Creation of AlkB Mutants for Protection of Bone Marrow

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DESCRIPTION (provided by applicant): Methylating agents are mainstay drugs for the treatment of cancer and are amongst the few agents available against brain tumors. Cells responsible for keeping the blood cell supply, "blood progenitor cells", are highly sensitive to these agents, which results in severe side-effects, including bleeding and infection. Blood progenitor cells can be protected from the toxic effects of methylation by resistance genes. The most effective one is AGT, which directly removes toxic modifications caused by methylation of the cell's genetic material (DNA). However, AGT does not recognize certain toxic DNA modifications. Here I propose the use of a different gene (AlkB), which targets some of these alternative lesions, to improve protection against the toxic effects of methylation. This gene, which has only recently been characterized, should therefore enhance AGT protection. Moreover, unlike AGT, AlkB remains active after mediating a repair reaction, which implies that AlkB repair should be more amenable to modulation. Here I propose a powerful approach to identify variants of one human homologue of AlkB (ABH2) with improved repair performance. Specifically, I want to enhance the enzyme's ability to recognize lesions generated by agents used in the clinic. Selected variants will be tested in human progenitor cells in culture and in a mouse animal model for protection against treatment with high doses of these agents. Ultimately, the mutants identified in this work should improve the management of side effects of methylating agents and would also allow the use of higher doses for more effective treatment.