Epigenetic evolution and lineage histories of chronic lymphocytic leukemia

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer. In addition to genetic mutations, chronic lymphocytic leukemia (CLL) undergoes diversification through stochastic DNA methylation changes – epimutations. To measure the epimutation rate at single-cell resolution, we applied multiplexed reduced representation bisulfite sequencing (MscRRBS) to healthy donors B cells and CLL patient samples. We observed that the common clonal CLL origin results in consistently elevated epimutation rate (i.e., 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 sample. 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. To validate the inferred tree topology, we integrated MscRRBS with single-cell transcriptomes and genotyping, which 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 CLL samples prior to and during ibrutinib-associated lymphocytosis. Lineage trees revealed divergent clades of cells preferentially expelled from the lymph node with ibrutinib therapy, marked by distinct transcriptional profiles. These data offer direct single-cell integration of genetic, epigenetic and transcriptional information in the study of leukemia evolution, providing deeper insight into its lineage topology and enabling the charting of its evolution with therapy. We profiled the single-cell DNA methylomes of 831 normal B cells from six healthy donor samples and 1,821 cells from 12 primary IGHV mutated and unmutated CLLs. Single-cell methylome profiling was performed with multiplexed single-cell reduced representation bisulfite sequencing (MscRRBS), an adaption of a previous scRRBS protocol that allows to increase throughput through the addition of cell barcodes early in the scRRBS protocol. We also integrated single-cell DNAme and whole transcriptome sequencing with targeted sequencing of known somatic mutations in the cDNA.