Abstract B25: Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia.
Authors: Oakes Christopher C. , Seifert Marc , Yassen Assenov , Gu Lei , Przekopowitz Martina , Ruppert Amy , Serva Andrius , Koser Sandra , Brocks David , Lipka Daniel , Bogatyrova Olga , Mertens Daniel , Zapatka Marc , Lichter Peter , Doehner Hartmut , Kueppers Ralf , Zenz Thorsten , Stilgenbauer Stephan , Byrd John , Plass Christoph 
Published: August 2015

Abstract
Knowledge of the cell-of-origin is essential for the full understanding of the causes of a malignant disease and for the rational design of targeted therapies. The B cell compartment is composed of a highly complex mixture of subtypes, each with distinct phenotypes and roles within the immune system. In chronic lymphocytic leukemia (CLL), heterogeneity in the biology and clinical course of the disease is thought to be linked to divergent cellular origins. We and others have previously shown that the epigenome of CLL, as measured by the global pattering of DNA methylation, is highly clonal and remarkably stable over time and thus represents a powerful approach to trace founder subtype populations. Here we combine epigenomic and transcriptomic analysis using next-generation sequencing approaches to compare CLL cells to highly purified and specific B cell subpopulations at various stages of maturation. We find that B cell maturation involves substantial unidirectional epigenetic programming that occurs as a continuum throughout the transition between naïve to fully-mature memory B cell subpopulations. Combining 258 CLL cases using Illumina 450K analysis reveals that all CLLs arise from a discrete window within the spectrum of B cell maturation that is more similar to mature B cells, with the majority of cases clustering at two distinct points correlating with unmutated IGHV versus highly mutated (&lt;95% homology) IGHV genes; however, a significant number (~20%) of cases arise at various points between these two clusters. Next we show that using RNA-seq, broad differences in global expression patterns mirror the degree of epigenetic programming achieved by individual CLLs. Progressively further programming is paralleled by a transition from an aggressive to indolent expression pattern, indicated by the decrease in the expression levels of genes with known roles in promoting CLL cell survival, such as ZAP70, BTK, TCL1a, MCL1, miR-155 and others. Using DNA methylation and ChIP-seq data to compare the sequence and chromatin features of genomic regions that are programmed in normal B cell maturation versus CLL, reveals that although a myriad of transcription factors and pathways connected to immune cell function are involved in normal epigenetic programming in B cells, aberrant CLL-specific alterations involve excess activity of NFAT and EGR gene families and, paradoxically, a reduction of AP-1 activity. To further investigate the role of immediate-early genes, RNA-seq analysis of in vitro-activated CLL cells revealed a strong association between the degree of epigenetic programming and the specific inducibility of EGR2 and c-FOS, supporting a functional role of these genes in aberrant DNA methylation programming. Finally, in an independent clinically well-annotated cohort of 349 CLL cases, we demonstrate that the degree of epigenetic programming is significantly associated with time to treatment and overall survival in patients. Collectively, this work demonstrates that instead of a distinct cell(s)-of-origin, CLL is rather derived from a continuum of possible programming states, and that the degree of programming achieved by a particular CLL at the time of transformation dictates its global gene expression pattern and clinical outcome. Furthermore, a parallel assessment of B cell maturation with CLL development permits a refinement of the disease-specific, early molecular events, highlighting the dysregulation of particular transcription factors and pathways in the pathogenesis of aggressive versus indolent disease.

Citation Format: Christopher C. Oakes, Marc Seifert, Assenov Yassen, Lei Gu, Martina Przekopowitz, Amy Ruppert, Andrius Serva, Sandra Koser, David Brocks, Daniel Lipka, Olga Bogatyrova, Daniel Mertens, Marc Zapatka, Peter Lichter, Hartmut Doehner, Ralf Kueppers, Thorsten Zenz, Stephan Stilgenbauer, John Byrd, Christoph Plass. Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia. [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 B25.