Transcriptomic and Epigenomic Landscape of Hairy Cell Leukemia at Single-Cell Resolution
Authors: Eton Elliot O. , Ganesan Saravanan , Murray Rebecca M. , Durham Benjamin H. , Park Jae H. , Abdel-Wahab Omar , Landau Dan A. 
Published: December 2024

Classic hairy cell leukemia (HCL) is an indolent lymphoid malignancy characterized by the accumulation of neoplastic mature B-like cells in the peripheral blood, bone marrow, and spleen. Leukemic cell infiltration of the bone marrow typically causes bone marrow failure and fibrosis as well as splenomegaly. Virtually all cases of HCL feature the somatic BRAFV600E mutation, which originates in hematopoietic stem cells (HSCs). Despite advances in treatment and bulk gene expression studies, HCL remains incurable, and its pathogenesis remains incompletely understood. Hence, we sought to comprehensively analyze the gene expression and chromatin accessibility of HCL cells at single-cell resolution and in concert with BRAFV600E mutational status within individual cells.
We applied single cell RNA sequencing to bone marrow mononuclear cells from patients who were newly diagnosed with HCL and previously untreated (n = 24,181 cells across 3 patients) and compared to age-matched bone marrow samples from healthy controls (n = 41,853 across 4 patients). HCL cells (n = 11,756 cells) formed a distinct, relatively homogeneous cluster, as confirmed through expression of HCL-defining immunophenotypic markers, scoring of a 17-gene expression signature specific to HCL, light chain monoclonality, and copy number aberrations. In 2 out of 3 HCL patients, we noted two distinct subgroups of hairy cells that were IgM+IgD+IgGlow or IgM-IgD-IgGhigh, suggesting differences in class switching. Compared to normal memory B cells, HCL cells showed an increased inflammation signature (IL2/STAT5 signaling, IFNg response) and were predicted to interact more extensively with T and NK cells through inference of receptor-ligand interactions with CellPhoneDB. This analysis recapitulated known interactions involved in HCL growth or bone marrow fibrosis including FLT3L-FLT3, FGF2-FGFR1, TGFB1-TGFB receptors, and identified pathways involved in general immune dysregulation and inflammation including CD86-CTLA4 and ICOSLG-ICOS, respectively. We also noted that CD8+ T effector cells had increased gene expression signatures of dysfunction and exhaustion, which may contribute to HCL pathogenesis. In addition to analyzing HCL cells, we examined HSCs from HCL samples, which, compared to HSCs from control samples, showed a significantly increased signature of BRAF-mutant kinase activity and were more primed towards the lymphoid (p &lt; 0.001) lineage than to the myelo-monocytic (p &lt; 0.001) or myelo-erythroid (p &lt; 0.001) lineages.
We next applied single-cell ATAC sequencing to bone marrow mononuclear cells from patients who were newly diagnosed with HCL and previously untreated (n = 41,247 cells across 4 patients). Hairy cells formed a single relatively homogeneous cluster. Differential transcription factor motif activity analysis between HCL (n = 430) and normal (n = 946) memory B cells revealed increased activity of transcription factors that promote memory B and suppress plasma cell development such as BACH2 (q &lt; 0.001) and BHLHA15 (MIST1) (q &lt; 0.001) and decreased activity of transcription factors that drive plasma cell development such as IRF4 (q &lt; 0.001).
To link genotype to phenotype at single-cell resolution in these primary HCL samples, we also performed BRAFV600E mutation identification in single cells with Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoTChA). With GoT, we observed expected enrichment of BRAFV600E in hairy cells and depletion in T, natural killer, and myeloid cells. This pattern was also observed through GoTChA, which enabled 8% genotyping.
Altogether, we have analyzed HCL at single-cell resolution and shown that hairy cells are relatively homogeneous transcriptionally and epigenetically, resembling memory B cells. In the inflammatory and fibrotic microenvironment, HCL cells have extensive interactions with other immune cells. HCL HSCs showed an increased BRAF-mutant pathway activity signature and an increased lymphoid and decreased myeloid lineage potential, contributing to HCL pathogenesis. This work is ongoing, and future studies are warranted to examine the effect of cladribine or BRAF inhibitor treatment on cell type-specific lineage bias as well as immune dysfunction and their association with clinical outcomes.