The broad objectives of this proposal are to understand how the Bcr/Abl protein induces chronic myeloid leukemia (CML), to create improved murine model systems that will facilitate the study of CML, and to develop an experimental framework for identifying and studying genes responsible for related myeloproliferative disorders.
The specific aims outlined below will address how Bcr/Abl induces bone marrow proliferation by activating cytokine-signaling pathways. Normal hematopoiesis is regulated by cytokines, but the hallmark of myeloproliferative disease is autonomous hematopoiesis, a pathology most clearly documented for erythroid progenitors, which form colonies in culture in the absence of erythropoietin (EPO). A prevailing hypothesis to explain the origin of this autonomous hematopoiesis is that somatic mutations arise in post-receptor signaling proteins, deregulating mitogenic pathways normally controlled by cytokine receptor signal transduction. Of the four adult myeloproliferative disorders, CML is the only one for which the activated signaling molecule is known. The CML-specific Bcr/Abl oncoprotein can abrogate growth factor requirements for established cell lines in culture and induce a CML-like myeloproliferative syndrome in mouse models, but which pathways are crucial to disease in vivo is unknown. The observation the EPO-independent erythropoiesis in CML patients requires stem cell factor (SCF), and recent insights into the cooperation of the EPO and SCF receptor pathways, suggest that Bcr/Abl functionally substitutes for the EPOR requirement in the SCF signaling pathway. Preliminary data confirm this by demonstrating that Bcr/Abl expression can rescue erythropoiesis in fetal liver progenitors of mice lacking the EPOR. This system affords a very direct assessment of the role of Bcr/Abl in a defined cytokine pathway. He proposes to determine which disease-related forms of Abl (P210 and P185 Bcr/Abl, Tel/Abl, and v-Abl) will rescue erythropoiesis from fetal liver progenitors and embryonic stem cells from EPOR(-/-) mice, using in vitro hematopoietic colony assays and in vivo reconstitution experiments. Through mutational analysis of Bcr/Abl and strategies for inhibiting the function of downstream signaling molecules, the p.i. will determine which domains of Bcr/Abl mediate signaling and what pathways are critical for rescue of erythropoiesis. He will then extend these studies to determine whether Bcr/Abl will induce myeloid colony formation in mice deficient in the IL-3, GM-CSF, and thrombopoietin receptors. Longer term objectives include expressing Bcr/Abl in ES cells under conditional promoters to develop a breedable strain of mice with regulated Bcr/Abl expression, and identifying genes relevant to other myeloproliferative disorders through expression cDNA cloning.
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