Micro-RNA and Gene Expression In CLL: Network Analysis Elucidating the Complex Role of the NF-κB Signaling Pathway In Disease.

Abstract 3642

NF-κB is a family of transcription factors associated with increased cell proliferation and evasion of apoptosis. NF-κB has been reported to be constitutively active in chronic lymphocytic leukemia (CLL), but neither the underlying mechanism of this activation or genes regulated by this important survival transcription factor are fully understood. To further define the mechanism(s) by which NF-κB promotes survival in CLL, we investigated the effect on this signaling pathway on the regulation of both mRNA (Affymetrix Human Genome U133 Plus 2.0 Array) and microRNA (miR) (custom 8K human microRNA expression Array; V 2.0) expression in CLL cells (n=10) following treatment with several therapeutic agents either currently in use or previously described for CLL. Additionally we compared these drug mediated gene expression signatures with those of CD40L and CpG oligodeoxynucleotide (ODN), factors known to induce CLL cell survival at least in part via NF-κB. In each case chosen, activation was demonstrated by up-regulation of known NF-κB target genes and also enhanced EMSA binding. One attractive therapeutic target currently being explored in CLL is the chaperone protein HSP90, and our group has recently described the inhibitory effect of the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) on NF-κB signaling in primary CLL cells. We showed that 17-DMAG inhibits both IKKα and IKKβ, the two main activating kinases of the NF-κB pathway. In the current study, we compare the effect of 17-DMAG to that of BAY-11, a kinase inhibitor known to specifically interfere with IKKβ and therefore classical NF-κB signaling. Microarray analysis reveals that these two agents differ in the regulation of several groups of NF-κB target genes, suggesting specific downstream consequences of targeting the alternative versus the classical NF-κB signaling pathway. Furthermore, we analyzed miR expression using miR array analysis and find an inverse relationship between the expression of several miRs and their target gene transcripts, including MCL1, an oncogene known to contribute to CLL pathogenesis. Interestingly, while 17-DMAG has a much broader and more potent effect on gene expression, BAY-11 exhibits more regulation on miR expression. This suggests different roles for alternative and classical signaling, as well as HSP90, in the regulation of miRs compared to mRNA transcripts. Finally, in order to address the potential role of NF-κB and the tumor microenvironment in the regulation of CLL cell survival, we analyzed the gene expression profile following in vitro treatment of CLL cells with two different activators of NF-κB, CD40L and CpG ODN. CD40L is able to activate both alternative and classical NF-κB signaling, while CpG ODN is more specific to classical signaling. Pathway analysis reveals that while CpG ODN regulates substantially more genes than CD40L, both treatments control similar pathways such as inflammatory response, cell growth and differentiation and cell death, and as expected both treatments are closely connected with immune disease and cancer. Interestingly, CpG ODN, unlike CD40L, increases the expression of DLEU2, a gene associated with the miR-15a/16-2 cluster commonly deleted in CLL and associated with disease progression. This suggests that CpG ODN, while typically considered pro-survival, may contribute to cell death through up-regulation of specific target genes. Overall, these array analyses help to elucidate diverse NF-κB signaling pathways and their importance in CLL cell survival. Additionally, this study explores the relationship between miR expression and gene expression following NF-κB manipulation in CLL cells.