Frequently in human cancers, dysregulated expression of transcription factors occurs. In T cell acute lymphoblastic leukemia (T-ALL), a particularly devastating disease in humans, the majority of cases ectopically express the basic helix-loop-helix (bHLH) transcription factor SCL/tal. Normally, SCL/tal promotes hematopoietic stem cell development and erythroid differentiation but is not expressed in thymocytes. A critical step in the function of SCL/tal consists of its heterodimerization with a subfamily of bHLH proteins known as E proteins. This dimerization step represents a compelling target for the design of novel treatment for T-ALL. This project will focus on the dimerization step. Initially, the structural determinants of SCL/tal dimerization with E proteins will undergo detailed dissection. The purpose will be to understand which specific amino acids influence the affinity and specificity of this interaction. The approach will involve in vitro molecular evolution, using random mutagenesis and yeast two-hybrid screening, to create an array of SCL/tal mutants with interesting properties: a) alterations in dimerization affinity, b) alterations in dimerization specificity, c) temperature sensitive dimerization, and d) complementary mutants of SCL/tal and E protein partners. Dimerization will be measured in multiple independent systems: yeast two-hybrid, radiolabelled affinity chromatography, and surface plasmon resonance. Computational modeling will be used to predict the structural basis for the altered dimerazation. In a complementary approach, site-directed mutagenesis will be used to test the predictions of computer generated models of wild type SCL/tal dimerization with E proteins. For functional characterization, selected SCL/tal mutants with known dimerization properties will undergo assays for induction of erythroid differentiation. Inhibitors of SCL/tal promote apoptosis in T-ALL cells. Accordingly, a reiterative in vitro molecular evolution protocol will be exploited to create ultra-high-affinity peptide antagonists of SCL/tal. The HLH dimerization domain of E2-2, an E protein partner of SCL/tal, will be subjected to repeated rounds of random mutagenesis followed by yeast two--hybrid screening for high affinity mutants. Candidate antagonists of SCL/tal will be characterized first in in vitro interaction assays and then tested for the induction of apoptosis in T-ALL cells.
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