Tumor microenvironment in CLL and MCL: pathogenesis, targets, and therapy

CLL 和 MCL 的肿瘤微环境：发病机制、靶点和治疗

• 批准号: 8746563
• 负责人: adrian u wiestner
• 金额: $ 113.18万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746563
• 关键词: 17p; Agammaglobulinemia; Antigens; Auranofin; B-Lymphocytes; Behavior; Biological Assay; Biological Models; Biology; Biopsy; Blood; Bone Marrow; CCND1 gene; Cell Line; Cell Proliferation; Cells; Cellular Assay; Chemicals; Chromosomal translocation; Chronic Lymphocytic Leukemia; Clinic; Clinical; Clinical Protocols; Clinical Trials; Collaborations; Conduct Clinical Trials; Correlative Study; Cyclin D1; Cyclins; Data; Defect; Development; Disease; Disease Progression; Enrollment; Environment; Event; FDA approved; Family; Flow Cytometry; Gene Expression Profiling; Genomics; Goals; Heavy-Chain Immunoglobulins; Hematologic Neoplasms; Heterogeneity; Homologous Transplantation; Human; Immune; Immunotherapeutic agent; Innovative Therapy; Integration Host Factors; Intervention; Kansas; Label; Lead; Ligands; Lymphocyte; Malignant Neoplasms; Mantle Cell Lymphoma; Mature B-Lymphocyte; Measures; Molecular; Mus; National Human Genome Research Institute; Ohio; Outcome; Pathogenesis; Pathway interactions; Patients; Play; Population; Pre-Clinical Model; Proliferating; Receptor Signaling; Receptors, Antigen, B-Cell; Recruitment Activity; Resistance; Rheumatoid Arthritis; Role; SYK gene; Sampling; Shapes; Signal Pathway; Signal Transduction; Site; Spleen; Systems Biology; T-Lymphocyte; TNFRSF5 gene; Testing; Therapeutic; Therapeutic Intervention; Tissue Sample; Tissues; Toll-like receptors; Tumor Necrosis Factor-alpha; United States National Institutes of Health; Universities; Water; Xenograft Model; Xenograft procedure; autoreactive B cell; base; cell type; chemotherapy; design; experience; immune function; improved; in vivo; inhibitor/antagonist; insight; loss of function mutation; lymph nodes; mouse model; novel; novel strategies; older patient; peripheral blood; phase 1 study; promoter; repository; research study; response; small molecule; therapeutic target; treatment response; tumor; tumor microenvironment; 17p; Agammaglobulinemia; Antigens; Auranofin; B-Lymphocytes; Behavior; Biological Assay; Biological Models; Biology; Biopsy; Blood; Bone Marrow; CCND1 gene; Cell Line; Cell Proliferation; Cells; Cellular Assay; Chemicals; Chromosomal translocation; Chronic Lymphocytic Leukemia; Clinic; Clinical; Clinical Protocols; Clinical Trials; Collaborations; Conduct Clinical Trials; Correlative Study; Cyclin D1; Cyclins; Data; Defect; Development; Disease; Disease Progression; Enrollment; Environment; Event; FDA approved; Family; Flow Cytometry; Gene Expression Profiling; Genomics; Goals; Heavy-Chain Immunoglobulins; Hematologic Neoplasms; Heterogeneity; Homologous Transplantation; Human; Immune; Immunotherapeutic agent; Innovative Therapy; Integration Host Factors; Intervention; Kansas; Label; Lead; Ligands; Lymphocyte; Malignant Neoplasms; Mantle Cell Lymphoma; Mature B-Lymphocyte; Measures; Molecular; Mus; National Human Genome Research Institute; Ohio; Outcome; Pathogenesis; Pathway interactions; Patients; Play; Population; Pre-Clinical Model; Proliferating; Receptor Signaling; Receptors, Antigen, B-Cell; Recruitment Activity; Resistance; Rheumatoid Arthritis; Role; SYK gene; Sampling; Shapes; Signal Pathway; Signal Transduction; Site; Spleen; Systems Biology; T-Lymphocyte; TNFRSF5 gene; Testing; Therapeutic; Therapeutic Intervention; Tissue Sample; Tissues; Toll-like receptors; Tumor Necrosis Factor-alpha; United States National Institutes of Health; Universities; Water; Xenograft Model; Xenograft procedure; autoreactive B cell; base; cell type; chemotherapy; design; experience; immune function; improved; in vivo; inhibitor/antagonist; insight; loss of function mutation; lymph nodes; mouse model; novel; novel strategies; older patient; peripheral blood; phase 1 study; promoter; repository; research study; response; small molecule; therapeutic target; treatment response; tumor; tumor microenvironment

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.

