CD49d expression is epigenetically regulated in chronic lymphocytic leukemia: Insights from CD49d bimodal cases

Abstract CD49d (integrin α4) is a key adhesion molecule in chronic lymphocytic leukemia (CLL) with prognostic relevance. While most cases show uniform CD49d expression, ~20% display a bimodal pattern with CD49d+ and CD49d– subpopulations originating from the same leukemic clone. In bimodal cases, CD49d+ cells expand over time, especially upon treatment, display increased proliferation, bone marrow enrichment and increased representation within the CXCR4dim/CD5bright proliferative CLL compartment. Clinically, CD49d bimodal CLL have a negative prognostic impact, resembling CD49d+ cases. Here we investigated the molecular mechanisms -whether genetic or epigenetic- driving CD49d modulation in CD49d bimodal CLL. First, we performed whole-genome sequencing (WGS) on FACS-sorted CD49d+ and CD49d- fractions from 8 bimodal CLL at 2-3 timepoints (average follow-up 3.3years). We identified a median of 4,815 heterozygous SNVs and 61 driver mutations across 22 genes. Phylogenetic analysis revealed branching evolutionary patterns shared between CD49d+ and CD49d- subsets, with no clone-specific segregation. Longitudinal tracking showed directional transitions mainly from CD49d- to CD49d+ cells, supporting a model of phenotypic plasticity within genetically stable clones. This finding was confirmed by single-cell DNA genotyping (Tapestri platform, Mission Bio) in 7 cases (~5,000–10,000 cells/sample) using WGS-driven custom panels (50-60 mutations/case). Epigenetic analyses linked CD49d expression to transcriptional permissiveness at the ITGA4 locus. DNA methylation profiling of the ITGA4 promoter in homogeneous cases (n=20) revealed significantly reduced methylation in CD49d+ versus CD49d- cells. Sorted fractions from CD49d bimodal cases (n=8) confirmed this trend, although inter-patient variability suggested additional regulatory layers. Chromatin accessibility profiling by ATAC-seq in 10 CD49d homogeneous and 7 CD49d bimodal cases showed increased chromatin accessibility in 14 upstream regions from the ITGA4 locus spanning ~170kb, which were enriched for transcription factor motifs relevant to B-cell regulation, including CTCF, EBF1, PU.1, and RUNX3. Chromatin immunoprecipitation in the CD49d+ HG-3 CLL cell line confirmed binding of CTCF, EBF1, PU.1, and RUNX3 in the same regions. These sites also recruited BRD4 and RPB1, suggesting functional enhancer/promoter interactions. Ongoing studies are investigating the roles of these factors in primary CLL cells, particularly EBF1, whose functional relevance is supported by RNA-seq data showing significantly higher EBF1 transcript levels in CD49d+ versus CD49d- cells from homogeneous (n=20) or bimodal (n=8) cases. Histone modification profiling by CUT&RUN (H3K4me3, H3K27me3, H3K27Ac, H3K4me1) in CD49d bimodal and CD49d+ or CD49d- homogeneous cases (n=7+4+4) focused on the same ITGA4 upstream enhancer/promoter regions highlighted by ATAC-seq. CD49d+ cells showed enrichment of active marks (H3K4me3, H3K27Ac, H3K4me1), consistent with a transcriptionally active chromatin state, thus reinforcing the regulatory relevance of these ITGA4 upstream elements. In contrast, CD49d- subsets were enriched for the repressive mark H3K27me3, indicating chromatin silencing. Homogeneous CD49d+ cases exhibited stronger active enhancer marks compared to CD49d+ fractions from bimodal cases, suggesting that in bimodal CLL the enhancer landscape is more plastic and dynamic, enabling phenotypic transitions from CD49d- to CD49d+, while homogeneous CD49d+ cells display a more stable, transcriptionally permissive enhancer configuration consistent with consolidated CD49d expression. Given the enrichment of histone acetylation at ITGA4 regulatory regions and the known roles of bromodomain proteins like BRD4 and histone acetyltransferases (HATs) in maintaining active chromatin states, we tested their functional relevance. Treatment of HG-3 cells with the BRD4 inhibitor PLX51107 (1 μM) and the HAT inhibitor Anacardic Acid (50 μM) for 48 hours significantly reduced CD49d transcript and surface protein levels, confirming their role in regulating CD49d expression. In summary, the longitudinal expansion of CD49d+ cells in CD49d bimodal CLL is driven by both proliferative advantage and a permissive chromatin landscape at and upstream the ITGA4 locus. These findings provide insight into chromatin-based mechanisms controlling CD49d expression and highlight the therapeutic potential of targeting this regulatory axis with epigenetic inhibitors.