MicroRNA Expression Profiling Identifies Activated B Cell Status in Chronic Lymphocytic Leukemia Cells Shuqiang Li1, Howell F. Moffett1,2, Jun Lu3¤, Lillian Werner4, Hao Zhang3, Jerome Ritz5,6, Donna Neuberg4, Kai W. Wucherpfennig1,7, Jennifer R. Brown5,6, Carl D. Novina1,3,8* 1 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America, 2 Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 4 Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America, 5 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America, 6 Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America, 7 Department of Neurology, Harvard Medical School, Boston, Massachusetts, United States of America, 8 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70+ and IgVH unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL. Citation: Li S, Moffett HF, Lu J, Werner L, Zhang H, et al. (2011) MicroRNA Expression Profiling Identifies Activated B Cell Status in Chronic Lymphocytic Leukemia Cells. PLoS ONE 6(3): e16956. doi:10.1371/journal.pone.0016956 Editor: Venugopalan Cheriyath, Cleveland Clinic, United States of America Received August 30, 2010; Accepted January 18, 2011; Published March 8, 2011 Copyright: 2011 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders of the study include NIH grant (PO1-CA081534), NIH grant (K23 CA115682), NRSA Fellowship (DFCI/NCI T32 CA70083), Carreras International Leukemia and Doris Duke Clinical Scientist Development Awards. Competing Interests: The authors have declared that no competing interests exist. * E-mail: carl_novina@dfci.harvard.edu ¤ Current address: Yale Stem Cell Center and Department of Genetics, Yale School of Medicine, New Haven, Connecticut, United States of America Introduction CLL is the most common form of adult leukemia in the western world accounting for approximately 30% of all leukemias in Caucasians. Contrary to its earlier description as a relatively homogeneous disease, CLL more recently has been viewed as a heterogeneous disease with variable clinical course that correlates with several biologic markers of prognosis [1] The most clinically significant prognostic markers are cytogenetics determined by fluorescence in situ hybridization (FISH) and IgVH followed by ZAP70 status. Patients with CLL demonstrating deletion of 11q or 17p, high expression of ZAP70 or CD38, or relative absence of V region somatic hypermutation have markers that indicate more aggressive disease. CLL is characterized as a disease of mature B cells. CLL cells typically express an anergic B cell receptor (BCR) and demonstrate dysregulated apoptotic programs. mRNA expres- sion profiling has been used to classify CLL [2,3,4,5]. Though it is not generally considered a disease of activated B cells, mRNA expression profiling in one study characterized CLL cells as similar to activated B cells [2] and in another as similar to memory B cells [3]. In normal B cells, the nuclear translocation of NF-kB is associated with B cell activation. Constitutive nuclear localization of NF-AT (nuclear factor of activated T cells) and NF-kB2/p52 characterizes CLL cells [6], suggesting an activated B cell state. Moreover, CLL cells demonstrate higher NF-kB DNA binding activity than untransformed B cells, the RelA subunit of NF-kB has been shown to be associated with clinical disease progression, and RelA binding activity is inversely correlated with apoptosis in CLL cells [7]. Recently, CLL cells were shown to express activated cell surface markers and intracellular phenotypes [8]. CLL has also been classified by miRNA expression profiling [9,10,11,12,13,14]. Interestingly, none of these miRNA expression profiles for CLL are identical. Dysregulation of particular miRNAs in some CLL signatures have been implicated in the CLL cell apoptotic defect. For example, downregulated miR-15a and miR- 16-1 fail to repress Bcl-2 [15] and downregulated miR-29 fails to repress Mcl-1 [16]. Using miRNA expression profiling, we identified a miRNA signature in untransformed B cells induced soon after activation. This activated B cell miRNA signature is also present in CLL cells indicating an activated B cell phenotype for CLL. Our data imply that individual miRNAs involved in B cell activation may participate in the B cell transformation process and could be targets for therapeutic gene silencing in CLL. PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e16956