Survival rates for patients with acute myeloid leukemia (AML) remain low, despite the fact that knowledge of the molecular events that cause AML has evolved rapidly in the last decade. This is in part due to a distinct lack of understanding of the response to chemotherapy at the cellular and molecular levels. Previous work has been directed toward the long term goal of developing novel, rational combination-therapies to improve the outcomes of patients with AML. The objective of this specific project is to provide pre-clinical validation of one such therapeutic strategy, combining antimetabolite chemotherapeutics with inhibition of Wee1, a cell cycle checkpoint protein. The central hypothesis of this project is that Wee1 is a critical mediator of leukemia cell survival, acting inS phase to ensure accurate DNA replication, and that combining inhibition of Wee1 with antimetabolites will be more effective in eliminating leukemia cells than antimetabolites alone. This hypothesis emanates from our preliminary data from a genome-wide shRNA screen, in which we identified Wee1 as a critical mediator of AML cell death after cytarabine treatment. We have validated these findings in a broad panel of AML cell lines and found that the effect of inhibiting Wee1 in combination with cytarabine involves abrogation of the S phase checkpoint induced by cytarabine. Notably, preliminary data show that cells with Class I leukemogenic mutations are quite sensitive to Wee1 inhibition alone and suggest that Wee1 may contribute to leukemogenesis. The hypothesis will be tested with three specific aims: 1) Determine whether Wee1 contributes to leukemogenesis, 2) Confirm the efficacy and tolerability of cytarabine and Wee1 inhibition in vivo and 3) Identify the mechanism of enhanced AML cell killing with cytarabine and Wee1 inhibition. To achieve Aim 1, we will examine the consequences of Class I and II mutation expression and Wee1 inhibition as they relate to oncogenic stresses in myeloid progenitor cells and perform immunohistochemistry for Wee1 expression in AML bone marrow biopsy specimens. To achieve Aim 2, mouse models of AML, including direct patient xenografts will be used to test the pharmacodynamics, tolerability and efficacy of combining cytarabine with Wee1 inhibition. To achieve Aim 3, pharmacologic and genetic inhibition will be used to test the functional significance of proteins upstream, downstream and in parallel with Wee1 in cell cycle checkpoint cascades to identify the key molecules involved;complementary cellular and molecular assays will be used to further define the mechanism of combinatorial cell killing. The project is innovative in 1) the study of Wee1 contributing to leukemogenesis, 2) the study of non-oncogene vulnerabilities of FLT3-ITD+ cells, 3) the development of a novel therapeutic target in AML, 4) translational modeling of leukemia with direct patient xenografts, and 5) high-throughput methodology to survey the role of DNA damage repair proteins in specific contexts. The proposed research is significant because it is expected to advance the understanding of a novel therapeutic target in AML that can be rapidly translated into clinical trials combining standard and targeted therapy.
The proposed research is relevant to public health because new treatment strategies to improve the survival rates of patients with acute myeloid leukemia are desperately needed. Current treatment protocols have maximized the doses of chemotherapy to the cusp of tolerability. The proposed studies will guide the development of novel targeted drug/chemotherapy combination strategies designed to potentiate the effects of the most important drugs for AML. Thus, the proposed research is relevant to a goal of the NCI's Division of Cancer Treatment and Diagnosis to decrease the time necessary to bring anticancer drugs and biomarkers to the clinic while enhancing the ability to predict treatments that will be most useful for each patient.
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