Chronic Lymphocytic Leukemia (CLL) Research Center
Research Faculty
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Lynn Wang, M.D.
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Project Title: "Role of innate immune signals in chronic lymphocytic leukemia"
The broad, long-term goal of our studies is to elucidate the role of innate immune signals in CLL. We propose that CLL may originate from the unrestrained expansion of yet undefined "innate" B cell subset specialized in immune responses to highly conserved microbial or “self” molecular patterns. This hypothesis is based upon recent observations showing that malignant CLL B cells express multiple innate antigen receptors, including germline gene-encoded Toll-like receptors (TLRs) and somatically recombined immunoglobulin (Ig) receptors, including polyreactive Ig receptors encoded by stereotyped V(D)J genes. We argue that microbial and/or autologous molecular patterns stimulate CLL B cells through two distinct innate pathways. In the first of these pathways, molecular patterns directly stimulate CLL B cells through dual engagement of TLRs and Ig receptors. These structures are powerful activators of NF-kB, a transcription factor typically dysregulated in CLL. In the second pathway, microbial and/or autologous molecular patterns indirectly stimulate CLL B cells by inducing stromal cell release of BAFF and APRIL, two TLR-inducible B cell-activating factor. By engaging TACI, BCMA and BAFF-R on CLL B cells, TLR-induced BAFF and APRIL would further stimulate CLL B cell survival, expansion and gene diversification. This possibility implies that soluble BAFF/APRIL decoy receptors (e.g., TACI-Ig) could be used to treat CLL.
In the past year, we made the following progress. We found that malignant CLL B cells expressed TLR9, an innate ligand for microbial and autologous hypomethylated CpG-rich DNA. Expression of TLR9 RNA and protein was documented in CLL B cells both ex vivo and in vivo. Of note, TLR9 as well as other TLRs seemed to be more abundant in the so-called proliferation centers, which are follicle-like areas of malignant B cell accumulation in the bone marrow of CLL patients. Within proliferation centers, CLL B cells also expressed more TACI, BAFF-R and phosphorylated Zap70, a kinase involved in Ig and perhaps TLR signaling.We also found that CLL B cells were proximal to stromal cells expressing BAFF and APRIL, including endothelial cells lining high endothelial venules. These small vessels expressed TLR9 and were often in close proximity of proliferation centers. Thus, malignant B cells may enhance their growth and survival by taking advantage of a TLR-induced BAFF/APRIL-dependent circuit within proliferation centers. Consistent with this possibility, endothelial cells augmented BAFF and APRIL production in response to TLR9 ligands. Of note, also malignant B cells expressed some BAFF and APRIL, indicating that BAFF and APRIL function via both paracrine and autocrine pathways.
Future studies will analyze the functional interaction between Ig, TLR, TACI and BAFF-R receptors in CLL B cells as well as the role of endothelial cells in the survival, proliferation and differentiation of CLL B cells. Additional studies will verify the ability of soluble BAFF/APRIL decoy receptors (e.g., TACI-Ig) and anti-angiogenic drugs, including Thalidomide derivatives, to attenuate CLL B cell accumulation as induced by TLR-activated endothelial cells.
Project Title: "Evaluation of therapeutic potential of IKK-NFkB inhibitors in CLL"
Our project is to evaluate potential therapeutic benefit of IKK-NFkB/Rel inhibitors in CLL. The IKK and NF-kB signaling pathway is involved in the regulation of cell growth and survival of many cell types and tumors. A landmark work published by Drs. Richard Furman and Elaine Schattner in 2000 demonstrated that majority of B cells derived from CLL patients express constitutively high levels of nuclear NF-kB/Rel activity. They further showed that CD40 likely contributed to NF-kB/Rel activation and blocking the CD40 pathway abolished NF-kB/Rel activation and induced apoptosis of CLL tumor cells. In light of the observation that CLL cells have an acquired survival advantage in vivo, we hypothesize that the constitutive NF-kB/Rel activity may contribute to CLL survival in vivo. Thus, the main focus of this project is to evaluate therapeutic potential of blocking IKK and NF-kB/Rel pathway for CLL.
