scholarly journals Single-Cell Multi-Omics Reveals Distinct Paths to Survival of Admixed BTKC481 Mutant Vs. Wild-Type Cells in Clinically Progressing Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-42
Author(s):  
Andrew Lipsky ◽  
Danny Luan ◽  
Shirley Chen ◽  
Ronan Chaligne ◽  
Neville Dusaj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Single Cell ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Publicly Traded ◽  
Cell Identity ◽  
Single Cell Sequencing ◽  
Bcr Signaling

Mutations in the kinase binding domain of BTK at position C481 are associated with resistance to BTK inhibitor (BTKi) therapy in chronic lymphocytic leukemia (CLL). Nearly half of patients manifesting clinical progression with these alterations exhibit a subclonal burden of resistance. Intriguingly, measured BTKC481 variant allelic fractions (VAF) are commonly lower than 10% [Ahn et al, Blood 2017]. This raises the important question of how BTKC481-mutated (MUT) and wildtype (WT) cells differ in their response to therapeutic challenge, and how low-burden MUT subclones facilitate escape from therapy. While the admixture of MUT and WT cells within the same individual presents an opportunity to directly study the downstream effects of subclonal resistance mutations, these cells cannot be separated through sorting. To overcome this limitation, we utilized Genotyping of Transcriptomes (GoT)-a strategy to jointly capture genotyping of a locus of interest together with whole transcriptomes at the single cell level (Fig. 1A). Importantly, GoT eliminates patient-specific and technical confounders, enabling direct linkage of BTK genotypes to transcriptional phenotypes. We applied GoT to 64,099 CD19+ cells across a cohort of seven patients with clinically progressive CLL found to have low-burden BTKC481 subclones (Fig. 1B). Samples were obtained at the time of progression and were screened for mutations in PLCG2. We genotyped 33.3% of cells, consistent with 34.0% of cells expressing BTK in whole transcriptome data. Clustering of the gene expression profiles showed that MUT and WT clones did not segregate by genotype in most (6/7) patients (Fig. 1C), implying a large degree of transcriptional similarity between MUT and WT cells, and further highlighting the need for multi-omics single-cell sequencing to directly link genotype to phenotype in this context. We note that the single exception with distinct genotypic clustering (CLL06, Fig. 1D) was driven by co-occurring large chromosomal aberrations within the MUT cells, including del(8p), which is associated with BTKi resistance [Burger et al, Nat Comm 2016]. CLL cells are known to cycle between the peripheral blood and protective microenvironmental niches, which is a process modulated by BTKi. We applied CXCR4/CD5 expression as a read out to these migration patterns [Chen et al, Leukemia 2016], and observed that MUT cells were comparatively enriched in CXCR4lowCD5hi CLL, which is associated with recent emigration from the stromal niche and increased BCR signaling (Fig. 1E). Conversely, WT cells were the majority of CXCR4hiCD5low CLL, which is associated with a resting peripheral cellular state and BCR downregulation, suggesting WT quiescence with limited stromal support. To explore the phenotypic changes associated with BTK mutations, we first measured the activity of reported BTKi response expression signatures. WT cells showed higher mean response scores compared to MUT (Fig. 1F), demonstrating that the WT cells preserve BTKi responsiveness. To unbiasedly interrogate transcriptional differences, we applied a set of 336 predefined gene modules representing B-cell cellular functions and processes. Enriched modules in MUT cells implicated restoration of B-cell receptor (BCR) signaling and recovery of CLL cell identity (NF-kB, IRF4, CD40, Fig. 1G), a finding also supported by de novo differential gene expression analysis (Fig. 1H), and gene set enrichment of differentially expressed genes (Fig. 1I). In contrast, WT cells showed increases in modules related to quiescence, hypoxia, cellular stress (HIF-1α, XBP1) and terminal B-cell development (Blimp-1). Intriguingly, changes in gene targets associated with restoration of NOTCH1 and IL4 activity were also observed. As these pathways are impaired in patients responding to BTKi and implicated in resistance [Del Papa et al, CCR 2019; Chen et al, ASH 2019], their re-emergence may reflect a cytoprotective strategy. In summary, we utilized multi-omics single-cell sequencing to identify the distinct transcriptional programs of admixed MUT and WT cells in subclonal BTKC481 progression. MUT cells showed robust escape from BTKi inhibition via increased immune receptor signaling and restoration of CLL cell identity program, while WT cells demonstrated a signature of hypoxia and stress response, with NOTCH1 and IL4 activation implicated as mechanisms of clonal persistence. Disclosures Trisal: Celgene: Current Employment, Current equity holder in publicly-traded company. Gandhi:Celgene: Current Employment, Current equity holder in publicly-traded company. Wiestner:Pharmacyclics LLC, an AbbVie Company, Acerta, Merck, Nurix, Verastem, and Genmab: Research Funding; NIH: Patents & Royalties: NIH. Allan:Acerta, Genentech, Abbvie, Sunesis, Ascentage, Pharmacyclics, Janssen, AstraZeneca, BeiGene: Consultancy; Celgene, Genentech, Janssen, TG Therapeutics: Research Funding; Abbvie, Janssen, AstraZeneca, Pharmacyclics: Honoraria. Furman:Genentech: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy, Research Funding; Acerta: Consultancy; Verastem: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy, Research Funding; Incyte: Consultancy; Janssen: Consultancy, Speakers Bureau; Oncotarget: Consultancy; Loxo Oncology: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Shanye Yin ◽  
Gregory Lazarian ◽  
Elisa Ten Hacken ◽  
Tomasz Sewastianik ◽  
Satyen Gohil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Binding ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Experimental Group

