Functional Deregulation Of Nf-Kb And Abnormal Tnfa Response In Acute Promyelocytic Leukemia

Abstract Acute promyelocytic leukemia (APL) accounts for approximately 10% of acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in patient bone marrow and peripheral blood. APL is associated with balanced chromosomal translocations involving retinoic acid receptor alpha (RARA), giving rise to fusion oncoproteins referred to as X-RARA. As deregulation of retinoid signaling is insufficient for leukemia development, our studies aim to determine other signaling pathways involved in APL by assessing the gene expression profiles and cell biology of X-RARA. We previously determined, using gene expression microarray analysis, common downstream targets of the variant APL fusion proteins NPM- and NuMA-RARA. We observed an over-representation of NF-κB target genes within this dataset. In these cells, a number of NF-κB target genes were commonly over-expressed. A subset of commonly deregulated genes were validated in our APL cell lines and 23 primary APL patient samples by real-time quantitative RT-PCR (RQ-PCR). 13/16 genes that were tested were significantly altered in APL compared to normal BM (n=11) p<0.05. The majority of these deregulated genes showed a progressive trend towards normal expression levels in post-treated samples. These data indicate defects in NF-κB-mediated gene expression in APL pathogenesis. We next examined NF-κB activity by assessing the expression levels of NF-κB target genes by quantitative real-time RT-PCR, in the presence and absence of TNFα. We observed that there was sustained activation of NF-κB in cells expressing NPM-RARA, as evidenced by the increased expression of NF-κB transcriptional target genes after induction by TNFα. Western analysis of protein derived from U937-NPM-RARA and NB4 cells demonstrated over-expression of NF-κB (p65) protein, as well as its transcriptional target IκBα. The increased pool of NF-κB localized to both the cytoplasm and the nucleus in U937-NPM-RARA cells as visualized by immunofluorescent confocal microscopy. Having observed deregulated expression of downstream targets of TNFα, we sought to examine the ability of X-RARA to confer resistance to TNF-mediated apoptosis. Our results in colony formation assays indicated that NPM-RARA expressing cells formed significantly more colonies, in the presence of 0-100 ng/mL TNFα, in a dose-dependent manner, compared to U937 control cells. These data suggested a greater ability on the part of NPM-RARA+ cells to survive and proliferate in the presence of TNFα. Our data provides the first evidence of the functional deregulation of the NF-κB-mediated signaling pathway and the TNFα response in cells expressing the variant APL fusion proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1240.