Progressive Epigenetic Programming during B Cell Maturation Is Reflected in a Continuum of Epigenetic Disease Phenotypes in Chronic Lymphocytic Leukemia

Abstract The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner. It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes. Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL. Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.

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Upregulation of Human Pim-2 in B-CLL, Involves Interactions between Oct-1, Oct-2, Bob-1 and NF-kB.

Blood ◽

10.1182/blood.v104.11.1916.1916 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1916-1916

Author(s):

Yosef Dicken ◽

Amos M. Cohen ◽

Hanna Bessler ◽

Daphna Levi-Hirsh ◽

Ariela Arad ◽

...

Keyword(s):

Transcription Factor ◽

B Cells ◽

B Cell ◽

Promoter Region ◽

Lymphocytic Leukemia ◽

Proximal Promoter ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Cell Extracts ◽

Hodgkin's Lymphomas

Abstract hPim-2 is a proto-oncogene that encodes a serine/threonine kinase and inhibits apoptosis by phosphorylation of BAD. We have shown that hPim is upregulated in human non-Hodgkin’s lymphomas (NHL) and in chronic lymphocytic leukemia (B-CLL) and its cellular transcript levels in B-CLL correlates with lymphocyte doubling time. We found no mutations in the promoter region of hPim-2 in B-cells of 30 patients with CLL (~2000 bp upstream). The proximal promoter region of hPim-2 (600 bp) contains two adjacent NF-kB-binding elements, two adjacent Oct-binding elements and an SP1 element by bioinformatic analysis. Studies have recently shown that the transcription factor Oct-2 and the B-cell specific Oct cofactor Bob-1 are overexpressed in certain large B-cell lymphomas, whereas increased expression of Bob-1 has also been observed in T-cell neoplasms. Shift assays (EMSA) analysis, using nuclear extracts from B-CLL cells and various fragments of hPim-2 promoter region used as probes, revealed that complexes containing an Oct elements were consistently heavier in B-CLL extracts compared with control B-cells. Accordingly, Oct-1, Oct-2 and Bob-1 protein levels were significantly higher in B-CLL compared to healthy extracts. Moreover, chromatin immunoprecipitation (Chip) assays confirmed that in-vivo Oct-1+2 and Bob-1 are indeed physically attached to the hPim-2 promoter, and that this interaction is significantly more intensive in B-CLL cells than in control B-cells. Furthermore, we have found in addition that the p52 isoform subunit of NF-kB predominates the interaction with the kB element in the hPim-2 promoter in B-CLL cells, as compared to the p50 isoform observed in control B-cells. To determine whether these interactions are transcriptionaly significant, we fused the luciferase reporter gene to various promoter fragments, and monitored luciferase expression in-vitro after incubation with either B-CLL or normal B-cell extracts. Luciferase expression was consistently higher when Oct element-containing fragment was incubated with B-CLL cell extracts. Together, these results suggest that the upregulation of hPim-2 in B-CLL is due to enhanced expression and transcriptional activity of the Oct-1+2 and Bob-1 complex and that it might synergistically act with the p52 containing NF-kB transcription factor.

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DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Blood ◽

10.1182/blood.v110.11.685.685 ◽

2007 ◽

Vol 110 (11) ◽

pp. 685-685 ◽

Cited By ~ 1

Author(s):

Rita Shaknovich ◽

Leandro Cerchietti ◽

Maria E. Figueroa ◽

Ari Melnick

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Dna Methyltransferase ◽

Critical Role ◽

Epigenetic Programming ◽

Differential Digestion ◽

Epigenetic Signatures

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

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Different Expression of FcgammaRIIb in Chronic Lymphocytic Leukemia and Human Normal B Lymphocytes

Blood ◽

10.1182/blood.v112.11.3134.3134 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3134-3134

Author(s):

Carol Moreno ◽

Rajendra Damle ◽

Sonia Jansa ◽

Gerardo Ferrer ◽

Pau Abrisqueta ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocytes ◽

Surface Membrane ◽

Memory B Cells ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

B Cell Subsets

Abstract The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p<0.001]. Significant differences were also observed between CD5− and CD5+ normal B cells. In contrast, FcγRIIb expression was lower in CLL cells than in CD5+ and CD5− normal B lymphocytes [median MFI: 6901(1034–42600), 10180 (5856–14820) and 12120 (7776–16040); p<0.05)]. Interestingly, FcγRIIb expression was variable within individual CLL clones, this being higher in CD38+ and CD49d+ cells than in CD38− and CD49d− cells (p<0.05). Furthermore, the highest density of FcγRIIb was observed on those cells which coexpressed CD38 and CD49d. In contrast, no significant differences were observed between FcγRIIb and the expression of the activation antigen CD69. Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with <30% CD49d+ cells (p=0.006). The findings presented in this study suggest a hierarchy of FcγRIIb expression in normal B-cells, CLL cells and their subpopulations: circulating normal CD5− B cells > circulating normal CD5+ B cells > circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

