Abstract 773: WT1 regulation of Cyclin A1 in leukemia.

Abstract

Abstract We have observed high levels of Wilms Tumor 1 (WT1) expression in acute leukemias, suggesting that expression of this zinc finger transcription factor might contribute to leukemia development. WT1 has also been used as a marker for minimal residual disease, and patients who relapse often show high WT1 expression in peripheral blood lymphocytes. WT1 levels in the bone marrow of leukemia patients are found to be higher than in normal marrow or peripheral blood. Thus, we predict that WT1 plays a leukemogenic role by regulating expression of growth control genes. Cyclin A1 (CCNA1) is a cell cycle protein expressed in highly proliferative tissues such as the testis and bone marrow, where it is found in both hematopoietic stem cells and leukemia cells. We hypothesized that WT1 up-regulates the expression of CCNA1, potentially enhancing proliferation of leukemic blasts. To determine whether WT1 regulated CCNA1 expression we analyzed the CCNA1 promoter region using the MatInspector software and identified two possible WT1 binding sites. WT1 binding at the CCNA1 promoter in chromatin of K562 leukemic cells was demonstrated by Chromatin Immunoprecipitation (ChIP) analysis using both end point and quantitative real-time PCR (qRT-PCR). Transcriptional activation of the CCNA1 gene by WT1 was tested by transfection of K562 cells with the transcriptionally active WT1 isoform (-KTS) and analysis of RNA expression. Analysis of cDNA by qRT-PCR showed an upregulation of CCNA1 transcripts in WT1 transfected cells. However, upregulation was not observed following transfection with a mutant form of the WT1 that lacks the DNA binding domain (due to a frameshift mutation in exon 7). CCNA1 up-regulation may be relatively specific for leukemia cells, as prostate cancer cells transfected with WT1 showed no changes in CCNA1 levels. To address the clinical relevance of WT1 expression in pediatric acute leukemia, qRT-PCR was used to measure WT1 and CCNA1 transcript levels in leukemic bone marrow, relative to non-neoplastic bone marrow samples. Results showed higher levels of WT1 expression in Acute Myeloid Leukemia (AML) in comparison to Acute Lymphoid Leukemia (ALL). In particular, an examination of AML-M3 bone marrow samples showed high expression of both WT1 and CCNA1, consistent with WT1 upregulation of CCNA1. Additionally, in pediatric ALL samples with elevated CCNA1 levels, we also observed elevated WT1 levels. Overall these findings support the hypothesis that WT1 may up-regulate CCNA1 expression in bone marrow progenitors potentially promoting oncogenic transformation. WT1 may also contribute to leukemogenesis through transcriptional regulation of other growth control genes (such as VEGF, bcl2, and myc) shown to be regulated by WT1 in other tumor types. Ultimately this approach of identifying WT1 target genes up-regulated in leukemic blasts could reveal new biomarkers and identify biologically-defined therapeutic targets. Citation Format: Sony Pandey, Mustafa Moazam, Douglas Snyder, Steven J. Kuerbitz, Gail C. Fraizer. WT1 regulation of Cyclin A1 in leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 773. doi:10.1158/1538-7445.AM2013-773