Integrated Single-Cell Detection of Genotype and Phenotype in SF3B1-Mutated Chronic Lymphocytic Leukemia Cells

Abstract Cancer cell phenotype is controlled by both genetic composition and gene expression. Recent large-scale cancer sequencing studies have revealed extensive intratumoral genetic heterogeneity and have demonstrated its potential impact on clonal evolution and clinical outcome. The most direct approach to uncovering the impact of genetic heterogeneity on cellular phenotype requires integration of genetic and transcriptomic profiles of single cells. Currently, however, RNA and DNA cannot be reliably isolated from the same cell. Here, we demonstrate the feasibility for linking single-cell somatic mutation data with cellular transcriptional heterogeneity through a targeted RNA-based approach. By leveraging a microfluidic platform (Fluidigm BioMarkTMHD system) to perform multiplexed targeted amplification of RNA derived from hundreds of single cells, we have generated a versatile approach for the integrated detection of somatic mutations in relation to specific gene transcripts. We focused on a series of chronic lymphocytic leukemia (CLL) B cells that were previously characterized by bulk whole-exome (WES) and RNA-sequencing (RNA-Seq). We developed 2 classes of assays. First, we generated multiplexed nested quantitative RT-PCR assays of 96 genes with known involvement in CLL biology. Second, to simultaneously detect patient-specific somatic mutations in the same cell, we devised multiplexed pre-amplification primers targeting transcribed regions containing somatic point mutations. These regions were then amplified using paired nested primers, for detection of the wild-type or mutant alleles. We focused on those somatic mutations with detectable expression in bulk CLL RNA (> 5 FPKM by RNA-seq). When applied to either artificial oligonucleotide templates or bulk patient cDNA, these paired wild-type and mutant allele detection assays reliably demonstrated consistent differences in DCT values of >6 cycles. In total, we designed expression assays for 96 genes and 46 mutation detection applied to 5 CLL samples (median of 9 assays/sample, range 6-13). We examined up to 384 single cells from each of 5 samples and from normal CD19+ B cells. Based on expression of housekeeping genes ACTB and B2M, we observed viable expression in 1951 of 2112 cells (92.4%). We could clearly discern that expression of the 96 genes was heterogeneous across 354 single CLL-B cells and could discriminate CLL from 174 normal B cells by principal component analysis. 32 out of 46 (70%) mutation detection assays successfully distinguished between wild-type and mutant alleles and the mutant allele was consistently observed in the originating CLL cells, but not in unrelated CLL or non-leukemic B cells. Our RNA-based estimates of allele frequency agreed with single-cell targeted DNA-based detection of somatic mutations conducted for 3 of 5 CLL samples as well as with frequencies estimated from bulk WES-based cancer cell fraction (CCF) measurements. We applied our integrated assay design to 2 CLL samples known to harbor mutations in the putative CLL driver SF3B1: Patient 1 with bulk CCF of 17% (G742D) and Patient 2 with 87% (K700E). Mutation of this critical spliceosome component broadly changes RNA splicing profiles although the functional impact of these alternative splice variants on CLL biology remains unknown. We generated multiplex assays for SF3B1 mutation detection and for expression of mutation-associated alternative splice variants. Consistent with the bulk-sequencing results, we detected 50 of 373 (13.4%) single CLL cells from Patient 1 with SF3B1 mutation. Moreover, the subset of cells with SF3B1 mutation demonstrated high expression of splice variants relative to wild-type cells (GCC2 and MAP3K7, p< 0.000001). This SF3B1 mutated subclone also displayed reduced expression of RNA splicing factors (BTAF1, DDX17, SNW1, SRSF3, U2SURP; all p<0.05), cell cycle regulators (CDC27, PDS5A; p<0.015) and an inflammatory pathway gene (MALT1p=0.039), suggesting involvement of SF3B1 mutation in these biological processes. Analysis of Patient 2 is ongoing. Taken together, our study demonstrates the feasibility of linking genotype with gene expression at the RNA level. Furthermore, these analyses reveal the potential for single cell RNA-based analysis to directly uncover the effects of driver mutations on the leukemia cell phenotype. Disclosures Brown: Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

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Genome-Wide Analysis of a Novel Murine Model of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.967.967 ◽

2016 ◽

Vol 128 (22) ◽

pp. 967-967

Author(s):

Lili Wang ◽

Rutendo Gambe ◽

Jing Sun ◽

Sachet Shukla ◽

Jaegil Kim ◽

...

