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Single-Cell Methylation Analysis Reveals CLL Evolution, Lineages With Distinct Treatment Responses

NEW YORK (GenomeWeb) – A team led by investigators at the New York Genome Center, Weill Cornell Medicine, and the Broad Institute that tracked DNA methylation in individual chronic lymphocytic leukemia (CLL) cells has uncovered relatively consistent epimutation rates in CLL compared with healthy B cells.

"[T]he common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate," senior and corresponding author Dan Landau, a researcher affiliated with the New York Genome Center and Weill Cornell, and his co-authors wrote. "By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock."

The researchers did single-cell, reduced-representation bisulfite sequencing (RRBS) on more than 1,800 individual B cells from a dozen individuals with CLL, comparing the epigenetic profiles to one another and to DNA methylation patterns in individual B cells from six unaffected, healthy donors. Their findings, appearing online today in Nature, offered a look at the consistency of epimutation rates in specific CLL lineages and provided clues to lineage-specific treatment outcomes in CLL.

"Collectively, by leveraging the heritable information captured by epimutation, we have retraced the evolutionary histories of CLL and charted its evolution after therapy, demonstrating how different lineages may be preferentially affected by a therapeutic intervention, even in the absence of genetic sub-clonal drivers," the authors noted.

Although seemingly random DNA methylation changes contribute to the diverse forms of CLL that emerge as the blood cancer continues growing, the team explained, approaches used to measure these so-called epimutations in the past have made it difficult to discern the reproducibility of CLL DNA methylation shifts and their relationship to other CLL alterations.

"The CLL epigenome is also an important disease-defining feature, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations," the authors explained. "However, previous studies using bulk sequencing methods to analyze the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogenously."

The researchers did pooled, multiplex single-cell RRBS on Illumina instruments on 1,821 individuals B cells from 12 CLL patients, including some with immunoglobulin heavy-chain variable region mutations and some without. They also profiled 831 B cells from six healthy individuals.

The team noted that the B cells from CLL patients had more DNA methylation changes, on average, than the healthy B cells. In contrast, though, the epimutation rates were more homogenous from one cell to the next in the CLL samples than in the healthy samples.

With their data, the researchers also got a look at the CLL lineages and evolutionary trajectories within each patient, bringing in corresponding genetic and gene expression data to further contextualize the findings in relation to heritable epigenetic features and somatic mutations in the tumors.

"The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history," the authors reported. "Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic sub-clones mapped to distinct clades, as inferred solely on the basis of epimutation information."

In a subsequent analysis, meanwhile, the team used single-cell DNA methylation profiling and transcriptome sequencing to follow CLL samples collected over time from patients receiving ibrutinib, a treatment that targets B cell receptor signaling. Those findings indicated that the treatment may be particularly potent against specific CLL lineages, including those with enhanced expression of the proto-oncogene BCL11A and the NF-kappa-B pathway regulator-coding gene BCL10.

From their results so far, the authors suggested that, in the future, the "multi-modality single-cell sequencing will enable the annotation of intra-tumoral disparities in transcription in response to therapy with precise lineage information, as well as the integration of genetic, epigenetic, and transcriptional information at the atomic unit of somatic evolution — the single cell."