Luke Wojdyla

Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

Oligonucleotides homologous to 3′-telomere overhang (T-oligos) trigger inherent telomere-based DNA damage responses mediated by p53 and/or ATM and induce senescence or apoptosis in various cancerous cells. However, T-oligo has limited stability in vivo due to serum and intracellular nucleases. To develop T-oligo as an innovative, effective therapeutic drug and to understand its mechanism of action, we investigated the antitumor effects of T-oligo or T-oligo complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG), in a p53 null melanoma cell line both in vitro and in vivo. The uptake of T-oligo by MM-AN cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the T-oligo-PVBLG nanocomplex increased uptake by 15-fold. In vitro results showed a 3-fold increase in MM-AN cell growth inhibition by the T-oligo-PVBLG nanocomplex compared with T-oligo alone. Treatment of preformed tumors in immunodeficient mice with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. Moreover, T-oligo treatment downregulated procaspase-3 and procaspase-7 and increased catalytic activity of caspase-3 by 4-fold in MM-AN cells. Furthermore, T-oligo induced a 10-fold increase of senescence and upregulated the melanoma tumor-associated antigens MART-1, tyrosinase, and thrombospondin-1 in MM-AN cells, which are currently being targeted for melanoma immunotherapy. Interestingly, siRNA-mediated knockdown of p73 (4–10-fold) abolished this upregulation of tumor-associated antigens. In summary, we suggest a key role of p73 in mediating the anticancer effects of T-oligo and introduce a novel nanoparticle, the T-oligo-PVBLG nanocomplex, as an effective anticancer therapeutic.

Targeting the Telomere with T-Oligo, G-Quadruplex Stabilizers, and Tankyrase Inhibitors

All mammalian chromosome ends are capped by telomeres, specialized DNA structures that protect chromosomes from genomic instability [1]. Telomeres are comprised of 1000-2000 tandem base pairs repeats, and their 3’ ends extend beyond the 5’ terminus, forming an overhang region that is comprised of a repeating TTAGGG sequence. During each cycle of cell division telomeres are incompletely replicated, and consequentially, their ends are progressively shortened. When telomeres reach a critically short length, DNA damage responses (DDRs) such as senescence and/or apoptosis are triggered. Hence, telomeres are considered to be “biological clocks,” as they limit the proliferative potential of most normal cells [2].

Abstract 4128: Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma

T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 39-telomeric overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of action of T-oligo in cancer cells is largely unknown. Recent studies suggest that GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. This study aims to examine the G4-forming capabilities of T-oligo in vitro and investigates the molecular mechanism of single-stranded (SS) and G4-T-oligo induced DDRs in melanoma cells (MM-AN). G4-formation by T-oligo was confirmed using the SS-T-oligo and G4-T-oligo on a polyacrylamide gel under non-denaturing conditions. NMR studies for T-oligo in KCl confirmed that T-oligo forms G4 structures. Immunofluorescence studies conducted with an anti-G-quadruplex antibody (BG4), a G4 detecting antibody, showed 88.4% co-localization of T-oligo and BG4 in the nuclei of melanoma cells confirming the ability of T-oligo to form G-quadruplexes inside melanoma cells. While G4-T-oligo was found more stable in nuclease degradation assay by DNase I, it had a decreased anti-proliferative effects compared to SS-T-oligo. However, G4-T-oligo had similar cellular uptake as SS-T-oligo. Further, two shelterin complex proteins TRF2 and POT1 which are mainly found at the telomere ends were found to be upregulated (2.0 fold) by T-oligo suggesting TRF2 and POT1 mediated telomere overhang dissociation. We also found that T-oligo can co-localize with telomere binding proteins TRF2 and POT1 by 88.4±4.5% (n=12) and 84.5±8% (n=10) respectively. Western blot analysis results also showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively. To further confirm the involvement of p-JNK in T-oligo mediated apoptosis we used a specific JNK inhibitor SP600125. Western blot analysis showed that T-oligo mediated upregulation of p-JNK was reversed in presence of SP600125. Results from an MTT assay showed a 73.8% decrease in cell viability after T-oligo treatment alone; however, cell viability was decreased to 45.8%, and 25.3% when SP600125 was present at concentrations of 10 μM, and 12 μM respectively, in comparison to diluent. T-oligo also inhibited mRNA expression of hTERT; a catalytic subunit of telomerase by 50% .We further investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. In conclusion, these studies demonstrate that T-oligo can form G-quadruplexes and the anti-proliferative mechanism of T-oligo may be mediated through POT1 and TRF2 as well as via JNK-activation inducing hTERT-inhibition in melanoma cells. Citation Format: Gagan Chhabra, Luke Wojdyla, Ankita Sanjali, Mark Frakes, Marko Ivancich, Pooja Vinay, Zachary Schrank, Benjamin E. Ramirez, Neelu Puri. Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2017-4128