Keyword(s):

B Cells ◽

Murine Model ◽

Rna Splicing ◽

Double Mutant ◽

Splice Variants ◽

Genetic Alterations ◽

Lymphocytic Leukemia ◽

Mutant Mice ◽

Genome Wide

Abstract Large-scale cancer sequencing efforts worldwide have yielded numerous novel cancer drivers; however, how these genetic alterations functionally lead to cancer remains largely unknown. An indispensible approach for establishing the causal features of disease is through in vivo animal models. In chronic lymphocytic leukemia (CLL), only limited mouse models are currently available and most do not reflect the genetics of human CLL. Studies of whole-exome sequencing (WES) using CLL samples have consistently pointed to the common co-occurrence of mutations in the RNA splicing factor gene SF3B1 and mutations in the DNA damage response gene ATM or deletion of chromosome 11q (del(11q), whose minimally deleted region encompasses ATM). We therefore asked whether this combination of traits would be productive of CLL in mice. To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. We found that in vivo co-expression of these two mutations in B cells, but not of either single lesion alone, led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen (at low penetrance) in aged (18 to 24-month old) but not young mice. These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations. To better understand how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if RNA level changes are present in the double mutant mice. We performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, or singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease (n=2-6 samples, per group). Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that the splice variants in micewith mutated Sf3b1 alone (without CLL) were highly enriched at 3' splice sites (27 of 77 splice variants, t-test q<0.05, absolute ΔPSI >10%). On the other hand, mice with Atm single deletion displayed an RNA splicing pattern with enrichment of alternative first and last exons (11 and 12 of 52, chi-squared test, p=4.5 x 10-4). B cells with the combined Sf3b1 and Atm mutations displayed a combination of splicing patterns that comprised of both alternative 3' splice variants, as well as alternative first and last exons. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously demonstrated to be associated with CLL. We further investigated the differential gene expression between B cells from double mutant mice with and without CLL-like disease. We identified 1,875 CLL-specific genes (DESeq2, q<0.01). Gene set enrichment analysis (GSEA) of these genes indicated their involvement in cellular processes such as IL2-STAT5 signaling and the interferon gamma response, both pathways implicated in human CLL. In parallel, we asked if there are DNA level changes in the doubly mutant mice. We examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. Preliminarily, we have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to cells with only single mutation. In summary, we have generated a genetically-engineered murine model that faithfully recapitulates human CLL genetics. This is the first demonstration that expression of putative CLL driver events identified from unbiased genome-wide sequencing indeed initiates CLL-like disease. Genome-wide DNA and RNA analysis using this model has revealed that altered RNA splicing, dysregulation of gene expression, and genomic instability all contribute to CLL leukemogenesis. We anticipate that further dissection of this murine model will shed light on mechanistic understanding of cooperation between Atm deletion and SF3B1 mutation in CLL. Disclosures No relevant conflicts of interest to declare.

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Comprehensive Bulk and Single Cell Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.2906.2906 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2906-2906 ◽

Cited By ~ 1

Author(s):

Jean Fan ◽

Lili Wang ◽

Angela N Brooks ◽

Youzhong Wan ◽

Donna S Neuberg ◽

...

Keyword(s):

