The cellular and molecular responses to a novel nucleotide analogue, GS-9219
GS-9219 is a cell-permeable double-prodrug of the acyclic nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG). The conversion of GS-9219 to its active metabolite, PMEG diphosphate (PMEGpp), involves several intracellular enzymatic reactions which reduces the concentration of nephrotoxic PMEG in plasma. PMEGpp competes with the natural substrate, dGTP, for incorporation by DNA polymerases. The lack of a 3'-hydroxyl moiety makes PMEGpp a de facto DNA chain-terminator. The incorporation of PMEGpp into DNA during DNA replication causes DNA chain-termination and stalled replication forks. Thus, the primary mechanism of action of GS-9219 in replicating cells is via DNA synthesis inhibition. GS-9219 has substantial antiproliferative activity against activated lymphocytes and tumor cell lines of hematological malignancies. Tumor cell proliferation was significantly reduced as measured by PET/CT scans in dogs with advanced-stage, spontaneously occurring non-Hodgkin's lymphoma (NHL). The hypothesis of this dissertation is that the incorporation of PMEGpp into DNA during repair re-synthesis would result in the inhibition of DNA repair and accumulation of DNA damage in chronic lymphocytic leukemia (CLL) cells and activate signaling pathways to cell death. To test this hypothesis, CLL cells were treated with DNA-damaging agents to stimulate nucleotide excision repair (NER) pathways, enabling the incorporation of PMEGpp into DNA. When NER was activated by UV, PMEGpp was incorporated into DNA in CLL cells. Following PMEGpp incorporation, DNA repair was inhibited and led to the accumulation of DNA strand breaks. The combination of GS-9219 and DNA-damaging agents resulted in more cell death than the sum of the single agents alone. The presence of DNA strand breaks activated the phosphatidylinositol 3-kinase-like protein kinase (PIKK) family members ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). The activated ATM initiated signaling to the downstream target, p53, which was subsequently phosphorylated and accumulated to exert its apoptotic functions. P53-targeted pro-apoptotic genes, Puma and Bax, were upregulated and activated when DNA repair was inhibited, likely contributing to cell death.
Tsai, Cheng-Yu, "The cellular and molecular responses to a novel nucleotide analogue, GS-9219" (2009). Texas Medical Center Dissertations (via ProQuest). AAI3353995.