Abstract B50: An innovative technique for preventing telomere elongation and its associated immortalization of cancer cells
Authors: Laster Brenda , Isaacson Carol , Perets Ekaterina , Msamra Maha , Priel Esther , Kalef-Ezra John , Kost Joseph 
Published: May 2015

This work offers an innovative technique to inhibit the activation of the telomerase enzyme over the long term and improve the outcome of cancer treatments. Telomerase activation elongates telomeres, sequences of DNA at the ends of chromosomes. This action confers unlimited proliferation on cancer cells and increases their resistance to chemo- and radio- therapy. Telomeres also lengthen when cells undergo homologous recombination via an Alternative Lengthening of Telomeres (ALT) pathway. Our technique embodies an effective drug delivery system (DDS) that maximizes tumor uptake of the inhibitor and provides its long term continuous release. This is accomplished by the direct, intratumoral insertion of a biodegradable polymeric rod incorporating a telomerase-inhibiting drug. Long-term continuous release of the drug is critical because, if the inhibitor is cleared from the tumor cell, telomerase is rapidly reactivated and the telomeres re-lengthen. The drug is a well-documented telomerase-inhibiting porphyrin (TMPyP4) that is incorporated into a Poly(Lactic-co-Glycolic) Acid (PLGA) polymer and inserted directly into anatomically-accessible tumors. The properties of TMPyP4 are retained even when the porphyrin is tagged with a stable palladium atom (Pd), PdTMPyP4. The molecule binds to, and influences the configuration of G-quadruplex (GQ) regions in DNA. GQs are located, among other sites, in the promoter region of the c-myc oncogene and in the telomeres. By stabilizing the promoter region of c-myc, PdTMPyP4 prevents its over-expression and stimulation of the human telomerase reverse transcriptase (hTERT), a subunit of telomerase, thereby inhibiting the activation of telomerase. The intratumoral insertion of the rod increases tumor-loading of the drug and permits the gradual and continuous release of PdTMPyP4 over the long term.

ICP-MS measurements of Pd uptake in the DNA of Hodgkin's Lymphoma cells after a 24 or 48h incubation with PdTMPyP4 yielded &gt;109 atoms/cell, and telomerase activity was significantly reduced as shown by the TRAP assay. Daily spectrophotometric measurements of the optical density (OD) of PdTMPyP4 shows its gradual and constant release from rods in vitro for 52 days and ex vivo for &gt;30 days. The concentration of PdTMPyP4 released from the rods is proportional to the mass of the rods. After insertion of the rods into the KHJJ murine mammary adenocarcinoma borne on the BALB/c mouse thigh, tumor growth was 5-8 times slower than tumors without rods. Most importantly, based upon OD measurements of blood plasma, systemic uptake of PdTMPyP4 is negligible after intratumoral insertion of the rod into the KHJJ tumors.

Each individual component of PdTMPyP4 independently challenges telomere elongation mechanisms. TMPyP4 inhibits telomerase, and the DNA-bound Pd atoms can be exploited to continuously fragment telomeres lengthened by ALT. The simultaneous or sequential intratumoral implantation of iodine-125 brachytherapy seeds and PdTMPyP4 rods, in a single treatment procedure, can produce clustered, non-repairable damage at all Pd-bound DNA sites due to the dense ionization of cascading Auger electrons. The 27 keV photon energies of iodine-125 seeds are ideally suited for inducing a photoelectric effect at the 24.5 K absorption edge of Pd, thereby eliciting the emission of low energy, short range Auger electrons over a sphere of 25 nm. Thus, the combined, but independent advantage of each molecular component, offers a potentially useful comprehensive therapeutic modality. Further, the intratumoral insertion of only the PdTMPyP4-releasing rod offers the potential of increasing the sensitivity of malignant tumors to conventional cancer treatments through telomerase inhibition. Conclusions reached thus far in our study strongly suggest that our technique for inhibiting the activation of telomerase has substantial utility for addressing a broad range of cancer types, such as prostate, breast, brain, and head and neck.