Alternative Splicing ◽

Single Cell ◽

Single Cells ◽

Lymphocytic Leukemia ◽

Rna Seq ◽

Wild Type ◽

Mutation Status ◽

Total Rna ◽

Gene Sets

Abstract Large-scale sequencing efforts have identified SF3B1 as arecurrently mutated gene in chronic lymphocytic leukemia (CLL). While SF3B1 mutations have been associated with adverse clinical outcome in CLL, mechanistic understanding of its role in the oncogenic phenotype remains lacking. We therefore undertook a comprehensive transcriptomic characterization of CLL in relation to SF3B1 mutation status at both bulk and single cell levels. We first profiled bulk mature poly-A selected RNA by sequencing (RNA-seq) from 37 CLLs (13 SF3B1 wild-type, 24 mutated). After identifying and classifying splice alterations using the tool JuncBASE, we found SF3B1 mutation to be associated with increased alternative splicing, with the most pervasive changes in 3' splice site selection. 304 alternatively spliced events were significantly associated with SF3B1 mutation, 4 of which we validated by qRT-PCR in 20 independent CLL samples with known SF3B1 mutation status. We further identified 1963 differentially expressed genes (q < 0.2) associated with SF3B1 mutation. By gene set enrichment analysis, SF3B1 mutation appeared to impact a variety of cancer and CLL-associated gene pathways, including DNA damage response, apoptosis regulation, chromatin remodeling, RNA processing, and Notch activation (q < 0.01). ~20% of these gene sets were also found to be significantly enriched for genes exhibiting alternative splicing in association with SF3B1 mutation. As SF3B1 acts at the level of pre-mRNA, we also performed bulk RNA-seq with total RNA libraries generated from 5 CLLs (2 SF3B1 wild-type, 3 with the common K700E mutation). We again observed an enrichment of 3' splice site changes, along with ~30% overlap of differentially expressed genes, and ~16% overlap of enriched gene sets with the aforementioned poly-A data analysis. One differentially over-expressed gene associated with SF3B1 mutation unique to this total RNA data analysis and validated by total RNA qPCR of independent CLL samples was TERC, an essential RNA component of telomerase that serves as a replication template during telomeric elongation. TERC is a non-polyadenylated transcript and thus was undetected by our previous poly-A selected RNA-seq and by targeted qRT-PCR of oligo dT-generated cDNA. Recent reports have highlighted the involvement of the spliceosome in telomerase RNA processing, and shorter telomere length of CLLs with SF3B1 mutation. Thus, although further investigation will be needed, our analyses suggest a potential mechanism by which SF3B1 mutation contributes to aberrant regulation of telomerase activity. Since SF3B1 is commonly found as a subclonal mutation in CLL, and because signals obtained from bulk analyses reflect only the average characteristics of the population, we assessed the transcriptomic effects of SF3B1 mutation in single cells within a subset of CLL cases. We developed a novel and sensitive microfluidic approach that performs multiplexed targeted amplification of RNA to simultaneously detect somatic mutation status, gene expression (96 targets), and alternative splicing (45 targets) within the same individual cell for 96 to 288 cells from 5 patients with different SF3B1 mutations. From the same patient sample, single cells with SF3B1 mutation generally exhibited increased alternative splicing for events identified from the bulk analysis, thus confirming the association of SF3B1 mutation with altered splicing at the single cell level. Different SF3B1 hotspot mutations within the HEAT repeat domains exhibited similar patterns of alternative splicing while a mutation outside of the repeat domain did not. Furthermore, we confirmed significant changes in gene expression between SF3B1 wild-type and mutant cells of target genes involved in the Notch pathway (NCOR2), cell cycle (CDKN2A, CCND1) and apoptosis (TXNIP). Consistent with these analyses, functional studies with overexpression of full-length mutated SF3B1 in a hematopoietic cell lines confirmed the modulation of these pathways by this putative CLL driver. Our high-resolution single cell analysis further uncovered 2 transcription factors strongly associated with SF3B1 mutation but not previously appreciated (KLF3 and KLF8). Our comprehensive transcriptomic analysis thus highlights SF3B1 mutation as an efficient mechanism by which a complex of changes relevant to CLL biology are generated that can contribute to disease progression. Disclosures Kipps: Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor. Li:Fluidigm: Employment. Livak:Fluidigm: Employment.

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Single Cell Bisulfite Sequencing Defines Epigenetic Diversification in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.1047.1047 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1047-1047 ◽

Cited By ~ 1

Author(s):