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Impact Of The Number Of Prior Treatment Lines Received On The Distribution Of Peripheral Blood Leukocyte Subsets In Advanced-Stage B-Cell Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v122.21.4158.4158 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4158-4158

Author(s):

Georgiana Grigore ◽

Susana Barrena ◽

Martin Perez-Andres ◽

Miriam Fierro ◽

Marcos González ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Lymphocytic Leukemia ◽

Advanced Stage ◽

Absolute Count ◽

Purine Analogues ◽

Previously Treated Patients ◽

Previously Treated ◽

The Absolute ◽

B Cell Subsets

Abstract Introduction It is currently well-known that B-cell chronic lymphocytic leukemia (CLL) patients have an impaired immune function -particularly in advanced disease-, which significantly contributes to a higher risk of infections. The introduction of new effective therapeutic agents, such as the purine analogues (or alkylating agents with concomitant properties of purine analogues) plusanti-CD20 monoclonal antibodies, have significantly increased the rate of complete responses in CLL, but so far their impact on the overall immune function and the spectrum of infections occurring in CLL patients after therapy, remains to be fully understood. Aim To evaluate the effect of the number of treatment lines received on the different normal circulating leucocyte cell populations, including normal B-cell subsets, in advanced-stage treated CLL. Material and Methods The distribution of peripheral blood (PB) leukocytes was analyzed in 85 untreated CLL patients, and compared to that of 63 patients who had previously been treated with 1 line of treatment (n=39) or >1 line of treatment (n=24), and who failed to respond. Analysis was performed by 8-color flow cytometry with monoclonal antibodies against CD3, CD4, CD5, CD8, TCRgd, CD19, CD20, CD27, CD38, CD45, CD56, sIgM, sIgA, sIgG, sIgLambda and sIgKappa. Results The absolute count of circulating malignant B cells was not significantly different (p>0.05) in the untreated vs. previously treated patients who received 1 or >1 line of treatment (77,627±84,211 vs 67,994±72,087 vs 59,282±74,206 cells/uL; respectively). In contrast, as compared to untreated patients, PB normal B cells were found to be reduced in patients who had received either 1 line or >1 line of treatment (89±142 vs 36±57 and 23±32 cells/uL, p=0.004 and p<0.001, respectively), but at similar levels between the two groups of previously treated patients (p>0.05). When dissecting the normal B-cell subsets, therapy-related decreased B-cell numbers were mostly due to a reduced number of circulating memory B cells (67±98 vs 21±45 and 15±26 cells/uL; p=0.001 and p<0.001, respectively), including all memory isotypes: IgM (24±31 vs 5±13 and 5±6 cells/uL, p<0.001), IgG (25±59 vs 10±32 and 6±13 cells/uL; p=0.008) and IgA (18±30 vs 5±10 and 6±12 cells/uL; p=0.001 and p=0.003). No significant differences were found as regards the absolute count of immature (5±11 vs 8±26 and 3±11 cells/uL; p>0.05) and naïve (16±55 vs 6±18 and 4±8 cells/uL; p>0.05) B cells, nor for circulating plasma cells (3±18 vs 5±21 and 2±6 cells/uL; p>0.05), regardless of the therapy status. As compared to untreated patients, the absolute count of CD4+ T cells and CD4/CD8 double negative TCRαβ cells were significantly lower in patients with >1 line of treatment (1,836±1,340 vs 1,256±1,027 and 131±165 vs 56±97 cells/uL, p=0.03 and p=0.007 respectively) but not in those who had received only 1 line (1,477±1,349 and 201±747 cells/uL; p>0.05). In contrast, therapy did not show a significant impact on the absolute count of PB T CD8+ and TCRgd cells. No statistically significant differences were observed in the number of PB innate immune subpopulations including, neutrophils, eosinophils, basophils, monocytes, NK cells and dendritic cells. Conclusions While there are no differences regarding the number of leukemic cells, previously treated patients have significantly reduced counts of total and memory (all isotypes) normal B-cell subsets when compared to untreated patients. Together with this, CD4+ helper T cells could also be compromised after more than 1 line of treatment. Monitoring of these therapy-related immune defects could contribute to a better management of infectious complications in advanced-stage CLL patients. Disclosures: No relevant conflicts of interest to declare.

