Data availability. All of the sequencing data is available via Gene Expression Omnibus (GEO) under the accession number GSE117826.

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer 1 . Chronic lymphocytic leukemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution with therapy 2 , 3 . The CLL epigenome is also an important disease-defining feature 4 , 5 , and growing CLL populations diversify through stochastic DNA methylation (DNAme) changes – epimutations 6 . 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.

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities.