Long-read single-cell RNA sequencing enables the study of cancer subclone-specific genotypes and phenotypes in chronic lymphocytic leukemia

Bruton tyrosine kinase (BTK) inhibitors are effective for the treatment of chronic lymphocytic leukemia (CLL) due to BTK's role in B cell survival and proliferation. Treatment resistance is most commonly caused by the emergence of the hallmark <i>BTK</i> ^C481S mutation that inhibits drug binding. In this study, we aimed to investigate cancer subclones harboring a <i>BTK</i> ^C481S mutation and identify cells with co-occurring CLL driver mutations. In addition, we sought to determine whether <i>BTK</i>-mutated subclones exhibit distinct transcriptomic behavior when compared to other cancer subclones. To achieve these goals, we use scBayes, which integrates bulk DNA sequencing and single-cell RNA sequencing (scRNA-seq) data to genotype individual cells for subclone-defining mutations. Although the most common approach for scRNA-seq includes short-read sequencing, transcript coverage is limited due to the vast majority of the reads being concentrated at the priming end of the transcript. Here, we utilized MAS-seq, a long-read scRNA-seq technology, to substantially increase transcript coverage and expand the set of informative mutations to link cells to cancer subclones in six CLL patients who acquired <i>BTK</i> ^C481S mutations during BTK inhibitor treatment. In two patients who developed two independent <i>BTK</i>-mutated subclones, we find that most <i>BTK</i>-mutated cells have an additional CLL driver gene mutation. When examining subclone-specific gene expression, we find that in one patient, <i>BTK</i>-mutated subclones are transcriptionally distinct from the rest of the malignant B cell population with an overexpression of CLL-relevant genes.