Abstract 4577: Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Telomeres, the ribonucleoprotein complexes found at the ends of eukaryotic chromosomes, protect genomic DNA from end-to-end fusions and degradation by nucleases. During each round of cell division, the ends of telomeres are incompletely replicated, resulting in a net loss of telomeric DNA. When critically shortened, telomeres lose their ability to protect chromosomes from sustaining damage, thereby triggering cellular senescence or apoptosis. In numerous cancer types, administration of an oligonucleotide homologous to 3′-telomere overhang (T-oligo) induces potent DNA damage responses, presumably by mimicking or inducing telomere DNA damage, resulting in cell cycle arrest, senescence, or apoptosis. Remarkably, T-oligo induces minimal side-effects in normal cells, and thus, is actively being investigated as a novel anticancer therapeutic. However, as an oligonucleotide, T-oligos intrinsic instability makes it susceptible to degradation by intracellular and serum nucleases, which severely limits its clinical use. To develop T-oligo as an innovative, effective therapeutic drug, it was complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG-8). PVBLG has been shown to be highly effective in delivering plasmid DNA and siRNA to a variety of cell lines, however, it has not been investigated using oligonucleotides. Here, our studies focus on its mechanism of action, stabilization and improved delivery. We show that PVBLG effectively stabilizes T-oligo by forming a nanocomplex (TOP complex), and enhances its anticancer properties both in vitro and in vivo. In addition, we identify new proteins involved in T-oligos mechanism of action. The uptake of T-oligo by MM-AN melanoma cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the TOP complex increased uptake by 15-fold. In MM-AN cells, T-oligo complexed with the PVBLG (0.025-.25mg/ml) inhibited growth in vitro in a dose dependent manner by 4.5-8.3-fold, compared to 3-fold by T-oligo alone. Similar results were seen in H358 lung cancer cells. Preformed MM-AN tumors in immunodeficient mice treated with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. In addition, T-oligo treatment demonstrated downregulation of telomere repeat factor 1, a negative regulator of telomere length, in MU melanoma cells, indicating a role for TRF1 in T-oligo-induced DNA damage responses. In summary, our investigation further defines T-oligos mechanism of action, and demonstrates the potential of a novel nanoparticle, the TOP complex, as an effective anticancer therapeutic. Citation Format: Luke M. Wojdyla, Srijayaprakash B. Uppada, Neelu Puri. Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4577. doi:10.1158/1538-7445.AM2014-4577

Therapeutic Potential of T-oligo and its Mechanism of Action

Traditional chemotherapy is the first treatment option for the majority of cancer patients, but due to harsh and toxic side effects, more targeted therapies are needed. T-oligo is an oligonucleotide homologous to the 3’ overhang of the telomere. It induces several DNA damage and anti-cancer responses similar to experimental telomere loop disruption, including senescence, apoptosis and differentiation in malignant cells. To explore T-oligo’s anticancer potential, a panel of 6 malignant melanoma cell lines was treated with T-oligo. Melanoma cell lines with functional p53 or p73 exhibited cell death ranging from 11.80% to 31.73% after T-oligo treatment, with MU and MM-AN melanoma cells expressing the maximum response. There was no significant response in p53 and p73 null RPM-EP cells. Based on these results, MM-AN cells were chosen as a model system to study T-oligo’s effects in vitro and in vivo. To further elucidate its mechanism of action, pro-apoptotic and differentiation markers typically up regulated in responsive melanoma cell lines were studied in RPM-EP cells. FACS analysis and immune fluorescence studies confirmed uptake of fluorescein labeled T-oligo in both cell lines. Western blotting and confocal microscopy studies indicated up regulation of YH2AX after T-oligo treatment in MM-AN cells. In RPM-EP cells, expression of p73 and TRP-1 was not detected, nor was there up regulation of E2F1 and Tyrosinase. For in vivo experiments, SCID mice were injected with MM-AN cells to form tumors on their flanks, which were later treated with T-oligo and complementary oligo using Alzet pumps. Results demonstrated a 98% reduction in tumor size, as well as up regulation of differentiation markers important for anti-tumor immune responses. This study provides novel evidence which further establishes p53/p73 as crucial downstream signalling proteins and important players in T-oligo mediated anti-cancer effects in melanoma. Our results clearly demonstrate that T-oligo may be an effective and novel therapeutic for melanoma.