WT1 Mutations Prevent Normal Hematopoiesis in Acute Myeloid Leukemia

The Wilms Tumor 1 (WT1) gene is a tumor suppressor involved in regulating normal cell growth and differentiation. While WT1 is overexpressed in most acute myeloid leukemia (AML), approximately 10% of both pediatric and adult patients with AML have a mutation in the WT1 gene. WT1-mutated AML is associated with poor outcomes for patients. At this time, however, patients with mutated WT1 are not treated differently from patients with wild-type WT1. The WT1 protein is hypothesized to interact with the proteins TET2 and IDH1/2 in the WIT pathway to initiate the early steps in demethylation. Therefore, WT1-mutated AML should be hypermethylated compared to AML with wild-type WT1. Our work seeks to identify the role of WT1 in normal blood cell development and in leukemogenesis. We hypothesize that WT1 is crucial for normal hematopoiesis and that mutation of WT1 contributes to leukemogenesis by affecting the methylation landscape of the DNA. We initiated this work by exogenously expressing wild-type and mutant WT1 in cell lines that have no WT1 expression. In both the human AML cell line U937 and the mouse myeloid cell line 32D, wild-type WT1 expression results in differentiation by both flow cytometric and morphologic evaluation; however, expression of mutant WT1 does not result in differentiation. In addition, in the HL60 human AML cell line, which expresses wild-type WT1, the small molecule all-trans retinoic acid (ATRA) results in differentiation, again by both flow cytometric and morphologic evaluation. In order to identify the target genes of WT1 in the setting of wild-type WT1 expression, we have optimized chromatin immunoprecipitation (ChIP) for HL60 cells that have been stimulated to differentiate with ATRA. These samples are currently being sequenced to identify genes for which WT1 results in demethylation. These genes, which we anticipate will encode proteins already known to be involved in hematopoietic differentiation and development, will then be validated using quantitative real-time PCR (qRT-PCR) and Western blotting. We also plan to use targeted approaches to characterize the methylation landscape, or methylome, of WT1-wildtype and WT1­-mutant, focusing on the areas where we have shown WT1 to bind to DNA. Our expectation is that we will find hypermethylation of these regions in cells expressing mutant WT1. We will then investigate whether hypomethylating agents, already used as treatment for certain types of AML, can revert the DNA to levels of methylation observed in cells with wild-type WT1 expression; potentially this strategy could be used in the treatment of patients with mutations in the WT1 gene.