Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.

Abstract

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution with therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing CLL populations diversify through stochastic DNA methylation (DNAme) changes – epimutations6. However, previous studies based on bulk DNAme sequencing could not answer whether epimutations affect CLL populations homogenously. To measure epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced representation bisulfite sequencing (MscRRBS) to healthy donors B cell and CLL patient samples. We observed that the common clonal CLL origin results in consistently elevated epimutation rate, with low cell-to-cell epimutation rate variability. In contrast, variable epimutation rates across normal B cells reflect diverse evolutionary ages across the B cell differentiation trajectory, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed high-resolution lineage reconstruction with single-cell data, applicable directly to patient samples. CLL lineage tree shape revealed earlier branching and longer branch lengths than normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. MscRRBS integrated with single-cell transcriptomes and genotyping confirmed that genetic subclones map to distinct clades inferred solely based on epimutation information. Lastly, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells preferentially expelled from the lymph node with therapy, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts CLL’s lineage history and its evolution with therapy.