Dan A. Landau ◽

Kendell Clement ◽

Hongcang Gu ◽

Evan Biederstedt ◽

Divy Kangeyan ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Single Cell ◽

Bisulfite Sequencing ◽

Single Cells ◽

Lymphocytic Leukemia ◽

Absolute Difference ◽

Bisulfite Conversion ◽

Conversion Rates ◽

Cell Variation

Abstract Genetic and epigenetic heterogeneity of cancer cells fundamentally shapes cancer progression and relapse. In chronic lymphocytic leukemia (CLL), we previously reported that intra-leukemic epigenetic diversity in DNA methylation (DNAme) follows a stochastic pattern reminiscent of genetic 'trial and error' in cancer evolution. We measured stochastic DNAme from bulk sequencing by observing the proportion of discordantly methylated sequencing reads (PDR), and found that higher PDR was associated with greater cell-to-cell transcriptional heterogeneity and adverse clinical outcome. However, bulk DNAme sequencing does not allow to phase stochastic DNAme across distant genomic loci for a single cell, as it is limited to length of a short sequencing read. Thus, it can only provide the average PDR for a population of cells, rather than the cell-to-cell variation in this important epigenetic feature. Therefore, to define stochastic DNAme changes at the single cell level, we optimized a multiplexed single cell reduced representation bisulfite sequencing (MscRRBS) protocol to allow high-throughput single cell DNAme sequencing. This protocol significantly improves scalability by multiplexing cells with shorter inline-barcodes at an early stage and utilizing SPRI beads purification to eliminate adapter-dimers. We applied MscRRBS to 393 single CD19pos B cells from two healthy volunteers and 111 single cells from a CLL sample. 88% of cells were evaluable with greater than 100,000 covered CpGs (average of 436,230 CpGs per cell). We achieved bisulfite conversion rates of 99.7%+/-0.0001 (mean+/- SD), without a significant reduction in coverage. A downsampling analysis showed that 2.1 million reads per cell provided 85% of CpG coverage with only marginal increase in coverage with further sequencing. Biallelic coverage was observed in 4.6+/-2% of germline SNPs. With MscRRBS, we measure the PDR of each individual cell. As expected from our prior bulk RRBS analysis, we found that the average PDR across cells was higher in CLL compared with B cells from healthy adult volunteers (0.39+/-0.01 vs. 0.26+/-0.08, P <0.00001). Strikingly, CLL cells exhibited a uniformly high PDR, in contrast to normal B cells, which exhibited higher cell-to-cell variation. The absolute difference in PDR values between any two cells was ten-fold higher in normal B cells compared with CLL cells (0.08+/-0.06 vs. 0.008+/-0.006, respectively, P<0.0001). A multivariable regression model, which included potential technical confounders (bisulfite conversion rate, number of reads, number of covered CpGs), showed that higher PDR dispersion was independently associated normal B cells compared to CLL cells. The higher uniformity of PDR in CLL may reflect the relationship between epi-mutation rate (measured through PDR) and the evolutionary age of the cells. As additional stochastic DNAme changes are generated with each generation, we hypothesize that PDR estimates the number of generations in a cells' evolutionary history. Thus, CLL cells have uniformly high PDR reflecting a high but uniform number of generations in their history, consistent with a single common cell of origin. In contrast, normal B cells have diverse histories with newly formed naive cells intermixed with long-lived memory cells. Consistent with this hypothesis, healthy donor CD27neg cells showed lower PDR and less PDR cell-to-cell variation compared with CD27pos cells (mean absolute PDR difference 0.008+/-0.01 vs. 0.03+/-0.03, respectively, P<0.0001). As MscRRBS allows complete phasing of DNAme across distant genomic loci, we further calculated the odds ratio of concordance in methylation state between any two neighboring CpG as a function of their genomic distance, compared with any two randomly paired CpGs. Through this procedure we quantified the properties of DNAme concordance decay and uncovered different genomic scales of stochastically disordered methylation. Finally, the MscRRBS also allowed to reconstruct phylo(epi)genetic relationship between the cells, and provided accurate estimate of the rates of stochastic epi-mutation across the genome. Thus, single cell DNAme is a powerful novel tool to define epigenetic diversification and its impact on CLL evolution. Disclosures No relevant conflicts of interest to declare.

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Multimodal Single-Cell Profiling Defines the Epigenetic Determinants of Chronic Lymphocytic Leukemia Evolution

Blood ◽

10.1182/blood-2018-99-115596 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1312-1312

Author(s):

Ronan Chaligne ◽

Federico Gaiti ◽

Franco Izzo ◽

Steven Kothen-Hill ◽

Hongcang Gu ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Single Cell ◽

Molecular Clock ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Lineage Tree ◽

