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Significance. We have discovered a new therapeutic agent that is highly specific to a subtype of human leukemia cells. Normal cells do not appear to be impacted by our new treatment, but some types of leukemia cells are ablated. Targeted therapies that spare normal cells and avoid the harsh toxicity and long-term damage caused by current regimens are the next wave of cancer treatment. We believe we are on the cutting edge of cancer treatment with this new therapy, and that this treatment is likely to lead to a clinical trial. Background. We hypothesize that we have derived a novel therapy which cures a major fraction of human leukemia. microRNA are small RNA molecules that block the expression of proteins. We identified two microRNAs as targets of a pathway frequently activated in human acute myeloid leukemia. Specifically, we have shown that these microRNA are direct transcriptional targets of the HoxA9 transcription factor. HoxA9 is of critical interest in human acute myeloid leukemia because it is activated by multiple genetic changes (chromosome 11 translocations involving MLL1 or Nup98). Moreover, according to three independent studies, the expression level of HoxA9 is prognostic in human AML (lacking chromosome 11 translocations). Thus, one would expect that some downstream targets of HoxA9 would signal the leukemic transformation engendered by HoxA9 expression. To determine the relevance of the microRNA to HoxA9-related leukemia, we used antisense RNA oligonucleotides (antagomir) to treat mice transplanted with one million MLL-Af9- initiated leukemia cells. We also used control antagomirs that were mutated so that they had a similar sequence, but do not bind microRNA. Mice treated with these control antagomirs died rapidly. In contrast, only one of five mice treated with antagomirs died. Importantly, the four surviving antagomir-treated mice lived long after the pump ran out of antagomirs, meaning the the treatment had a lasting effect. Next, we treated primary human leukemia cells with antagomirs for 30 minutes, then assessed their ability to form multicellular colonies in an incubator (a surrogate for leukemia formation in the body). A 50% reduction in colonies was seen in a leukemia with abnormal HoxA9 signaling (MLL, 11q23), but not a leukemia which uses a different pathway (AML-ETO)(Fig. 1B). We don’t have room to show it, but this effect is very similar to the effect of the antagomirs on colony formation from murine bone marrow cells engineered to express these leukemia-causing proteins. Specifically, each time we treat leukemias with the antagomirs we get sequentially less and less colonies, suggesting that the treatment is blocking the stem cells that are causing the disease. Experimental Design: We need to confirm and expand these preliminary experiments. First, we will repeat the mouse modeling with at least two independent MLL-Af9 initiated leukemias. We will also characterize the effect of the treatment on growth, survival, and differentiation of both the bulk leukemia cells and the c-Kit+ leukemia stem cells. We want to know which cells within the leukemia are affected by the treatment. Second, we will treat three primary human 11q23 translocation leukemias and assess colony formation. If these leukemias are sensitive to treatment, we will initiate xenografts with these leukemias and treat with osmostic pump-delivered antagomirs. Dr. Martin Carol (U. Penn) already knows that these leukemias will engraft in immunodeficient mice. We want to know if the treatment affects human leukemias in the body. Finally, we will treat the leukemias with a different inhibitor (LNA oligos). We want to know the most efficacious way to target the microRNA. Future studies will examine the cancer causing biological pathways engendered by the microRNA.