Long-read single-cell RNA sequencing enables the study of cancer subclone-specific genotype and phenotype

Chronic lymphocytic leukemia (CLL) is a type of blood cancer that affects the body’s immune system, particularly the B cells. Bruton’s tyrosine kinase (BTK) inhibitors have revolutionized the treatment landscape for CLL by targeting BTK, a protein critical for B cell survival and proliferation. However, treatment resistance can emerge, often due to the acquisition of specific mutations, such as the BTKC481S mutation, which prevents the drug from binding effectively.

Researchers at the University of Utah aimed to understand how the presence of additional mutations in cancer subclones harboring the BTKC481S mutation influences their expansion and behavior. Additionally, they investigated whether BTK-mutated subclones exhibit distinct gene expression patterns compared to other cancer subclones.

To achieve these goals, the researchers employed advanced sequencing techniques, including bulk DNA sequencing and single-cell RNA sequencing (scRNA-seq). Traditional scRNA-seq methods often have limited transcript coverage, which can hinder the detection of mutations. However, the researchers utilized MAS-seq, a long-read scRNA-seq technology, to enhance transcript coverage and identify mutations across the entire length of transcripts.

Visualization of single-cell genotypes to identify subclone structures

A) The subclone structure of Patient 1 identified in the bulk DNA sequencing data. Subclones are depicted by the colored circles, with representative variant clusters inside each circle. B) The cell genotypes at subclone-defining variants in Patient 1, with green markers representing only reference alleles present in the scRNA-seq reads at the given variant location within the cell and red markers indicating at least one scRNA-seq read in the cell contains the somatic variant allele. Darker marker coloring indicates an increased number of reads supporting that genotype. Variants and cells are grouped by their subclone assignment.

The study focused on six CLL patients who developed BTKC481S mutations while undergoing treatment with BTK inhibitors. The findings revealed that BTK-mutated subclones tend to acquire additional mutations in CLL driver genes, which drives their rapid expansion within the cancerous cell population.

Moreover, when examining gene expression profiles, the researchers discovered that BTK-mutated subclones in one patient displayed distinct transcriptional patterns compared to other malignant B cell populations. These subclones exhibited overexpression of genes relevant to CLL, providing insight into the molecular mechanisms underlying BTK inhibitor resistance and disease progression.

Overall, this study sheds light on the dynamic interplay between cancer subclones and their response to targeted therapies. By elucidating the genetic and transcriptional changes associated with BTK inhibitor resistance in CLL, researchers can develop more effective treatment strategies and improve patient outcomes. The findings underscore the importance of personalized medicine approaches that consider the heterogeneity of cancer cells and their evolving genetic profiles.

Black GS, Huang X, Qiao Y, Moos P, Sampath D, Stephens DM, Woyach JA, Marth GT. (2024) Long-read single-cell RNA sequencing enables the study of cancer subclone-specific genotype and phenotype in chronic lymphocytic leukemia. bioRXiv [Online Preprint]. [article]

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