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Modeling of the Epigenome of the Cell-of-Origin Identifies Cancer-Specific DNA Methylation Patterns in CLL

Blood ◽

10.1182/blood-2018-99-115941 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3885-3885

Author(s):

Justyna Anna Wierzbinska ◽

Reka Toth ◽

Naveed Ishaque ◽

Jan-Phillip Mallm ◽

Karsten Rippe ◽

...

Keyword(s):

Dna Methylation ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

B Cell Differentiation ◽

Advisory Committees ◽

Normal Differentiation ◽

Methylation Patterns

Abstract Normal B cells undergo extensive epigenetic programming during normal differentiation and distinct B cell differentiation stages represent unique DNA methylation patterns. Chronic Lymphocytic Leukemia (CLL) originates from rapidly differentiating B cells and their DNA methylation signature is stably propagated in CLL. Consequently, CLL methylome data can be used to infer the putative cell-of-origin (COO) for each individual CLL case. We define the COO of CLL as the cell that has acquired a first oncogenic hit and which will initiate tumorigenic growth if one or more additional hits have been acquired. This means that two factors contribute to the epigenetic profile of CLL cells: first, the epigenetic profile of the founder B cell at the time of malignant transformation and second, CLL-specific epigenetic alterations that are acquired during leukemogenesis and progression of the disease. Previous studies using peripheral blood CD19+ B cells as a reference for aberrant methylation calls completely neglected the massive epigenetic programming that occurs during normal B cell differentiation. Thus, novel strategies aiming at identifying truly CLL-specific methylation changes considering the highly dynamic methylome during normal B cell differentiation were urgently needed. Here we outline a new analytical framework to delineate CLL-specific DNA methylation. We demonstrate how this approach can be applied to detect epigenetically deregulated transcripts in CLL. Firstly, we modeled the epigenome dynamics occurring during normal B cell differentiation using linear regression. The DNA methylomes of CLL cells were then precisely positioned onto the normal B cell differentiation trajectory to define the closest normal B cell methylome for every CLL patient, the COO. The epigenome of the COO then served as a reference for aberrant DNA methylation calls. We dissected two categories of CLL-specific methylation events: those occurring at sites undergoing epigenetic programming during B cell differentiation and those that normally do not change during B cell differentiation. The first group was further subdivided into class A and B, displaying exaggerated methylation loss or gain, respectively, and class C showing both hyper- and hypomethylation relative to the normal differentiation. The second group was classified into class D displaying hypo- and class E showing hypermethylation. Overall, only 1.6% of the CpG-sites (7,248 CpGs) represented on the Illumina 450k array were affected by disease-specific methylation programming, mostly hypomethylation (6,680 CpGs). Next, the molecular programs underlying the CLL-specific methylation patterns were investigated. We tested enrichment of chromatin states and of transcription factor binding sites (TFBS) as identified in an immortalized B cell line (GM12878). This indicated that disease-specific methylation events target transcriptionally relevant cis-regulatory elements in CLL (enhancers, weak and poised promoters and insulator regions). In line with this, CLL-specific differentially methylated regions affected TFBS associated with signaling pathways known to be important in normal B-cell differentiation (i.e. BATF, EBF1). We also observed altered methylation at CTCF binding sites suggesting their involvement in CLL pathogenesis. In the present work, we dissected CLL methylomes to distinguish between normal B cell differentiation-associated methylation patterns and CLL-specific methylation events. We showed that this approach is indispensable to identify key pathogenic events driving CLL pathogenesis. The relevance of our approach was demonstrated by contrasting the number of epigenetically deregulated miRNAs and protein-coding genes to those determined with a classic analysis using CD19+ B cells as controls. This highlights the extent of overcalling of CLL-specific methylation patterns in previous studies (~30-fold for protein-coding genes and ~10-fold for miRNAs) and stresses the importance to consider normal differentiation trajectories for the identification of aberrant DNA methylation events. Here we propose 11 protein-coding genes (e.g. DOK2, CLLU1) and 4 miRNAs (e.g. miR-486, miR-195) as being epigenetically deregulated in CLL. Our analytical approach provides a general framework for the identification of disease-specific epigenomic changes that should be applicable to other cancers in the future. Disclosures Küppers: the Takeda Advisory Board: Membership on an entity's Board of Directors or advisory committees. Stilgenbauer:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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SYK Inhibition Disrupts the Cross-Talk Between B-Cell Activation Factor (BAFF) and B-Cell Receptor (BCR) and Thereby Antagonizes Mcl-1 in Chronic Lymphocytic Leukemia (CLL) B-Cells

