Aberrant Methylation and Decreased Expression of NRIP1 in IGHV-Unmutated CLL

Aberrant DNA methylation patterning occurs in many cancers, including chronic lymphocytic leukemia (CLL). Methylation programming changes as B cells differentiate from immature progenitors to mature B cells. CLL clones originate from a continuum of B cell maturation states, which differ in their "methylome." Emerging evidence suggests that aberrant methylation programming occurs at numerous genes that are differentially expressed in IGHV-defined CLL subtypes. Nuclear receptor interacting protein 1 (NRIP1) reproducibly shows significant gene expression differences in IGHV unmutated (U-CLL) and mutated CLL (M-CLL), with lower transcript levels observed in U-CLL cells. Low expression of NRIP1 is associated with poorer overall survival and time-to-treatment. NRIP1 encodes a cofactor that interacts with various nuclear receptors and other proteins to regulate transcription of genes involved in cellular proliferation, survival, metabolism, inflammation, and other processes.

Published data reveal several CpG dinucleotides near the NRIP1 promoter that undergo increasing methylation during the maturation and differentiation of normal peripheral blood B cells (NBC), concordant with a stepwise decrease in transcript levels of NRIP1 over the course of maturation. Interestingly, our analysis of publically available expression data revealed significantly lower NRIP1 expression in U-CLL cells (the less mature CLL subtype) than NBC, while M-CLL cells (the more mature CLL subtype) express levels comparable to NBC. Given these observations, our objective was to interrogate the promoter and regulatory regions at the NRIP1 locus for differences in CpG methylation in genomic DNA isolated from U-CLL, M-CLL, and NBC. We hypothesized that the NRIP1 locus is more highly methylated in U-CLL than in M-CLL and NBC, and that this increased methylation represses NRIP1 gene transcription, contributing to its aberrantly low expression in U-CLL compared to M-CLL and NBC.

We bisulfite-converted genomic DNA from negatively-selected CD19+ treatment-naïve CLL samples (n=74 U-CLL, n=37 M-CLL) and NBC from healthy donors (n=5). We PCR-amplified regions in or near the CpG island of NRIP1 and used Agena Bioscience's EpiTyper kit to prepare the samples for analysis on a MassARRAY spectrometer. EpiTyper software was used to detect mass differences that indicate either non-methylated or methylated CpG-containing fragments, and to calculate their relative frequency in each sample. For each CpG locus that was assayed, we fit a beta-regression model to detect differential methylation between M-CLL and U-CLL. We also computed Pearson correlation coefficients comparing methylation levels to gene expression levels.

We assessed 47 CpGs mapping to the promoter and nearby regulatory elements of NRIP1 for methylation frequency and correlation with NRIP1 expression. From our comparison of methylation frequency in U-CLL and M-CLL, 3 adjacent CpG loci were significant at false discovery rate (FDR) =1% (CpG 58, p=0.00016; CpG 144, p=0.00062; and CpG 155, p=0.00287). The three most significant correlation coefficients arose from the same three CpG loci (CpG 58, R=0.677, p=2.2e-16; CpG 144, R=0.539, p=7.16e-10; CpG 155, R=0.651, p=5.77e-15). Unexpectedly, all 3 loci were methylated at higher levels in M-CLL and at lower levels in U-CLL (on average), and expression was positively correlated with methylation levels. These CpGs map near a documented binding site for the repressive transcription factor RUNX3, prompting us to hypothesize that differential methylation in U-CLL versus M-CLL may affect the binding affinity of RUNX3 and contribute to the robust NRIP1 expression differences in these CLL subtypes. Future work will test this hypothesis using gel-shift and luciferase reporter assays followed by chromatin immunoprecipitation experiments.

Few studies have focused on aberrant methylation patterning in CLL and the consequences of deregulated gene expression. We are the first to look at NRIP1 for differences in DNA methylation across IGHV subtypes. Future studies aimed at understanding how methylation at this locus affects NRIP1 transcription, as well as how levels of NRIP1 affect cell survival pathways, will enhance our understanding of the role of this gene in CLL, and the contributions of deregulated methylation to disease pathophysiology.