Abstract B33: The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells.

The Wilms tumor gene, WT1, encodes multiple splice isoforms with varying functions including both transcriptional and post-transcriptional regulation mediated by DNA, RNA and protein binding domains. Involvement of WT1 in regulation of various growth control genes has been widely studied in many solid tumor types. However, evidence of its role in leukemia has been contradictory; although WT1 is both overexpressed and/or mutated in different subsets of leukemia patients, and these aberrations are linked to poor or intermediate prognosis. Our working hypothesis is that in leukemia WT1 regulates expression of genes that play mitogenic roles, such as Cyclin A1 (CCNA1) and Vascular Endothelial Growth Factor (VEGF). To demonstrate that WT1 regulates these genes we first identified potential WT1 binding sites in both CCNA1 and VEGF promoters, using the MatInspector Software. Three potential WT1 binding sites were located within both the CCNA1 and VEGF gene promoters and their functional significance was validated by chromatin immunoprecipitation (ChIP) assays. ChIP analysis of chromatin from K562 cells revealed WT1 binding at 2 of 3 sites within the CCNA1 promoter; and in chromatin of 293 Kidney cells and LNCaP prostate cancer (PC) cells, WT1 binding was observed in VEGF promoter. To demonstrate that these functional sites are involved in modulating transcription from these promoters, we performed luciferase assays using reporter constructs containing CCNA1 (-1180/+145) and VEGF (-411/+50) promoter regions. Co-transfection of WT1 with these reporter constructs demonstrated that both gene promoters were activated by WT1 over-expression in K562 cells. Transcriptional regulation of the endogenous genes was confirmed by Quantitative PCR in K562 cells transfected with WT1. Although CCNA1 mRNA levels increased as expected, no significant changes in the VEGF mRNA levels were observed. This absence of VEGF mRNA up-regulation in K562 cells differs from that observed in kidney and PC cells, and could be attributed to many factors, including a lack of necessary co-activators. To determine the biological relevance of WT1 mediated regulation, we measured the levels of WT1, CCNA1 and VEGF mRNA levels in pediatric leukemia bone marrow (BM) samples using Quantitative PCR. Overall, WT1 levels were higher in Acute Myelogeneous Leukemia- M3 than in Acute Lymphoblastic Leukemia BM samples and WT1 levels were low or undetected in non-neoplastic BM. The AML-M3s samples with high WT1 levels also had higher expression of CCNA1. Conversely, in ALL samples variation was seen in the expression of WT1 and CCNA1. VEGF transcript levels in leukemia BM were generally near or below those in normal BM. Taken together, these results suggest that WT1 transcriptionally up-regulates CCNA1, but the regulation of VEGF may be cell specific. Since CCNA1 is primarily expressed in leukemias and normal testes, this suggests that WT1 could be a significant factor controlling expression of this proliferative gene. Conversely,VEGF is expressed in many different tissues and under many different conditions, including hypoxia, suggesting that WT1 may be part of the many factors contributing to the complex orchestration of VEGF expression. Nonetheless when these proliferative factors are up-regulated in leukemia, WT1 may contribute to their altered expression levels, and therefore be a leukemogenic factor. Citation Format: Sony Pandey, Shorog Al omair, Mustafa Moazam, Kurtis Eisermann, Steven J. Kuerbitz, Gail C. Fraizer. The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B33.