SUMMARY ? Project 1 Each year, tens of thousands of people in the United States are diagnosed, and die, as a result of acute myeloid leukemia (AML) and glioblastoma multiforma (GBM). Despite intensive therapies, the five-year survival rate for both tumors is abysmal and has remained unchanged for the last fifty years. Both of these malignancies are characterized by rapid cell division and genetic instability. The goal of Project 1 is to identify clonal and subclonal mutations, present in these malignancies prior to therapy, that may promote proliferation, treatment resistance, and relapse. We have developed a highly accurate and sensitive sequencing protocol, Duplex Sequencing, which takes advantage of the double- stranded nature of DNA to detect mutations, even when present at a frequency of less than one in 10 million. Using our established Duplex Sequencing platform, we have detected subclonal mutations in both AML and GBM that cannot be accurately identified by routine next-generation DNA sequencing. We will pursue the following Aims in Project 1: 1) determine the types of subclonal mutations in AML and GBM; 2) determine how the overall mutation load or presence of specific subclonal mutations correlate with prognosis, response to therapy, and likelihood of relapse (in the case of GBM, we will also determine the geographical heterogeneity of mutations within tumors); and 3) identify potent drugs that prevent the accumulation of these mutations by screening libraries of small molecules. In coordination with other the Projects and Cores, we will analyze how these mutations alter the functions of the encoded genes in these malignancies.