Single-cell Transcriptome Analysis Uncovers Heterogeneity and Key Regulators in Ibrutinib-resistant Chronic Lymphocytic Leukemia

Abstract

BackgroundIbrutinib as a widely used Bruton’s tyrosine kinase inhibitor has shown outstanding value in clinical therapy for chronic lymphocytic leukemia (CLL). However, the bottleneck of ibrutinib resistance has caused widespread concerns, necessitating the exploration of novel targets. MethodsSingle-cell RNA sequencing (scRNA-seq) was used to characterize the heterogeneity of ibrutinib-sensitive (IBS) and -resistant (IBR) CLL patients and single-cell stemness estimation and metabolic pathway enrichment analysis were performed. Lectin galactoside-binding soluble 1 (LGALS1) and lymphocyte-activating gene 3 (LAG3) were screened as key factors by analyzing the RNA-sequencing data at bulk and single cell levels. Subsequently, pseudo-time trajectory analysis and gene set enrichment analysis were conducted. In addition, an IBR CLL cell line (MEC1-IR) was generated and RT-qPCR, western blotting, and immunofluorescence were performed to detect the expression of LGALS1 and LAG3. OTX008, a selective inhibitor of galectin-1 (Gal-1, encoded by LGALS1) was assessed in CLL cells and CCK8 and apoptotic assays were conducted for functional analysis.ResultsIBR CLL showed significantly different characteristics from IBS in terms of transcriptome expression and energy metabolism. LGALS1 and LAG3 were gradually upregulated in B cells along the evolution trajectory from IBS to IBR. Their expression was verified to be closely related to the prognosis of CLL, as well as sensitivity to ibrutinib. OTX008 could effectively suppress the proliferation and induce apoptosis of CLL cells, especially for those with ibrutinib resistance.ConclusionsAn LGALS1 and LAG3 gene panel is a promising indicator of ibrutinib resistance and a prognostic marker for CLL. OTX008 displays pronounced performance against CLL cells, especially with IBR, and might represent a novel therapeutic strategy for CLL.