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

scholarly journals Cirmtuzumab Targets ROR1 to Inhibit Ibrutinib-Resistant, Wnt5a-Induced Rac1 Activation in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2034-2034 ◽  
Author(s):  
Jian Yu ◽  
Liguang Chen ◽  
Bing Cui ◽  
Christina Wu ◽  
Michael Y. Choi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Standard Dose ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Synergistic Activity ◽  
Bcr Signaling

Abstract Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting that ancillary pathways contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. We hypothesized that the effects of ibrutinib on blocking BCR-signaling might be offset by non-canonical Wnt-signaling via ROR1. If so, then inhibition of both ROR1- and BCR-signaling might have an enhanced anti-tumor effect. We examined the CLL cells of patients who were taking ibrutinib at the standard dose of 420 mg per day. Freshly isolated CLL cells had activated Rac1, which diminished over time upon culture in serum-free media, unless treated with exogenous Wnt5a, as noted for CLL cells of patients not taking ibrutinib. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated in vitro with ibrutinib, even at concentrations that exceeded those required to completely inhibit BTK and BCR-signaling. On the other hand, Wnt5a-induced activation of Rac1 was blocked by treatment of the CLL cells with cirmtuzumab (UC-961), a first-in-class humanized mAb specific for a functional extracellular epitope of ROR1; this mAb is being evaluated in a phase I clinical trial in patients with CLL. To examine the activity of ibrutinib and/or cirmtuzumab, alone or in combination, we transferred human CLL cells into the peritoneal cavity of immune-deficient Rag2−/−γc−/− mice, which subsequently were treated with ibrutinib via oral gavage and/or cirmtuzumab administered iv. Although either agent alone resulted in some leukemia-cell clearance, cirmtuzumab and ibrutinib had apparent synergistic activity when used together in clearing human leukemia cells. We also examined the activity of each agent, alone or in combination, against a ROR1+ mouse leukemia, which we had engrafted in Rag2−/−γc−/− mice. While the engrafted mice treated with cirmtuzumab or ibrutinib alone had significantly smaller spleens and lower proportions of leukemia cells than the engrafted animals that did not receive any treatment, the mice treated with the combination of cirmtuzumab and ibrutinib had significantly smaller spleens and synergistic clearance of leukemia cells. Collectively, this study demonstrates that cirmtuzumab and ibrutinib may have synergistic activity in the treatment of patients with CLL, providing the rationale for clinical trials using cirmtuzumab in combination with ibrutinib, or another inhibitor of BTK, such as acalabrutinib, for treatment of patients with CLL or other B-cell malignancies dependent on non-canonical Wnt5a/ROR1 signaling. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.

scholarly journals Single Cell Approaches - Drilling Down Deeper Using Technology and Integrated Data Analyses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-28-SCI-28
Author(s):  
Dan A. Landau
Keyword(s):  
High Resolution ◽  
Chronic Lymphocytic Leukemia ◽  
Chronic Lymphocytic Leukaemia ◽  
Single Cell ◽  
Research Funding ◽  
Somatic Mutations ◽  
Lymphocytic Leukaemia ◽  
Lymphocytic Leukemia ◽  
Cancer Evolution ◽  
Cell Identity

