Gutierrez, Albert

Abstract 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; and investigate 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.

Abstract Abstract 92 BACKGROUND: All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS: We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS: Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS: Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract 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.

Abstract The canonical Wnt signaling pathway is pathogenic in a variety of cancers. We previously identified aberrant expression of the Wnt pathway transcription factor and target gene lymphoid enhancer binding factor-1 (LEF1) in chronic lymphocytic leukemia (CLL). This suggested that the Wnt signaling pathway has a role in the biology of CLL. In this study, we performed a Wnt pathway analysis using gene expression profiling and identified aberrant regulation of Wnt pathway target genes, ligands, and signaling members in CLL cells. Furthermore, we identified aberrant protein expression of LEF-1 specifically in CLL but not in normal mature B-cell subsets or after B-cell activation. Using the T cell–specific transcription factor/LEF (TCF/LEF) dual luciferase reporter assay, we demonstrated constitutive Wnt pathway activation in CLL, although the pathway was inactive in normal peripheral B cells. Importantly, LEF-1 knockdown decreased CLL B-cell survival. We also identified LEF-1 expression in CD19+/CD5+ cells obtained from patients with monoclonal B-cell lymphocytosis, suggesting a role for LEF-1 early in CLL leukemogenesis. This study has identified the constitutive activation and prosurvival function of LEF-1 and the Wnt pathway in CLL and uncovered a possible role for these factors in the preleukemic state of monoclonal B-cell lymphocytosis.

Abstract Much of the ability of the MUC1 oncoprotein to foster tumorigenesis and tumor progression likely originates from the interaction of its cytoplasmic tail with proteins involved in oncogenic signaling. Many of these interactions are regulated by phosphorylation, as the cytoplasmic tail contains seven highly conserved tyrosines and several serine/threonine phosphorylation sites. We have developed a cell line–based model system to study the effects of tyrosine phosphorylation on MUC1 signaling, with particular emphasis on its effects on gene transcription. COS-7 cells, which lack endogenous MUC1, were stably infected with wild-type MUC1 or a MUC1 construct lacking all seven tyrosines (MUC1 Y0) and analyzed for effects on transcription mediated by the extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathways. COS.MUC1 Y0 cells showed heightened active ERK1/2 with increased activator protein-1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) transcriptional activity; there was also a simultaneous decrease in NF-κB transcriptional activity and nuclear localization. These changes altered the phenotype of COS.MUC1 Y0 cells, as this line displayed increased invasion and enhanced [3H]thymidine incorporation. Analysis of the three lines also showed significant differences in their cell cycle profile and bromodeoxyuridine incorporation when the cells were serum starved. These data support the growing evidence that MUC1 is involved in transcriptional regulation and link MUC1 for the first time to the NF-κB pathway. (Mol Cancer Res 2006;4(7):489–97)