Survival Of pro-B Cells Research Articles

Chromodomain helicase DNA-binding 4 is required for proliferation, distal VH rearrangements and developmental progression of B cell progenitors (HEM1P.221)

Abstract Chromodomain Helicase DNA-binding protein 4 (CHD4, or Mi-2β) is a catalytic core subunit of Nucleosome Remodeling and Deacetylase (NuRD) complexes, which regulate chromatin structure and transcription in lymphocytes. CHD4 activities include ATP-dependent mobilization of nucleosomes, DNA binding and binding to histone tails. Here, we investigated requirements for CHD4 in B cell development in a mouse model system. We utilized Chd4flox/flox:Cd79a-Cre (Chd4 cko) mice, which inactivate Chd4 genes selectively in early B cell progenitors. These mice confirmed that CHD4 is essential for B lymphopoiesis. Following the loss of CHD4 expression, B cell development is arrested at the pro-B cell stage. Peripheral B220+ cells were nearly absent. To address the basis of the observed developmental arrest, we measured effects of the lack of CHD4 on proliferation and Igh gene rearrangements. CHD4-deficient pro-B cells fail to proliferate in response to IL-7. Furthermore, pro-B cells lacking CHD4 complete proximal VH to DJH rearrangements, but rearrange distal VH segments only rarely. Overall, our data demonstrate that CHD4 and NuRD complexes are essential for multiple aspects of early B cell development, including V(D)J recombination, proliferation and survival of pro-B cells.

Regulation of proliferation and survival of B-lymphocytes by Ebf1 - implications in leukaemia

Ebf1 is an important determinant of the specification of B lymphocytes. However, expression of this transcription factor is not limited to early B lymphopoiesis and constitutive deletion of Ebf1 results in developmental block and embryonic lethality. To gain insight into additional functions of Ebf1, we inactivated Ebf1 at various stages of differentiation using a Cre-lox-regulated conditional knockout allele. We found that Ebf1 is required for the proliferation and survival of pro-B cells and peripheral B-cell subsets. In Ebf1-deficient pro-B cells, overexpression of the pro-survival genes Bcl2 or BclXL was not sufficient for cell–survival. We found a remarkable clonal difference in the cell cycle response of transformed pro-B cells upon induced Ebf1-deletion: one cell type arrested in the G(1) phase and the other type died without arrest. Unexpectedly, the cycling cell-type survived upon c-myb overexpression, in contrast to the other transformed clone type or to primary pro-B cells, showing cell cycle arrest as well. This point to two different Ebf1 regulated cell-cycle-coupled cell survival pathways. As inactivating mutations of the EBF1 gene are frequent in acute lymphoblastic leukaemia, elucidating the role of Ebf1 in cell cycle and survival could provide novel means of intervention in the growth of leukaemia cells.

Aberrant Regulation of the LEF-1 Locus in Monoclonal B Cell Lymphocytosis (MBL) and Chronic Lymphocytic Leukemia (CLL): A Possible Role for Epigenetic Regulation.

Abstract 669 Background:Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. More recently, a widely prevalent premalignant condition termed monoclonal B cell lymphocytosis (MBL) has been defined and similarly involves expansion of a CD19+CD5+ population of B cells. MBL has been the focus of a wide number of recent studies in hopes it can provide insight into the early pathological events that lead to clonal expansion of a pre-leukemic CLL-like clone. Previously, we and others identified the transcription factor lymphocyte enhancer-binding factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells relative to blood CD19+ B cells from healthy adults. LEF-1 is crucial for the proliferation and survival of pro-B cells during B cell development and deregulated LEF-1 activation has been directly linked with leukemogenesis in a transgenic murine model. In the present study, we addressed three critical questions concerning LEF-1 and CLL: 1) do CD5+ normal B cells express LEF-1; 2) at what stage of transformation does LEF-1 expression first appear; and 3) what mechanism(s) underlies LEF-1 expression in leukemic B cells. Methodology:The goals of our study were to: 1) determine the LEF-1 expression status of normal CD 19+/CD5+ human B cells; 2) determine the expression status of LEF-1 in clonal B cells present in patients with MBL; and 3) identify regulatory mechanisms that control aberrant expression of LEF-1 in CLL B cells. In order to achieve the first two goals, a 3-color flow cytometry assay for CD19, CD5, and intracellular LEF-1 was developed. To achieve the third goal, we performed in silico analysis of the LEF-1 locus and correlated this with publicly available genome wide methylated CpG island recovery assay (MIRA) data describing the methylome of normal human B cells (Rauch, T. A. Proc Natl Acad Sci U S A. 2009; 106(3): 671-8). Results:Analysis of human umbilical cord blood B cells, a rich source of the CD19+/CD5+ B cell subset, revealed that all normal CD 19+/CD5+ human B cells are negative for LEF-1 protein expression. These data demonstrate LEF-1 expression by CLL B cells is truly aberrant and does not simply reflect the phenotype of CD5+ lineage B cells. We next tested if the CD19+/CD5+ cells obtained from patients with MBL express LEF-1 protein. In these analyses, we restricted our study to MBL patients with absolute B cell counts of less than 2.5 ×10 9 cells/(L) (range: 0.756 - 2.44 × 10 9 cells/(L)), which is significantly below the upper limit that defines the MBL to CLL transition. Of interest, each MBL sample analyzed (n=6) revealed the presence of two populations of cells: 1) CD19+/CD5+ B cells expressing LEF-1; and 2) CD19+/CD5- B cells lacking expression of LEF-1. To confirm the clonal nature of the CD19+/CD5+ cells from MBL patients we demonstrated that the cells were light chain restricted. These data clearly indicated that LEF-1 expression becomes deregulated in the premalignant state of MBL and may therefore represent an early event in CLL leukemogenesis. In order to identify differential pathways of LEF-1 regulation that may be altered in CLL and MBL B cells, in silico promoter analysis of LEF-1 was performed. We identified a number of potential transcription factor binding sites as well as a putative CpG island in the 5' promoter region of LEF-1. Using data from a human B cell genome wide methylation array, we were able to determine that this same putative LEF-1 promoter CpG island was highly methylated in normal human B cells. Methylation of promoter region CpG islands is known to play an important role in developmental regulation of gene expression and may be the operative mechanism underlying silencing of LEF-1 expression in normal B cells. Conclusions:LEF-1 expression is deregulated in MBL and appears to be an early and possibly key event in the transition of normal B cells into a premalignant/malignant state. Our data suggest that loss of epigenetic regulation of this developmentally important locus may play a role in aberrant LEF-1 expression in MBL and CLL B cells. Ongoing studies are aimed at determining the methylation status of the LEF-1 promoter in CLL B cells and the functional role of this protein in transcriptional regulation and survival of CLL and MBL cells. Disclosures:No relevant conflicts of interest to declare.