Cancer progression, relapse and resistance are the result of an evolutionary optimization process. Vast intra-tumoral diversity provides the critical substrate for cancer to evolve and adapt to the selective pressures provided by effective therapy. Our previous work has shown that genetically distinct subpopulations compete and mold the genetic makeup of the malignancy (1, 2). Additionally, we have shown that epigenetic changes in cancer may be similar to the process of genetic diversification, in which stochastic trial and error leads to rare fitness enhancing events (3). These studies demonstrate the need to integrate genetic, epigenetic and transcriptional information in the study of cancer evolution, specifically at the single-cell resolution - the atomic unit of somatic evolution. To enable this work, we have developed a single-cell multi-omics toolkit, and apply it to chart the evolutionary history and developmental topographies of normal and malignant blood cells. First, we have applied single-cell multi-omics to chronic lymphocytic leukaemia (CLL), a highly informative model for cancer evolution (4). We applied multiplexed single-cell reduced-representation bisulfite sequencing to healthy B and CLL cells, and demonstrated that epimutations serve as a molecular clock. Heritable epimutation information therefore allows to infer high-resolution lineages with single-cell data, directly in patient samples. CLL tree topography showed earlier branching and longer branch lengths than normal B cell trees. These features reflect rapid drift after malignant transformation and CLL's greater proliferative history. Multi-omic single-cell Integration of methylome sequencing with whole transcriptome and genotyping capture validated tree topology inferred solely on the basis of epimutation information. To examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles involving TLR pathway activation. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy. Second, charting the transcriptomes of clonally mutated cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To tackle this challenge, we developed Genotyping of Transcriptomes (GoT), a technology to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing(5). With GoT we profiled thousands of CD34+ cells from patients myeloproliferative neoplasms to study how somatic mutations corrupt the process of human hematopoiesis. These data allow to superimpose the two differentiation trees; the native wildtype tree and the one corrupted by mutation. High-resolution mapping of malignant versus normal progenitors showed increased fitness with myeloid differentiation with CALR mutation. We identified the unfolded protein response as a predominant outcome of CALR mutations, with dependency on cell identity. Notably, stem cells and more differentiated progenitors show distinct transcriptional programs as a result of somatic mutation, suggesting differential sensitivity to therapeutic targeting. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in blood neoplasms is dependent on the native cell identity. Landau, D. A., Carter, S. L., Stojanov, P. et al., Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell152, 714-726 (2013).Landau, D. A., Tausch, E., Taylor-Weiner, A. N. et al., Mutations driving CLL and their evolution in progression and relapse. Nature526, 525-530 (2015).Landau, D. A., Clement, K., Ziller, M. J. et al., Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia. Cancer Cell26, 813-825 (2014).Gaiti, F., Chaligne, R., Gu, H. et al., Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia. Nature569, 576-580 (2019).Nam, A. S., Kim, K. T., Chaligne, R. et al., Somatic mutations and cell identity linked by Genotyping of Transcriptomes. Nature571, 355-360 (2019). Disclosures Landau: Pharmacyclics: Research Funding; Celgene: Research Funding; Illumina Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2922-2922
Author(s):  
Elisa ten Hacken ◽  
Thomas Oellerich ◽  
Maria Gounari ◽  
Julia Hoellenriegel ◽  
Kuan-Ting Pan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Label Free ◽  
Bcr Signaling ◽  
Associated Proteins

Abstract Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined. Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells. Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands. Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases. Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment. Disclosures Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

scholarly journals Aspartic Aminopeptidase Is a Novel Biomarker of Aggressive Chronic Lymphocytic Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1876
Author(s):  
Pramath Kakodkar ◽  
Sanket More ◽  
Kinga András ◽  
Nikos Papakonstantinou ◽  
Sharon Kelly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Phosphatidyl Inositol ◽  
Cell Types ◽  
Cell Receptor ◽  
Gene Signature ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Treatment of chronic lymphocytic leukemia has advanced substantially as our understanding of the kinase signal transduction pathways driven by the B cell receptor (BcR) has developed. Particularly, understanding the role of Bruton tyrosine kinase and phosphatidyl inositol 3 kinase delta in driving prosurvival signal transduction in chronic lymphocytic leukemia (CLL) cells and their targeting with pharmacological inhibitors (ibrutinib and idelalisib, respectively) has improved patient outcomes significantly. The kinase signaling pathway induced by the BcR is highly complex and has multiple interconnecting branches mediated by tyrosine and serine/threonine kinases activated downstream of the BcR. There is a high level of redundancy in the biological responses, with several BcR-signaling kinases driving nuclear factor kappa B activation or inducing antiapoptotic Bcl-2 genes. Accordingly, common gene targets of BcR-signaling kinases may serve as biomarkers indicating enhanced BCR-signaling and aggressive disease progression. This study used a gene expression correlation analysis of malignant B cell lines and primary CLL cells to identify genes whose expression correlated with BCR-signaling kinases overexpressed and/or overactivated in CLL, namely: AKT1, AKT2, BTK, MAPK1, MAPK3, PI3KCD and ZAP70. The analysis identified a 32-gene signature with a strong prognostic potential and DNPEP, the gene coding for aspartic aminopeptidase, as a predictor of aggressive CLL. DNPEP gene expression correlated with MAPK3, PI3KCD, and ZAP70 expression and, in the primary CLL test dataset, showed a strong prognostic potential. The inhibition of DNPEP with a pharmacological inhibitor enhanced the cytotoxic potential of idelalisib and ibrutinib, indicating a biological functionality of DNPEP in CLL. DNPEP, as an aminopeptidase, contributes to the maintenance of the free amino acid pool in CLL cells found to be an essential process for the survival of many cancer cell types, and thus, these results warrant further research into the exploitation of aminopeptidase inhibitors in the treatment of drug-resistant CLL.