慢性淋巴细胞性白血病（CLL）和地幔细胞淋巴瘤（MCL）是成熟B细胞的肿瘤，在生物学上和临床行为上密切相关。目前两者都无法治疗化疗。由于许多患者年龄较大，因此同种异体移植的应用和侵袭性化学疗法的耐受性受到限制。 CLL患者的中位生存期为10年，MCL患者的范围​​为5至6年。因此，有必要开发新的治疗方法，尤其是靶向药物和更耐受的免疫治疗方法。认识到在CLL细胞上表达的B细胞受体（BCR）曲目高度偏斜的原因导致了这样的假设：抗原选择在疾病发病机理中起作用。 通常，这些抗原似乎是自动抗原，导致CLL是自动反应性B细胞的恶性肿瘤的概念。 MCL的标志是染色体易位T（11; 14），它将细胞周期蛋白D1基因置于免疫球蛋白重链重链启动子的控制之下，导致这种D型细胞周期蛋白的高表达，从而驱动肿瘤增殖。 MCL细胞中细胞周期蛋白D1的存在是CLL和MCL之间最独特的特征。 BCR信号已成为CLL发病机理中的关键途径。我组的主要贡献是在体内CLL患者中首次证明了主动BCR信号传导。 我们的发现支持BCR对疾病进展的重要性，并确定该途径是治疗干预的相关靶标。 此外，我们证明了BCR信号传导和随之而来的NF-B途径的激活主要发生在淋巴结微环境中，而不是在外周血或骨髓中。 因此，在关键方面，CLL的生物学是由其环境塑造的。一种改变我们的治疗方法，相关研究设计和模型系统发展的见解。 我们已经表明，CLL细胞中NF-B途径的BCR信号传导和激活主要发生在淋巴结微环境中。 我们假设BCR信号传导的这种分区化反映了在淋巴结中激活的共刺激途径的贡献。 基于我们的基因表达分析，Toll样受体（TLR）信号传导和肿瘤坏死因子（TNF）家族配体（例如CD40）可能在体内扮演这样的作用。通过TLR和类风湿关节炎中的BCR途径响应自动反应性B细胞的激活可能是先例。现在，我们正在使用原代细胞，途径特异性抑制剂和我们的小鼠模型的分子和细胞测定的结合来研究这些不同信号通路的贡献。我们对组织微环境对CLL生物学影响的最初研究的局限性是使用散装肿瘤种群。因此，我们开发了流式细胞术测定，可以剖析克隆异质性并确定具有较高增殖的CLL细胞的亚群（使用Ki67测量）并正在积极信号传导（通过其表达磷酸化信号传递分子的表达来鉴定）。此外，我们启动了使用氘化水的临床方案，以标记体内CLL克隆的增殖分数（NCT01117142），该分数为淋巴结中肿瘤增殖的增加提供了直接的体内证据。虽然几条证据表明，BCR信号传导和微环境在MCL的发病机理中与CLL同样重要，但MCL生物学的这一方面仍然不确定。 现在，我们将我们的经验应用于CLL的经验，以分析原发性MCL肿瘤样品的大量存储库。 在显示了淋巴结微环境对CLL细胞的重要性之后，我们开发了一个临床前模型系统，该系统可以在肿瘤和宿主微环境之间再现串扰。由于目前尚无良好的细胞系或CLL小鼠模型，我们改编了最近描述的小鼠异种移植模型，并验证了在鼠脾脏中植入的人类CLL细胞增殖并经历BCR和NF-B途径的激活，类似于我们以前在人类淋巴结中发现的东西。正如我们在人类淋巴结中所证明的那样，组织微环境对CLL细胞上的BCR充分激活的需求表明，体内其他细胞类型或共刺激分子的作用。 我们正在使用NSG CLL异种移植模型来研究特定的抗原或宿主因子或添加或消除不同人类细胞群体（例如T细胞）将调节异种移植的CLL细胞的存活和增殖。 BCR信号传导和PI3K的几个小分子抑制剂已进入CLL和其他血液系统恶性肿瘤。当前，Fostamatinib（Syk的抑制剂），Ibrutinib（BTK的不可逆抑制剂）和GS-11101（PI3K的抑制剂）已实现了最有希望的早期临床结果。我们最近使用从fostamatinib治疗的患者的血液中获得的CLL细胞完成了对BCR抑制剂在体内影响的第一个详细的生物学分析。我们通过证明抑制BCR信号传导并持续减少肿瘤增殖来验证靶向效应。 有趣的是，在循环的CLL细胞中，无论临床反应如何，BCR信号传导都受到同样抑制，这表明对组织样品的分析对于更充分地评估体内BCR靶向剂的影响和局限性可能很重要。因此，在我们的临床试验中，我们融合了淋巴结活检，这是CLL生物学的关键部位，对于NIH临床中心以外的研究非常困难。从2012年1月到2013年5月，我们将多达86名患者中有60多名纳入我们的单一特工Ibrutinib试验（NCT01500733）。我们招募有未满足的临床需求的患者，他们通常无法忍受当前的侵略性标准疗法，或者以17号染色​​体的短臂缺失（缺失17）的CLL。后一种患者的化学疗法结果特别差，并且最需要新颖的方法。这项研究的目的是建立对伊布鲁替尼的反应率，确定反应的耐用性，并探索使用单药的长期治疗的可行性。由于BTK中功能突变的丧失会导致严重的免疫缺陷，称为Brutons agammaglobloblobloblobloinemia，因此评估BTK抑制对免疫功能的影响将特别重要。我们采样了来自Ibrutinib研究的患者的外周血，骨髓和淋巴结。 我们正在使用基因表达分析，流式细胞仪和分子分析的组合分析伊布鲁替尼对肿瘤增殖，细胞激活，BCR和NF-B信号的影响。初步数据表明，不依赖BTK的信号通路可能会影响临床反应。 在一种互补的方法中，与NHGRI化学基因组学设施合作，我们启动了FDA批准的具有抗白血病活性的化合物的屏幕。我们鉴定了5种对CLL细胞高活性的化合物，但对正常淋巴细胞的毒性却没有或较少。我们已经完成了I阶段研究，与堪萨斯大学和俄亥俄州立大学合作，在诊所中测试了铅化合物Auranofin。不幸的是，该化合物在患者中并不活跃，我们目前正在分析本研究中获得的样品，以阐明治疗耐药性的机制。