Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are tumors of mature B cells and are closely related biologically and in clinical behavior. Both are currently incurable with chemotherapy. Because many patients are older, application of allogeneic transplantation and tolerability of aggressive chemotherapy is limited. Median survival for patients with CLL is 10 years and for MCL patients ranges between 5 and 6 years. Thus, there is a need to develop novel treatments, especially targeted agents and more tolerable immunotherapeutic approaches. The recognition that the B-cell receptor (BCR) repertoire expressed on CLL cells is highly skewed led to the hypothesis that antigen selection plays a role in disease pathogenesis. Frequently, these antigens appear to be auto-antigens leading to the concept that CLL is a malignancy of auto-reactive B-cells. The hallmark of MCL is the chromosomal translocation t(11;14) that places the cyclin D1 gene under the control of the immunoglobulin heavy chain promoter resulting in high expression of this D-type cyclin that drives tumor proliferation. The presence of Cyclin D1 in MCL cells is the single most distinctive feature between CLL and MCL. BCR signaling has emerged as the pivotal pathway in the pathogenesis of CLL. A major contribution from my group has been the first demonstration of active BCR signaling in CLL patients in vivo. Our findings support the importance of the BCR for disease progression and identify the pathway as a relevant target for therapeutic intervention. Furthermore, we showed that BCR signaling and the consequent activation of the NF-B pathway occurs primarily in the lymph node microenvironment rather than in the peripheral blood or bone marrow. Thus, in key aspects, the biology of CLL is shaped by its environment;an insight that changes our therapeutic approach, the design of correlative studies, and the development of model systems. We have shown that BCR signaling and activation of the NF-B pathway in CLL cells occurs primarily in the lymph node microenvironment. We hypothesize that this compartmentalization of BCR signaling reflects the contribution of co-stimulatory pathways activated in the lymph node. Based on our gene expression analysis, Toll-like-receptor (TLR) signaling and Tumor necrosis factor (TNF) family ligands such as CD40 may play such a role in vivo. Activation of autoreactive B-cells in response to additive signaling through TLR and BCR pathways in rheumatoid arthritis may serve as a precedent. We are now investigating the contribution of these different signaling pathways using a combination of molecular and cellular assays in primary cells, pathway specific inhibitors, and our mouse model. A limitation of our initial study on the effect of the tissue microenvironment on CLL biology was the use of bulk tumor populations. We therefore developed flow cytometric assays that can dissect clonal heterogeneity and identify subpopulations of CLL cells that have higher proliferation (measured using Ki67) and are actively signaling (identified through their expression of phosphorylated signal transduction molecules). In addition, we initiated a clinical protocol using deuteriated water to label the proliferative fraction of the CLL clone in vivo (NCT01117142), which provided direct in vivo evidence for increased tumor proliferation in the lymph node. While several lines of evidence indicate that BCR signaling and the microenvironment are equally important in the pathogenesis of MCL as they are for CLL, this aspect of MCL biology remains ill-defined. We now apply our experience in CLL to the analysis of a substantial repository of primary MCL tumor samples. Having shown the importance of the lymph node microenvironment for CLL cells, we developed a preclinical model system that can reproduce crosstalk between tumor and host microenvironment. As there are currently no good cell line or mouse models of CLL, we adapted a recently described mouse xenograft model and validated that human CLL cells engrafting in the murine spleen proliferate and undergo activation of BCR and NF-B pathways, similar to what we have previously found in the human lymph node. The requirement of the tissue microenvironment for full activation of the BCR on CLL cells, as we have demonstrated in the human lymph node, suggests a role for additional cell types or co-stimulatory molecules in vivo. We are using the NSG CLL xenograft model to investigate whether specific antigens or host factors or the addition or elimination of distinct human cell populations such as T cells will modulate the survival and proliferation of xenografted CLL cells. Several small molecule inhibitors of BCR signaling and of PI3K have entered clinical development in CLL and other hematologic malignancies. Currently, the most promising early clinical results have been achieved with fostamatinib (an inhibitor of SYK), ibrutinib (an irreversible inhibitor of BTK) and GS-1101 (an inhibitor of PI3K). We recently completed the first detailed biologic analysis on the impact of a BCR inhibitor in vivo, using CLL cells obtained from the blood of patients treated with fostamatinib. We validated the on-target effect by demonstrating inhibition of BCR signaling and a sustained reduction of tumor proliferation. Interestingly, in the circulating CLL cells BCR signaling was equally inhibited irrespective of clinical response, suggesting that analysis of tissue samples will likely be important to more fully assess the impact and limitations of BCR targeting agents in vivo. In our clinical trials we therefore incorporate biopsies of lymph nodes, a key site for the biology of CLL that is very difficult to sample for research studies outside of the NIH Clinical Center. From January 2012 to May 2013 we accrued over 60 of a total of up to 86 patients into our single agent ibrutinib trial (NCT01500733). We recruit patients with an unmet clinical need, either elderly patients who are often unable to tolerate current aggressive standard therapies, or CLL with a deletion of the short arm of chromosome 17 (deletion 17). The latter patients have a particularly poor outcome with chemotherapy and are in greatest need for novel approaches. The goal of this study is to establish response rates to ibrutinib, determine the durability of response, and explore the feasibility of long term therapy using single agent. Because loss of function mutations in BTK causes a severe immune defect known as Brutons agammaglobulinemia, assessing the impact BTK inhibition on immune function will be particularly important. We sample peripheral blood, bone marrow, and lymph nodes from patients enrolled in the ibrutinib study. We are analyzing the impact of ibrutinib on tumor proliferation, cellular activation, BCR and NF-B signaling using a combination of gene expression profiling, flow cytometry and molecular analyses. Preliminary data suggest that signaling pathways that are not dependent on BTK may influence the clinical response. In a complementary approach, in collaboration with the NHGRI chemical genomics facility, we initiated a screen for FDA approved compounds with anti leukemic activity. We identified 5 compounds highly active against CLL cells but not or less toxic to normal lymphocytes. We have completed a phase I study testing the lead compound auranofin in the clinic in collaboration with Kansas University and Ohio State University. Unfortunately, the compound is not active in patients and we are currently analyzing samples obtained on this study to elucidate mechanisms of treatment resistance.

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Project End
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Support Year
9
Fiscal Year
2013
Total Cost
$1,131,849
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
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Saba, Nakhle; Wiestner, Adrian (2014) Do mantle cell lymphomas have an 'Achilles heel'? Curr Opin Hematol 21:350-7
Fiskus, Warren; Saba, Nakhle; Shen, Min et al. (2014) Auranofin induces lethal oxidative and endoplasmic reticulum stress and exerts potent preclinical activity against chronic lymphocytic leukemia. Cancer Res 74:2520-32
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