Single Cell Profiling ◽

The Common

Abstract Genetic, epigenetic and transcriptional heterogeneity cooperate to fuel cancer's ability to evolve and adapt to therapy. In chronic lymphocytic leukemia (CLL), we have shown through bulk DNA methylation (DNAme) sequencing that the growing CLL populations diversify through stochastic DNAme changes (epimutations), impacting transcriptional heterogeneity, clonal evolution and clinical outcome. To directly integrate across epigenetic, genetic and transcriptional intra-leukemic cell-to-cell variation, we developed a high-throughput multi-modality platform that jointly interrogates the methylome, transcriptome and genetic driver mutations from the same single cell (Fig. A). We applied it to >2,000 B cells from 6 healthy donors and 12 CLL samples. We found that the common clonal CLL origin resulted in an elevated but uniform epimutation rate (i.e., low cell-to-cell epimutation variability). In contrast, in index sorted B cell subsets, ranging in maturity from naïve B cells (CD27-IgM+IgD+++IgG-) to memory B cells (CD27+IgG+), variable epimutation rates reflect cells with diverse evolutionary ages across the B cell differentiation trajectory (Fig. B). Thus, we posited that epimutation can serve as a "molecular clock", enabling high-resolution lineage reconstruction, applicable directly to patient samples. CLL lineage tree topology revealed earlier branching, and longer branch lengths than normal B cells, consistent with rapid drift after transformation, and a greater proliferative history (Fig. C). In contrast to CLL topologies, non-clonal normal B cell trees provided a smaller increase in clustering accuracy compared with parsimony-based trees (P < 0.001; Fig. D). To validate the inferred tree topology, we leveraged our multi-modal capture of DNAme and genetic drivers in single cells. Indeed, genetic subclones mapped accurately to distinct clades inferred solely based on epimutation information [e.g., a clade composed of SF3B1 mutants and another clade composed of SF3B1 wild-type cells (P = 7 x 10-9; Fig. E, F)]. Using the joint single-cell transcriptional profiling, we found that cells in SF3B1 mutated clade also displayed higher alternative 3' splicing than their wild-type counterparts (P = 0.01526; Fig. G). Cells belonging to SF3B1 mutated clade were marked by expression changes in genes related to DNA damage (e.g., KLF8) and Notch signaling (e.g., DTX4) (P < 0.05; Fig. H, I). The direct linking of single-cell transcriptional data with lineage identity also showed that transcriptional similarity between cells decreases as a function of their lineage distance (P < 0.05; Fig. K). Notably, the molecular clock feature of epimutations enabled precise timing of subclonal divergence event in the CLL's evolutionary history, estimated to have occurred 2180±219 days after the emergence of the parental clone (Fig. J). To examine potential lineage biases during therapy, we performed serial multimodal single-cell profiling of a CLL patient without subclonal genetic drivers, prior to and during ibrutinib-associated lymphocytosis. The lineage trees revealed a distinct clade of cells preferentially expelled from the lymph node, marked by a distinct transcriptional profile, likely representing ibrutinib-sensitive cells. Lastly, we hypothesized that frequent epigenetic modifier mutations seen in hematological malignancies may increase the epimutation rate promoting intra-leukemic cellular diversity. To test this hypothesis, we applied our multi-modality single-cell platform to cells from TET2 knock-out (KO) mouse models, and observed higher epimutation rates, closely associated with higher cell-to-cell transcriptional heterogeneity compared with wild-type cells (P < 0.0001; Fig. L, M). In summary, we revealed that CLL show uniformly elevated epimutation rates, reflecting the common evolutionary age of these cells. By leveraging the heritable information captured through epimutation data, we provided a native lineage tracing applicable directly to patient samples. With this approach, we showed that CLL lineage topology exhibit early branching and long-branch length, consistent with exponential growth. Finally, we demonstrated that multimodal single-cell profiling enables projection of genetic and transcriptional identity onto the lineage tree, providing a direct measurement of the heritability of transcriptional profiles as a function of lineage distance. Figure. Figure. Disclosures Wu: Neon Therapeutics: Equity Ownership.