This work offers an innovative technique to inhibit the activation of the telomerase enzyme over the long term and improve the outcome of cancer treatments. Telomerase activation elongates telomeres, sequences of DNA at the ends of chromosomes. This action confers unlimited proliferation on cancer cells and increases their resistance to chemo- and radio- therapy. Telomeres also lengthen when cells undergo homologous recombination via an Alternative Lengthening of Telomeres (ALT) pathway. Our technique embodies an effective drug delivery system (DDS) that maximizes tumor uptake of the inhibitor and provides its long term continuous release. This is accomplished by the direct, intratumoral insertion of a biodegradable polymeric rod incorporating a telomerase-inhibiting drug. Long-term continuous release of the drug is critical because, if the inhibitor is cleared from the tumor cell, telomerase is rapidly reactivated and the telomeres re-lengthen. The drug is a well-documented telomerase-inhibiting porphyrin (TMPyP4) that is incorporated into a Poly(Lactic-co-Glycolic) Acid (PLGA) polymer and inserted directly into anatomically-accessible tumors. The properties of TMPyP4 are retained even when the porphyrin is tagged with a stable palladium atom (Pd), PdTMPyP4. The molecule binds to, and influences the configuration of G-quadruplex (GQ) regions in DNA. GQs are located, among other sites, in the promoter region of the c-myc oncogene and in the telomeres. By stabilizing the promoter region of c-myc, PdTMPyP4 prevents its over-expression and stimulation of the human telomerase reverse transcriptase (hTERT), a subunit of telomerase, thereby inhibiting the activation of telomerase. The intratumoral insertion of the rod increases tumor-loading of the drug and permits the gradual and continuous release of PdTMPyP4 over the long term.

ICP-MS measurements of Pd uptake in the DNA of Hodgkin's Lymphoma cells after a 24 or 48h incubation with PdTMPyP4 yielded &gt;109 atoms/cell, and telomerase activity was significantly reduced as shown by the TRAP assay. Daily spectrophotometric measurements of the optical density (OD) of PdTMPyP4 shows its gradual and constant release from rods in vitro for 52 days and ex vivo for &gt;30 days. The concentration of PdTMPyP4 released from the rods is proportional to the mass of the rods. After insertion of the rods into the KHJJ murine mammary adenocarcinoma borne on the BALB/c mouse thigh, tumor growth was 5-8 times slower than tumors without rods. Most importantly, based upon OD measurements of blood plasma, systemic uptake of PdTMPyP4 is negligible after intratumoral insertion of the rod into the KHJJ tumors.

Each individual component of PdTMPyP4 independently challenges telomere elongation mechanisms. TMPyP4 inhibits telomerase, and the DNA-bound Pd atoms can be exploited to continuously fragment telomeres lengthened by ALT. The simultaneous or sequential intratumoral implantation of iodine-125 brachytherapy seeds and PdTMPyP4 rods, in a single treatment procedure, can produce clustered, non-repairable damage at all Pd-bound DNA sites due to the dense ionization of cascading Auger electrons. The 27 keV photon energies of iodine-125 seeds are ideally suited for inducing a photoelectric effect at the 24.5 K absorption edge of Pd, thereby eliciting the emission of low energy, short range Auger electrons over a sphere of 25 nm. Thus, the combined, but independent advantage of each molecular component, offers a potentially useful comprehensive therapeutic modality. Further, the intratumoral insertion of only the PdTMPyP4-releasing rod offers the potential of increasing the sensitivity of malignant tumors to conventional cancer treatments through telomerase inhibition. Conclusions reached thus far in our study strongly suggest that our technique for inhibiting the activation of telomerase has substantial utility for addressing a broad range of cancer types, such as prostate, breast, brain, and head and neck.

Citation Format: Brenda Laster, Carol Isaacson, Ekaterina Perets, Maha Msamra, Esther Priel, John Kalef-Ezra, Joseph Kost. An innovative technique for preventing telomere elongation and its associated immortalization of cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B50.