scholarly journals Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
Author(s):  
Andreas Rosenwald ◽  
Ash A. Alizadeh ◽  
George Widhopf ◽  
Richard Simon ◽  
R. Eric Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Germ Line ◽  
Gene Expression Signature ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hanyin Wang ◽  
Shulan Tian ◽  
Qing Zhao ◽  
Wendy Blumenschein ◽  
Jennifer H. Yearley ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Expression Profiles ◽  
Lymphocytic Leukemia ◽  
B Cell Activation ◽  
Advisory Committees ◽  
Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

High ZAP-70 and Differential Expression of B-Cell Receptor Related Genes in Chronic Lymphocytic Leukemia with V3-21 Gene Usage.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 773-773
Author(s):  
Dirk Kienle ◽  
Alexander Kröber ◽  
Dirk Winkler ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Bcr Signaling ◽  
Gene Usage ◽  
Lower Expression

Abstract V3-21 gene usage defines a distinct genetic subgroup of chronic lymphocytic leukemia (CLL) characterized by a poor clinical outcome regardless of the VH mutation status. V3-21 cases exhibit a highly characteristic B-cell receptor (BCR) structure as demonstrated by homologous CDR3 sequences and a restricted use of VL genes implicating a common antigen involved in tumor pathogenesis of this specific CLL subgroup. To investigate the role of antigenic stimulation in the pathogenesis of V3-21 using CLL, we analyzed the quantitative expression of genes involved in BCR signaling (ZAP-70, SYK, BLNK, LYN, PI3K, PLCG2, FOS), B-cell activation (TRAF3, STAT6, NFKB), and cell cycle or apoptosis control (ATM, BCL-2, BAX, CDK4, CCND1, CCND2, CCND3, p27, E2F1, MYC) in V3-21 cases in comparison to VH mutated (VH MUT) and VH unmutated (VH UM) cases not using the V3-21 gene. To obtain native expression signatures we studied a non-CD19-purified (nPU) cohort (V3-21: 18 cases, equally divided into VH mutated and VH unmutated cases; VH MUT: 17; VH UM: 19) and, for verification, a CD19-purified (PU) cohort (V3-21: 10 cases, equally divided into VH mutated and unmutated; VH MUT: 12; VH UM: 16) to exclude a contamination of the results by non-tumor cells. All cases were analyzed by FISH for +3q, 6q-, +8q, 11q-, +12q, 13q-, 17p-, and t(11;14) to avoid major imbalances of genomic alterations between the subgroups under study. As expected, ZAP-70 expression was higher in VH UM as compared to VH MUT cases in the nPU (p=0.007) as well as the PU cohort (p=0.009). V3-21 cases showed a higher ZAP-70 expression as compared to VH MUT (nPU: p=0.033; PU: p=0.038). This applied also when restricting this comparison to V3-21 mutated cases (nPU: p=0.018). Median ZAP-70 expression in the PU cohort was 1.15 in VH MUT vs. 7.69 in VH UM cases, as compared to 7.05 in V3-21 cases (V3-21 mutated cases: 10.69; V3-21 unmutated: 6.7). Other genes differentially expressed between the V3-21 and VH MUT subgroups in nPU cases were PI3K (p=0.048), PLCG2 (p=0.007), CCND2 (p=0.003), p27 (p=0.003), BCL-2 (p=0.025), and ATM (p=0.006). In addition, a set of genes was detected with a differential expression between V3-21 and VH UM (nPU) including PLCG2 (p=0.014), NFKB (p=0.023), CCND2 (p=0.001), p27 (0.002), and BAX (p=0.028). Notably, except for ZAP-70, all of the differentially expressed genes showed a lower expression in V3-21 as compared to the other subgroups. When comparing the V3-21 mutated and V3-21 unmutated subgroups (nPU), there were no significant gene expression differences except for CDK4, which showed a lower expression in V3-21 unmutated cases. Therefore, cases with V3-21 usage appear to show a rather homogeneous gene expression pattern independently of the VH mutation status, which can be distinguished from VH MUT and VH UM cases not using V3-21. The expression differences observed suggest a role of differential BCR signaling in the pathogenesis of this distinct CLL subgroup. Deregulation of cell cycle, apoptosis, and candidate genes such as ATM indicate the involvement of additional pathways in the pathogenesis of CLL cases using V3-21.

