Long-Read Single-Cell RNA Sequencing Enables the Study of Cancer Subclone-Specific Genotypes and Phenotypes in Chronic Lymphocytic Leukemia

Authors

Gage S Black
Xiaomeng Huang
Yi Qiao
Philip Moos
Deepa Sampath
Deborah M Stephens
Jennifer A Woyach
Gabor T Marth

Language

English

Publication Date

4-14-2025

Journal

Genome Research

DOI

10.1101/gr.279049.124

PMID

39965935

PMCID

PMC12047255

PubMedCentral® Posted Date

4-1-2025

PubMedCentral® Full Text Version

Post-print

Abstract

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 BTKC481S mutation that inhibits drug binding. In this study, we aimed to investigate cancer subclones harboring a BTKC481S mutation and identify cells with co-occurring CLL driver mutations. In addition, we sought to determine whether BTK-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 BTKC481S mutations during BTK inhibitor treatment. In two patients who developed two independent BTK-mutated subclones, we find that most BTK-mutated cells have an additional CLL driver gene mutation. When examining subclone-specific gene expression, we find that in one patient, BTK-mutated subclones are transcriptionally distinct from the rest of the malignant B cell population with an overexpression of CLL-relevant genes.

Keywords

Humans, Leukemia, Lymphocytic, Chronic, B-Cell, Agammaglobulinaemia Tyrosine Kinase, Single-Cell Analysis, Sequence Analysis, RNA, Mutation, Genotype, Phenotype, Transcriptome

Published Open-Access

yes

Recommended Citation

Black, Gage S; Huang, Xiaomeng; Qiao, Yi; et al., "Long-Read Single-Cell RNA Sequencing Enables the Study of Cancer Subclone-Specific Genotypes and Phenotypes in Chronic Lymphocytic Leukemia" (2025). Faculty, Staff and Student Publications. 6464.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/6464