Blood ◽

10.1182/blood.v128.22.303.303 ◽

2016 ◽

Vol 128 (22) ◽

pp. 303-303

Author(s):

Cody Paiva ◽

Taylor Rowland ◽

Olga Danilova ◽

Bhargava Sreekantham ◽

Stephen E Spurgeon ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Cell Survival ◽

Research Funding ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Mutational Status ◽

Pathway Gene ◽

Bcr Signaling

Abstract Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Soluble mediators emanating from the tumor microenvironment perpetrate CLL cell survival and may account for resistance to BCRi. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses.The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied. We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling, uncovering the functional significance of the BCR-associated kinases and Bcl-2 family proteins in this setting. Peripheral blood mononuclear cells were isolated from patients with CLL. We established a novel BAFF-expressing stromal co-culture model and referenced it to control, CD40L-expressing stroma and soluble BAFF. We employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib) and measured CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry. CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X. BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYKand LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi's fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. We found that entospletinib, but not other BCRi's, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma. Unlike in IgM-stimulated cells, entospletinib did not promote Mcl-1 protein degradation. By contrast,, targeting SYK in BAFF-stimulated cells abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript, an effect mimicked by ruxolitinib, a JAK/STAT inhibitor. BAFF receptor signals via the TRAF3/NIK/IKK1 axis to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that NIK (NFκB-inducing kinase) or IKK1 could be directly responsible for SYK activation by BAFF. Indeed, genetic knockdown of NIK resulted in decreased SYK activation, whereas IP experiments demonstrated that NIK directly complexed with SYK in BAFF-stimulated neoplastic B-cells, confirming NIK role in activation of BCR signaling. Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL. Disclosures Spurgeon: Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Danilov:Prime Oncology: Honoraria; Dava Oncology: Honoraria; ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Takeda: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy.

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Opposite Effects of Bruton Tyrosine Kinase (BTK) Inhibitor Therapy with Ibrutinib and FCR Chemo-Immunotherapy on the Normal B Lymphocyte Repertoire in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.4402.4402 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4402-4402

Author(s):

Simon Schliffke ◽

Mariela Sivina ◽

Ekaterina Kim ◽

Benjamin Thiele ◽

Nuray Akyüz ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Research Funding ◽

Treatment Initiation ◽

Lymphocytic Leukemia ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Btk Inhibitor ◽

V Gene ◽

Immunoglobulin Levels

Abstract Disease-inherent and treatment-related immune dysfunction remain leading causes for morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). The advent of kinase inhibitors that target B cell receptor (BCR) signaling, which lack myelo- and T lymphocyte toxicity, raised hopes that these new agents may be less immunosuppressive and allow for better immune reconstitution when compared to chemo-immunotherapy (CIT). The effects of the BTK inhibitor ibrutinib or CIT with fludarabine, cyclophosphamide and rituximab (FCR) on the normal B cell repertoire have not been well characterized. Here, we used state-of-the-art immunosequencing technology to investigate how ibrutinib treatment affects the regeneration of non-malignant B-cells when compared to patients treated with FCR. Clinical data on infection rates and immunoglobulin levels was analyzed from 40 CLL patients treated with ibrutinib (median number of two pre-treatments) or frontline CIT with FCR at MD Anderson Cancer Center. In a representative subset of 20 patients, flow cytometry and next generation sequencing (NGS) of the immunoglobulin heavy chain (IGH) gene locus was used to monitor non-malignant B-cell immune reconstitution for 24 months after start of treatment with ibrutinib or FCR. Comparison of ibrutinib treatment with CIT revealed that immunoglobulin levels remained stable and relatively low in both cohorts, except for an increase in IgA during ibrutinib treatment, as previously reported. NGS results showed that ibrutinib treatment significantly decreased the non-malignant B-cells count after 24 months of treatment, while the counts were quantitatively stable in the FCR cohort. Next, we determined the dynamics of non-malignant B-cell immune repertoire composition over treatment. Based on the mutational status of the V gene, non-malignant B-cells were classified as IGH hypermutated (<98% identity to the corresponding germline V gene, corresponding to antigen-experienced B-cells) or IGH unmutated (≥98% identity to the corresponding germline V gene, corresponding to antigen-naïve B-cells). Before treatment initiation, the mean percentage of antigen-experienced B-cells did not significantly differ between the groups (ibrutinib 39%, FCR 48%). After 24 months, a significant decrease of antigen-experienced B-cells was observed in the FCR cohort, while the ratio of antigen-experienced and antigen-naïve B-cells remained unchanged in ibrutinib treated patients (ibrutinib 39%, FCR 22%, p=0.01). Analysis of the IGH clonotype repertoire using the Shannon-Wiener and the inverse Simpson diversity indices confirmed these results, showing that the non-malignant IGH repertoire was composed of balanced numbers of antigen-experienced and antigen-naïve medium sized clones before treatment initiation in both cohorts. In line with the IGH repertoire shift towards antigen-naïve B-cells in FCR treated patients, the medium-sized clones disappeared after treatment, with large numbers of small-sized unmutated clones dominating after 24 months (p<0.0001). In ibrutinib treated patients, the repertoire diversity remained stable throughout the course of treatment. Taken together, our data indicate that continuous treatment with ibrutinib preserves preexisting (partially antigen-experienced) B-cells but impairs de-novo generation of naive B-cells. In contrast, FCR leads to a deletion of memory B-cells but also a subsequent substantial renewal of the B-cell repertoire. Both patterns may differentially affect immune-competence towards infections. Disclosures Bokemeyer: Karyopharm: Research Funding. Jain:Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Genentech: Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria, Research Funding; BMS: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding. Wierda:Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Genentech: Research Funding. Burger:Pharmacyclics: Research Funding.