The first aim of this project is to investigate the effect of IKK-beta inhibitor on CLL B cell survival, apoptosis, and drug sensitivity. An experimental IKK-beta inhibitor will be tested on B cell tumors derived from CLL patients. The purpose is to assess any potential therapeutic value by using IKK-beta inhibitors either as a single agent or in combination with current CLL therapeutic agents. The second aim of the project is to explore and develop novel NF-kB inhibitors for CLL. We have begun a high throughput screening approach with the goal to identify NF-kB-member specific small molecular inhibitors. Initial screen has identified two classes of compounds that have potential inhibitory activity on c-Rel/NF-kB. Current effort is to improve potency and physical chemical property for the two scaffolds. Lead compounds with high potency and selectivity will be tested on CLL tumor cells. Our ultimate goal is to identify and develop novel IKK and/or NF-kB inhibitors as potential drug candidates for further clinical evaluation in the CLL Research Center at the New York Presbyterian Hospital.
Project title: "Enhancing Apoptotic CLL-loaded Dendritic Cell Vaccination by Targeting GC-binding Protein, a Novel Regulator of Interleukin-12 Production"
It is commonly believed that the key factor produced by DCs in such a system that drives the potent anti-tumor immunity is Interleukin-12 (IL-12), which is the most potent cytokine that promotes T helper 1 (TH1) differentiation and cell-mediated immunity against intracellular pathogens and malignant cells. Moreover, IL-12 receptor expressed on B cells has been shown recently to function as a “tumor suppressor” to limit the development of chronic B cell lymphoproliferative disorders and B cell-mediated autoimmunity. Thus, the production of IL-12 by DC during antigen take up and presentation is a critical event that determines whether a potent tumor-specific immune response can be generated.
Paradoxically, phagocytosis of apoptotic cells by APC usually results in an anti-inflammatory state with inhibition of proinflammatory cytokines such as IL-12. Recently, our group demonstrated that phagocytosis of autologous apoptotic cells by macrophages and DC induces strong inhibition of IL-12 production through phosphatidylserine (PS)-mediated signaling, leading to the activation by tyrosine dephosphorylation of a novel zinc finger nuclear protein, which we identified, cloned, and named GC binding protein (GC-BP). GC-BP selectively inhibits IL-12 p35 gene transcription by binding to its promoter in vitro and in vivo, thus decreasing IL-12 production by APC and IFNg production by activated T cells. Therefore, GC-BP may represent a major regulator of IL-12 production during phagocytosis of apoptotic cells by APC.
We hypothesize that the functional capacity of APC, i.e. activation of CLL-specific T cells, following phagocytosis of apoptotic CLL can be significantly enhanced by elevating the level of IL-12 production via blocking the expression of GC-BP in APC.
Thus far, the following experiments have been done:
We have compared CLL patient-derived myeloid dendritic cells (mDCs) with normal donors’ mDCs in terms of their capacity to produce IL-10 and IL-12. The data, although preliminary, indicates that CLL cells, unlike normal B cells, have lost the ability to suppress IL-12 production by LPS-activated mDCs. This loss is not related to IL-10, a potent inhibitor of IL-12 synthesis. IL-12 was recently described to have a protective activity against spontaneous apoptosis of CLL cells. The loss of the IL-12-inhibiting activity by CLL cells may be a way to promote their own survival.
Currently, we are performing experiments to determine if the loss of the IL-12-inhibiting activity by CLL cells is dependent on TGF-b, another potent inhibitor of IL-12 production and immunosuppressor.
We are also trying various strategies to knockdown GC-BP expression in mDCs that are fed with apoptotic CLL cells to see if that can lead to greater IL-12 production and more potent activity of the DCs to stimulate autologous T cell activation.
Project Title: "Angiogenesis in CLL"
Angiogenesis, the growth of new blood vessels, requires dynamic expansion, assembly and stabilization of vascular endothelial cells in response to pro-angiogenic stimuli. Anti-angiogenic strategies have become an important therapeutic modality for solid tumors. Neo-angiogenesis has increasingly been recognized to play potentially important pathogenic roles in lymphomagenesis, by mediating autocrine stimulation of proliferation and survival of lymphoma tumor cells via the VEGF-VEGF receptor axis, and through recruitment of bone marrow-derived progenitors to support neovascular assembly and metastasis.
Angiogenic mechanisms and anti-angiogenic therapies have been explored in CLL/SLL. Both vascular endothelial growth factor (VEGF) and VEGF receptors are expressed by CLL cells. VEGF signaling via VEGFR-1 and VEGFR-2 confers resistance to apoptosis and promotes survival. Disruption of this autocrine pathway in CLL by epigallocatechin-3-gallate (EGCG), a receptor tyrosine kinase (RTK) inhibitor, leads to apoptosis and cell death. Anti-angiogenic compounds, particularly the immunomodulatory drugs (iMiDs), have demonstrated therapeutic efficacies in CLL patients. In combination with fludarabine, thalidomide was associated with significant therapeutic efficacy in both upfront and relapsed settings. A newer generation iMiDs, lenalidomide has shown marked activity in relapsed CLL patients.
