RHPS4 and BIBR1532 in tumor cells were about 100 times higher than that of 15b (Figures 3C and S13). To further validate the selective cytotoxicity of TeloTACs towards tumor cells rather than normal cells, the authors tested the effect of TeloTAC 15b on a co-culture system consisting of GFP-labeled HeLa cells (tumor cells) and mCherry-labeled LF1 cells (non-cancerous normal cells) (Figure 3D). The results showed that TeloTAC 15b selectively killed tumor cells, while having no effect on non-cancerous normal cells, while the telomerase inhibitor RHPS4 showed non-selective cytotoxicity to both cell types. These results demonstrate that TeloTAC 15b has selective cytotoxicity towards tumor cells and has potential application value.
Result 4: TeloTAC Induces Apoptosis and Cellular Senescence Signals
The authors verified the mechanism by which TeloTAC induces cell death in both tumor and non-cancerous normal cells. Studies have shown that the high expression of TERT, TRF1, and TRF2 plays an important role in cancer cell proliferation, while non-cancerous normal cells do not rely on telomerase for survival, indicating that TRF1 and TRF2 are promising anti-cancer targets. Therefore, the authors compared the expression levels of TRF1/2 mRNA and protein in various tumor and non-cancerous control groups. The results showed that TRF1/2 mRNA and protein levels were higher in tumor cells than in non-cancerous normal cells (Figure 4A). Since TeloTAC significantly shortens telomere length in cancer cells (Figure S8), the authors investigated whether TeloTAC treatment would affect cellular senescence and apoptosis signaling pathways in tumor and non-cancerous normal cells. The results showed that TeloTAC treatment (6b, 14b, and 15b) increased the levels of the aging markers p53 and p27 in tumor cells, while there was no such effect in non-cancerous normal cells (Figure 4B). In addition, TeloTAC triggered apoptosis-related pathways in tumor cells, inducing the expression of caspase 3 and PARP, while normal cells were unaffected (Figure 4C). These results suggest that TRF1/2 degradation and telomere shortening triggered by TeloTAC rebuild the aging and apoptosis signaling pathways in tumors, inhibiting tumor cell proliferation without harming normal cells (Figure 4D).