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Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression

Journal of Experimental Medicine ◽

10.1084/jem.20160712 ◽

2016 ◽

Vol 213 (13) ◽

pp. 3007-3024 ◽

Cited By ~ 24

Author(s):

Kyoko Hayakawa ◽

Anthony M. Formica ◽

Joni Brill-Dashoff ◽

Susan A. Shinton ◽

Daiju Ichikawa ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Subset ◽

Lymphocytic Leukemia ◽

Transgenic Expression ◽

Bcr Signaling ◽

B Cell Subsets ◽

A Minor ◽

B1 B Cells

In mice, generation of autoreactive CD5+ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eμ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.

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Genome-wide promoter methylation of hairy cell leukemia

Blood Advances ◽

10.1182/bloodadvances.2018024059 ◽

2019 ◽

Vol 3 (3) ◽

pp. 384-396 ◽

Cited By ~ 4

Author(s):

Alberto J. Arribas ◽

Andrea Rinaldi ◽

Giorgia Chiodin ◽

Ivo Kwee ◽

Afua Adjeiwaa Mensah ◽

...

Keyword(s):

Dna Methylation ◽

B Cells ◽

B Cell ◽

Hairy Cell Leukemia ◽

Methylation Profile ◽

Hairy Cell ◽

Cell Leukemia ◽

Dna Methylation Profiling ◽

B Cell Subsets ◽

Methylation Profiling

Abstract Classic hairy cell leukemia (HCL) is a tumor of mature clonal B cells with unique genetic, morphologic, and phenotypic features. DNA methylation profiling has provided a new tier of investigation to gain insight into the origin and behavior of B-cell malignancies; however, the methylation profile of HCL has not been specifically investigated. DNA methylation profiling was analyzed with the Infinium HumanMethylation27 array in 41 mature B-cell tumors, including 11 HCL, 7 splenic marginal zone lymphomas (SMZLs), and chronic lymphocytic leukemia with an unmutated (n = 7) or mutated (n = 6) immunoglobulin gene heavy chain variable (IGHV) region or using IGHV3-21 (n = 10). Methylation profiles of nontumor B-cell subsets and gene expression profiling data were obtained from public databases. HCL had a methylation signature distinct from each B-cell tumor entity, including the closest entity, SMZL. Comparison with normal B-cell subsets revealed the strongest similarity with postgerminal center (GC) B cells and a clear separation from pre-GC and GC cellular programs. Comparison of the integrated analysis with post-GC B cells revealed significant hypomethylation and overexpression of BCR–TLR–NF-κB and BRAF-MAPK signaling pathways and cell adhesion, as well as hypermethylation and underexpression of cell-differentiation markers and methylated genes in cancer, suggesting regulation of the transformed hairy cells through specific components of the B-cell receptor and the BRAF signaling pathways. Our data identify a specific methylation profile of HCL, which may help to distinguish it from other mature B-cell tumors.

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Axl Regulates Fibroblast Growth Factor Receptor Signaling in B-Cell Chronic Lymphocytic Leukemia: Dual Targeting in CLL Therapy

Blood ◽

10.1182/blood.v124.21.831.831 ◽

2014 ◽

Vol 124 (21) ◽

pp. 831-831

Author(s):

Sutapa Sinha ◽

Justin Boysen ◽

Charla Secreto ◽

Steven L. Warner ◽

Neil E. Kay ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

High Affinity ◽

Fgfr Signaling ◽

Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells. Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells. To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition. In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

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