Abstract
The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner.
It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes.
Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL.
Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program.
Disclosures
Kipps: Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.
Breaking the Silence: The Interplay Between Transcription Factors and DNA Methylation