The Role of Wilms’ Tumor Protein 1 (WT1) in Chronic Myelogenous Leukemia Progression.

Wilms’ tumor protein 1 (WT1) is a key regulatory gene whose function is required for proper development and differentiation of the heart, retina, genito-urinary, olfactory and hematopoietic systems. WT1 over expression is a characteristic observed in blasts of Chronic Myeloid Leukemia (CML). Despite this apparent co-relation, a direct link between WT1 over expression and progression of CML to blast crisis has never been established. The two major isoforms of WT1 are generated by alternative splicing; an event that involves the use of two alternative splice sites at the end of exon 9. The outcome is the insertion or not of only 3 amino acids (lysine, threonine and serine; KTS) at the c-terminus of the protein. The ratio between these isoforms is conserved among tissues and disruption of this balance can end up causing developmental abnormalities. Our group is testing if the over expression of WT1 in hematopoietic precursors can lead to progression in a murine model of CML. Over expression of WT1 has been shown to block differentiation in myeloid cell lines. However, over expression of WT1 in primary hematopoietic cells has variable effects on differentiation and proliferation. These differences may reflect differences in vector design and/or assays to evaluate. We have utilized the Murine Stem Cell Vector (MSCV), a retroviral construct that has high efficiency of transducing murine hematopoietic stem cells, to generated retroviral constructs. The vector contains an internal ribosomal entry site allowing expression of WT1+KTS or WT1-KTS isoforms with and without Bcr-Abl/GFP, the causative protein in CML. Retroviral transduction of hematopoietic progenitors was performed for 48 hours and FACS sorted GFP⁺Sca⁺lin⁻ cells were utilized for colony forming assays in methylcellulose and in liquid culture. Our results have shown that primary murine hematopoietic progenitors transduced with Bcr-Abl/GFP and WT1+KTS demonstrate dramatically greater cytokine independent growth than progenitors transduced with Bcr-Abl/GFP alone. In contrast, progenitors transduced with Bcr-Abl/GFP and WT1-KTS demonstrate impaired growth.

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In addition transduced cells will be used to rescue lethally irradiated mice what will provide an excellent model to determine if WT1 cooperates with BCR-ABL to induce blast crisis. The exact function of each WT1 isoform is still unknown. However, there is clear data that they have different properties and are involved in different functions. Several lines of evidence suggest a role for the +KTS isoform in splicing. During hematopoietic cell differentiation, levels of WT1 protein were determined to decrease drastically. This fluctuation in WT1 expression level may play a critical role in the process of differentiation. In conclusion, our results strongly suggest that WT1 expression influences Bcr-Abl induced growth with the isoforms WT1+KTS and WT1-KTS displaying differential effects. Our data indicate that +KTS enhances Bcr-Abl dependent cellular proliferation while -KTS isoform impairs proliferation.