Overexpression of the LEF-1 and TCF4 Transcription Factors in B-CLL: Further Evidence for a Role of the Wnt Signaling Pathway in B-CLL Biology and Leukemogenesis

Background: B-chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. Our strategy was to employ gene expression profiling (GEP) to discover genetic differences between CLL B cells and blood B cells from healthy adults. Previously, we and others identified the transcription factor (TF) lymphocyte enhancer factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells suggesting a role for the oncogenic Wnt signaling pathway. LEF-1 acts as a central mediator of Wnt signaling and is crucial for the proliferation and survival of pro-B cells during development. Recently, deregulated LEF-1 activation has been directly linked with leukemogenesis.Methodology: The goals of our study were to:validate LEF-1 over expression in CLL B cells in a new cohort of B-CLL patients and normal controls;study CLL B cell LEF-1 protein expression; andinvestigate CLL B cell expression levels of other genes linked to Wnt signaling.To accomplish these goals, Affymetrix U133 GEP was performed on B cells from 41 B-CLL patients who were enrolled on a trial of combined pentostatin, cyclophosphamide, and rituximab and 11 healthy adults over age 60. Only the perfect match data were utilized for all analyses; the non-background corrected perfect match data were normalized using an intensity-dependent procedure and analyses were done using the base-2 logarithm transformed normalized values.Results: GEP analysis confirmed CLL B cells expressed the LEF-1 gene at ~28-fold higher levels than normal B cells (p<.0001). These results were validated by quantitative PCR and all CLL B cells studied expressed LEF-1 mRNA. By contrast, LEF-1 mRNA expression was undetectable in blood B cells from healthy adults both before and after in vitro mitogenic stimulation suggesting aberrant LEF-1 expression in B-CLL. Moreover, LEF-1 protein was readily detected by flow cytometry in CLL B cell samples and all clonal B cells expressed a uniform level of LEF-1. Full length LEF-1 is more oncogenic and predominates in certain cancers, while a dominant negative short isoform is found at greater levels in normal lymphocytes. Of interest, western blot analysis revealed that CLL B cells predominantly expressed full length oncogenic LEF-1. Ongoing studies are focused on silencing LEF-1 expression to determine LEF-1 target genes. In this regard, IGFBP4 (IGF binding protein 4) mRNA expression levels are 19-fold higher in CLL vs control B cells (p<0.0001) and the IGFBP4 protein was recently shown to antagonize Wnt signaling. A scan of the IGFBP4 promoter element identified 3 possible LEF/TCF consensus sites. Thus, there is a possible negative feedback loop in CLL, with activation of the Wnt pathway leading to expression of a negative Wnt regulator. Finally, we queried the GEP data for evidence of differential expression of 63 other Wnt-related genes. Surprisingly, in addition to LEF1, only five genes met our selection threshold of ≥1.5 fold change in expression between CLL and control B cells and p value <.001. These genes were CSNK1D (casein kinase, delta 1; 1.9-fold higher in control vs CLL; p<0.0001); CCND2 (cyclin D2; 2.4-fold higher in CLL vs control; p<0.0001); JUN (3.2-fold higher in control vs CLL); WNT3 (6.4-fold higher in CLL vs control; p<0.0001); and TCF4 (transcription factor 4, alias ITF-2; 4-fold higher in CLL vs control; p<0.0001). To our knowledge, we are the first to report that the TCF4 (ITF-2) gene is over expressed in B-CLL. This TF is of great interest because it is a known downstream target of the Wnt/TCF pathway, is activated in human cancers with b-catenin defects, and it promotes neoplastic transformation. Using Spearman correlation analysis to explore the relationship between LEF1, TCF4, and WNT3 expression levels, we observed a marginal correlation between WNT3 expression and LEF1 (ρ=0.31; p value=0.05) and TCF4 (ρ=0.29; p value=0.07). Summary: These studies add new support for a critical role of the Wnt pathway in CLL. The WNT3 results are consistent with the literature; however, it is notable that we failed to corroborate other reports demonstrating that other Wnts and Fzd genes are differentially expressed in CLL B cells vs normal B cells. It is striking that CLL B cells over express two TFs in this pathway that have been linked with neoplastic transformation. Ongoing studies are aimed at further elucidating the roles of these genes in B-CLL.