Abstract
Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez-García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development.
First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014).
To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18).
Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition.
Disclosures
Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.
methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data