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High throughput single-cell detection of multiplex CRISPR-edited gene modifications

Genome Biology ◽

10.1186/s13059-020-02174-1 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Elisa ten Hacken ◽

Kendell Clement ◽

Shuqiang Li ◽

María Hernández-Sánchez ◽

Robert Redd ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Single Cell ◽

High Throughput ◽

Gene Editing ◽

Somatic Mutations ◽

Single Cells ◽

Lymphocytic Leukemia ◽

Cell Detection ◽

Loss Of Function ◽

Single Cell Detection

Abstract CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

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Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes

Blood ◽

10.1182/blood.v84.3.708.708 ◽

1994 ◽

Vol 84 (3) ◽

pp. 708-715 ◽

Cited By ~ 83

Author(s):

J Tamaru ◽

M Hummel ◽

M Zemlin ◽

B Kalvelage ◽

H Stein

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Hodgkin's Disease ◽

Somatic Mutations ◽

Hodgkin’S Disease ◽

Receptor Gene ◽

High Sensitivity ◽

Cell Phenotype ◽

Igh Genes

Abstract The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.

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Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

Journal of Bacteriology ◽

10.1128/jb.00460-18 ◽

2018 ◽

Vol 200 (23) ◽

Cited By ~ 1

Author(s):

Griffin Chure ◽

Heun Jin Lee ◽

Akiko Rasmussen ◽

Rob Phillips

Keyword(s):

Cell Survival ◽

Single Cell ◽

Osmotic Shock ◽

Single Cells ◽

Mechanosensitive Channel ◽

Membrane Tension ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

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Loss of NFAT2 Leads to an Acceleration of Clonal Evolution in CLL

Blood ◽

10.1182/blood.v128.22.4369.4369 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4369-4369

Author(s):

David J Müller ◽

Melanie Märklin ◽

Lothar Kanz ◽

Martin R Mueller ◽

Stefan Wirths

Keyword(s):

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Conditional Knockout ◽

Wild Type ◽

Vdj Recombination ◽

Aggressive Disease ◽

Dismal Prognosis ◽

Pcr Products

Abstract Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of patients Richter's transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell-specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter's syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered downstream signaling. Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/-mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied. A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, lead to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency - indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans. In summary, the loss of NFAT2 signaling in CLL prefers the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease. Disclosures No relevant conflicts of interest to declare.

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Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽

10.1182/blood-2018-99-110626 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3887-3887

Author(s):

Moosa Qureshi ◽

Fernando Calero-Nieto ◽

Iwo Kucinski ◽

Sarah Kinston ◽

George Giotopoulos ◽

...

Keyword(s):

Dendritic Cell ◽

Single Cell ◽

Cell Line ◽

Single Cells ◽

Mutant Form ◽

Rna Seq ◽

Wild Type ◽

Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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Self-controlled Sequencing Suggests that Somatic Mutations of Signaling, Transcription and Tumor Suppression Genes are a Precondition for AML Transformation in MDS

10.21203/rs.3.rs-1142924/v1 ◽

2022 ◽

Author(s):

Feng Xu ◽

Ling-Yun Wu ◽

Juan Guo ◽

Qi He ◽

Zheng Zhang ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Tumor Suppressors ◽

Rna Splicing ◽

Somatic Mutations ◽

Target Genes ◽

Single Cell Sequencing ◽

Target Sequencing ◽

Paired Samples ◽

Whole Exome

Abstract Background The transformation biology of secondary AML from MDS is still not fully understood. Here, we performed a large cohort of paired self-controlled sequences including target, whole-exome and single cell sequencing to search AML transformation-related mutations (TRMs). Methods 39 target genes from paired samples from 72 patients with MDS who had undergone AML transformation were analyzed by next generation target sequencing. Whole exome and single-cell RNA sequencing were used to verify the dynamics of transformation. Results The target sequencing results showed that sixty-four out of the 72 (88.9%) patients presented presumptive TRMs involving activated signaling, transcription factors, or tumor suppressors. Of the 64 patients, most of TRMs (62.5%, 40 cases) emerged at the leukemia transformation point. All three of the remaining eight patients analyzed by paired whole exome sequencing showed TRMs which are not included in the reference targets. No patient with MDS developed into AML only by acquiring mutations involved in epigenetic modulation or RNA splicing. Single-cell sequencing in one pair sample indicated that the activated cell signaling route was related to TRMs which take place prior to phenotypic development. Of note, target sequencing defined TRMs were limited to a small set of seven genes (in the order: NRAS/KRAS, CEBPA, TP53, FLT3, CBL, PTPN11 and RUNX1, accounted for nearly 90.0% of the TRMs). Conclusions Somatic mutations involving in signaling, transcription factors, or tumor suppressors appeared to be a precondition for AML transformation from MDS. The TRMs may be considered as new therapy targets.

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