Project Summary/ Abstract Telomere maintenance at chromosome ends is essential for the growth of cancer cells. In humans, telomeres end in a single strand overhang consisting of GGTTAG repeats that form the substrate for telomerase. Telomerase is a reverse transcriptase composed of two subunits: a protein component (TERT) and an RNA component (TR) which contains an integral template used to reverse transcribe multiple telomeric repeats during a single telomere binding event. Telomerase is an attractive target for cancer therapies because it is expressed in over 85% of cancer cells, while most adult somatic cells lack telomerase. Thiopurines are a class of nucleoside analogs used to treat leukemia and some pediatric cancers but are highly toxic. The prodrug 6- thio-2'-deoxyguanosine (6-thio-dG) was developed to reduce non-specific thiopurine toxicity. This drug was shown to successfully reduce the growth of mouse tumor xenografts for multiple cancer types. 6-thio-dG treatment can also cause telomere shortening and dysfunction, however, the mechanism was unknown until recently. In biochemistry experiments, I discovered that human telomerase can readily add the 6-thio-dG metabolite, 6-thio-dGTP, to a growing telomere chain, but this insertion then strongly inhibits telomerase’s ability to add additional telomeric repeats. I found that POT1-TPP1, which normally enhances telomerase binding and the addition of multiple repeats, cannot restore telomere elongation in the presence of 6-thio- dGTP. In addition, 6-thio-dGTP has a low micromolar IC50 for human telomerase due to telomerase’s inability to discriminate between dGTP and 6-thio-dGTP but does not inhibit DNA polymerase progression, reducing its off-target impact. My preliminary data for this project demonstrate that 6-thio-dG can disrupt telomerase binding when the modified base is located at the 3’ end of the telomeric overhang. I hypothesize that 6-thio-dG addition impairs telomerase’s interaction with the telomere, thereby disrupting cycling for repeat addition synthesis and inhibiting telomerase elongation of telomeres in cells. I will use complementary biochemical, single-molecule, and cellular approaches to test my hypothesis. Aim 1 will elucidate the mechanism by which 6-thio-dG disrupts the telomerase catalytic cycle. Aim 2 will determine whether telomerase can synthesize DNA and extend telomeres in cancer cells treated with 6-thio-dG. Aim 3 will determine if 6-thio-dG efficacy in cancer cells is enhanced by inhibiting the thiopurine sanitase, NUDT15. Completing this project will improve our understanding of how 6-thio-dG impacts telomerase activity and telomere maintenance in cancer cells. It will also enhance our understanding of how 6-thio-dGTP nucleotide impacts telomerase catalysis and its potential utility as a therapeutic for halting cancer cell proliferation. This fellowship will allow me to acquire new skills related to the project’s aims and outlines specific mentors for each. Ca...