B-Cell Receptor Signaling Enhances Migration of B-Cell Chronic Lymphocytic Leukemia Cells in Response to the Chemokine Stromal Cell-Derived Factor-1 (SDF-1/CXCL12).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1187-1187
Author(s):  
Jan A. Burger ◽  
Myriam Krome ◽  
Andrea Bürkle ◽  
Tanja N. Hartmann
Keyword(s):  
Dendritic Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Stromal Cells ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Follicular Dendritic Cells ◽  
Accessory Cells ◽  
Bcr Signaling

Abstract There is growing evidence that the microenvironment confers survival signals to Chronic Lymphocytic Leukemia (CLL) B-cells that may result in disease progression and resistance to therapy. In the marrow or secondary lymphoid tissues, CLL cells are in close contact with non-tumoral accessory cells, such as mesenchymal stromal cells or nurselike cells. We previously characterized SDF-1 (CXCL12) as a central mediator for CLL cell migration and interaction with the protective microenvironment. Constitutive secretion of CXCL12 attracts CLL cells to stroma or NLC through its cognate receptor, CXCR4. These accessory cells protect CLL cells from spontaneous or drug-induced apoptosis, which is contact-dependent and partially mediated by CXCL12. B-cell receptor (BCR) signaling has been considered another important regulator of CLL cell survival. Typically, CLL cell that lack somatic mutations in the immunoglobulin (Ig) variable region (V) genes and display high levels of the tyrosine kinase ZAP-70 strongly responds to anti-IgM stimulation. Because both, CXCL12 stimulation and BCR signaling may represent important mechanism for maintenance of CLL cell within the microenvironment, we examined whether anti-IgM stimulation affects CXCL12 responses in correlation with the ZAP-70 status. BCR signaling was modulated either by crosslinking the BCR with IgM or by blocking the tyrosine kinase Syk. Effective BCR cross-linking with anti-IgM antibodies was demonstrated by phosphorylation of Syk and p44/42 MAP kinase. In ZAP-70 positive cells, BCR crosslinking resulted in a robust activation of Syk, p44/42 MAP kinases, and protein kinase B (Akt). ZAP-70 negative CLL cells displayed a weaker activation of p44/42 upon IgM crosslinking. Pretreatment of CLL cells with anti-IgM resulted in an enhanced calcium mobilization upon CXCL12 stimulation. This was not due to changes in surface expression of CXCR4. Accordingly, Syk inhibition by piceatannol resulted in a loss of calcium response upon CXCL12 stimulation. Furthermore, anti-IgM stimulation significantly increased CLL cell chemotaxis towards CXCL12 1.4 ± 1.2fold (n=9, p=0.027), and Syk inhibition by piceatannol decreased chemotaxis to 0.6 ± 0.2fold of controls (n=8). In these experiments, we could not detect differences between ZAP-70 positive or negative cells. However, there was a strong difference regarding the spontaneous, CXCL12-dependent migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis). BCR crosslinking significantly increased pseudoemperipolesis of ZAP-70 expressing CLL cells 13.4 ± 21.0fold (n=7, p=0.043), whereas there was no significant increase in pseudoemperipolesis of ZAP-70 negative cells (1.4 ± 0.2fold increase, n=8). Syk inhibition by piceatannol significantly decreased the pseudoemperipolesis of ZAP-70 positive as well as ZAP-70 negative CLL cells to 0.4 ± 0.07 of controls (n=5, p=0.043). Interestingly, spontaneous migration of CLL cells beneath follicular dendritic cells (HK cells) was also significantly enhanced by anti-IgM stimulation, in particular in ZAP-70 positive cases. In summary, BCR signaling enhances calcium mobilization, CLL cell migration to CXCL12, and pseudoemperipolesis beneath marrow stroma or follicular dendritic cells. These data suggest that BCR stimulation co-operates with CXCL12 for localization and/or maintenance of CLL cells within distinct tissue microenvironments.

ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 178-178
Author(s):  
Stefania Gobessi ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Dimitar G. Efremov
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Progressive Disease ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

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