Novel Therapeutic Approaches for Cancer Therapy Based on Targeting the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

• Author / Creator

  Shire, Zahra

• The relentless growth of tumors is triggered by a complex array of molecular changes such as DNA damage, disruption of cell-cycle progression, uncontrolled proliferation and escaping cell death. Various therapies have been developed to treat cancer, many of which kill cancer cells by damaging their DNA. DNA damage in cells, including DNA strand breaks, are caused by endogenous agents, mainly reactive oxygen species (ROS), and exogenous sources such as ionizing radiation (IR) and topoisomerase poisons, such as irinotecan. Clinical evidence indicates that DNA repair is a major cause of cancer resistance. Therefore, attack on DNA repair processes renders cancer cells more sensitive to radiotherapy and DNA damage chemotherapy.
  Targeting DNA repair enzymes is one approach to overcome resistance in cancer. DNA strand breaks, major lesions generated by ROS, IR and irinotecan, are lethal to cells if not repaired. The 3′- and 5′- termini of the DNA strand breaks are often modified and do not present the correct termini for completion of DNA repair. Among the frequently generated modifications are 3′-phosphate and 5′-hydroxyl termini. Human polynucleotide kinase/phosphatase (PNKP), a bifunctional DNA repair enzyme which phosphorylates DNA 5′-termini and dephosphorylates DNA 3′-termini, can process the unligatable DNA termini. Moreover, cancer cells depleted of PNKP show significant sensitivity to ionizing radiation and chemotherapeutic drugs such as irinotecan. Initial screening for the first generation of small molecule inhibitors of PNKP phosphatase activity identified A12B4C3, an imidopiperidine compound, which enhanced the radio- and chemosensitivity of lung and breast cancer cells. Based on these findings, we intended to identify more potent PNKP phosphatase inhibitors than A12B4C3 and design suitable nanoparticles to target inhibitors to cancer cells.
  First, we developed a novel fluorescence-based assay in order to screen a second generation of imidopiperidine compounds. This resulted in the identification of A12B4C50 and A83B4C63, which are more potent inhibitors than A12B4C3. In addition, we screened new compounds from a natural derivative library, which resulted in the identification of two new promising 3′-phosphatase inhibitors, N12 and O7. The novel assay was used to determine the IC50 values of the newly identified inhibitors. Kinetic analysis revealed that A83B4C63 acts as a non-competitive inhibitor, whereas N12 acts as an uncompetitive inhibitor.
  To test the hypothesis that nano-encapsulation would enhance the effectiveness of the newly identified imidopiperidine-based 3′-phosphatase inhibitors in a cellular context, a series of experiments was carried out with A12B4C50 and A83B4C63. First we examined the retention of the inhibitors by polymeric micelles of different poly(ethylene oxide)-b-poly(ester) based structures to determine suitable encapsulation media for each inhibitor. Cellular studies revealed that encapsulated A12B4C50 and A83B4C63 sensitized HCT116 cells to γ-radiation and irinotecan. Furthermore, the encapsulated inhibitors were capable of inducing synthetic lethalilty in phosphatase and tensin homolog (PTEN)-deficient HCT116 cells. In addition, actively targeted delivery of nano-encapsulated inhibitors to colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR) was achieved by attachment of the peptide GE11 on the surface of polymeric micelles. Preliminary studies with a human xenograft model in nude mice indicated that encapsulated A83B4C63 has the capacity to treat PTEN deficient tumors as a monotherapeutic agent.
  
  Finally, investigation of the potential site of binding of 3′-phosphatase inhibitors to PNKP was determined by photoaffinity crosslinking method coupled with liquid chromatography-mass spectrometry technique (LC/MS/MS). The photoactivatable PNKP inhibitors A95B4C50, A95B4C3 and A12B4C67 revealed three distinct binding sites located in both the kinase and phosphatase domain of PNKP.

• Subjects / Keywords

  • Polynucleotide kinase phosphatase
  • Synthetic lethality
  • Polymeric micelles
  • DNA repair

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3J38M05R

• License

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