The goal of this proposal is to establish a novel mouse transformation model in which the human T cell leukemia virus type I (HTLV-1) Tax oncoprotein is conditionally expressed in T cells, and to use this model to determine the transforming activity of wild-type and mutant Tax proteins. HTLV-1 is a human retrovirus that currently infects approximately 15 million people in the world and is the cause of the aggressive malignancy, adult T cell leukemia/lymphoma. Although the Tax oncoprotein has been shown to transform cells in culture and to induce tumors in a variety of transgenic mouse models, the mechanism by which Tax transforms cells is not well understood. A large number of Tax mutants have been generated and their biological activities have been thoroughly characterized primarily in cell culture systems. A major problem currently facing the field is that the transforming activity of Tax mutants cannot be compared using available transgenic models due to random transgene integration sites, variable transgene copy number and inconsistent transgene expression levels. Thus, it is very difficult to link the biological activities of Tax mutants with their transforming potential. To solve this problem we will develop an innovative mouse model in which to study Tax transformation using vectors containing wild-type or mutant Tax genes that are silenced by a preceding floxed stop cassette. These vectors will be knocked in to the Rosa26 locus of recipient mice by recombination. By crossing these mice with Lck-CRE mice, the stop cassette will be specifically excised in developing thymocytes where the Lck promoter is active, allowing conditional expression of wild-type or mutant Tax proteins in T cells, the natural target of HTLV-1 infection. Insertion into the Rosa26 locus will eliminate inconsistent integration sites and standardize gene copy number resulting in consistent levels of wild-type and mutant Tax protein expression. The mouse model will be established in Aim 1 by creating targeting vectors containing silenced wild-type or mutant Tax genes. Targeting vectors will be knocked in to the Rosa26 locus of C57BL/6 mice, which will then be crossed with homozygous Lck-CRE mice, thereby excising the stop cassette to generate mice expressing wild-type or mutant Tax specifically in T cells. Aim 2 will examine the effect of mutations that disable specifi biological functions of Tax on Tax- mediated tumorigenesis. Tax can bind to and regulate the activity of members of the SRF, CREB, NF-kB and PBM protein families, each of which has been implicated in oncogenesis. Mice established in Aim 1 will be used to compare for the first time the tumorigenic potential of wild-type and mutant Tax proteins in an effort to identify pathways that are required for Tax tumorigenesis. The proposed studies will establish a new mouse model that will overcome current limitations and provide greater insight into the mechanism of HTLV-1 Tax transformation, knowledge that is currently lacking and that promises to yield novel insights into viral and cellular biology.

R21CA175404

2013-04-01

2016-03-31