In search for novel cellular and molecular mechanisms involved in the assembly of tumor neo-vessels, our group investigated the impact of perivascular accessory cells and stromal cells on lymphoma angiogenesis. We have shown that perivascular composition of different human lymphoma subtypes reflects differential recruitment and assembly of either hemangiogenic (CD68+VEGFR-1+) cells or mesenchymal (a-SMA+) cells. In CLL/SLL, the perivascular compartment is marked by diffuse a-SMA+ pericytic coverage, leading to a more mature vascular composition. In contrast, the perivascular apparatus was marked by VEGF-producing CD68+VEGFR1+ myelomonocytic hematopoietic cells in aggressive subtypes of Burkitt’s lymphoma and DLBCL, lending structural and paracrine support to nascent vasculature which was largely devoid of a-SMA+ pericyte coverage in response to rapid neoplastic growth. Thus, perivascular accessory cells in lymphoma subtypes reflect differential angiogenic responses to distinct proangiogenic growth factor / cytokine milieu within the tumor microenvironment, providing potentially unique targets for anti-angiogenic intervention.
We are currently studying the function of VEGFR-1+CD68+ hematopoietic progenitor cells and a-SMA+ mesenchymal stromal cells in supporting neo-angiogenesis including lymphangiogenesis and tumor growth in CLL/SLL, and explore the clinical prognostic utility of these angiogenesis biomarkers with regards to disease status and response to anti-angiogenic treatment. Specifically, we aim to 1) determine the origin and functional contribution of a-SMA+ stromal cells and VEGFR-1+CD68+ hematopoietic progenitors in supporting neo-angiogenesis in mouse models of non-Hodgkin’s lymphoma; 2) characterize the levels of tissue and circulating VEGFR-1+CD68+ hematopoietic progenitor cells and a-SMA+ mesenchymal stromal cells in patients with CLL/SLL, and to correlate with CLL immunoglobulin heavy chain mutational status and clinical outcome.
Project Title: "MicroRNAs in Chronic Lymphocytic Leukemia: Prognostic Markers and Oncogenic Regulators"
Our group is interested in elucidating the role of microRNAs (miRNA) in chronic lymphocytic leukemia (CLL). MicroRNAs are small RNAs which regulate gene expression by inhibiting translation or promoting mRNA degradation via specific interaction with binding sites in 3’ untranslated regions of target genes. As critical gene regulators, they have important functions in a variety of physiological and patho-physiological processes, including tumorigenesis. MicroRNAs are frequently deregulated in cancers and/or located in chromosomal fragile sites. Moreover, a few of them have been demonstrated directly by animal or cell culture studies to have oncogenic or tumor suppressor functions.
CLL is the first human cancer in which the involvement of miRNAs was documented. MiR-15 and miR-16 were identified as tumor suppressor genes in CLL. In addition, a small set of miRNAs were shown to be differentially expressed between prognostic subgroups in CLL. Our group focuses on studying one of these miRNAs called miR-155, which appeared to be more highly expressed in CLLs with unmutated immunoglobulin heavy chain gene and more aggressive clinical course. MiR-155 is the mature miRNA product of the host gene BIC, which was originally identified as a common integration site in avian leukosis virus-induced lymphomas. MiR-155 has vital functions in germinal center reaction and innate immunity. MiR-155 can be considered an oncogenic miRNA. It is frequently over-expressed in different hematopoietic malignancies. Over-expression of BIC RNA co-operates with c-myc in the pathogenesis of lymphomas and leukemias in chickens. MiR-155 transgenic mice develop abnormal lymphoproliferation and frank lymphomas.
We plan to address the contribution of miR-155 to the pathogenesis and clinical behavior of CLL. Specific questions include: (1) Is there a gene signature associated with CLL with high miR-155 expression? (2) What is the biologic relationship between this gene signature and miR-155 expression? (3) What the some of the gene targets of miR-155 in CLL? We are currently searching for answers to these questions using global gene expression analysis, bio-informatics and functional studies on CLL cells in vitro. Through these investigations, we hope to dissect the pathologic roles of miR-155 and provide a prototypic model for studies of other miRNAs in CLL. In addition, this work should be useful for the identification of additional biologically relevant subgroups and novel potential therapeutic targets in CLL.
