Myelodysplasia

骨髓增生异常

• 批准号: 8149587
• 负责人: NEAL S YOUNG
• 金额: $ 114.22万
• 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/8149587
• 关键词: Affect; Age; Aneuploid Cells; Apoptosis; Apoptotic; CD34 gene; CSF3 gene; Cell Cycle Regulation; Chromosomal Instability; Chronic; Chronic Myeloid Leukemia; Clinical; Clonal Expansion; Cyclin D1; Cyclosporine; Cyclosporins; Dasatinib; Data; Deferoxamine; Diagnosis; Diploid Cells; Diploidy; Disease; Drug usage; Dysmyelopoietic Syndromes; Elderly; Environment; Enzymes; Erythrocytes; Exhibits; Failure; Flow Cytometry; Gene Dosage; Gene Transfer; Genes; Growth; Growth Factor; Hematopoietic; Hematopoietic stem cells; Hemolysis; Human; Immunotherapy; In Vitro; Individual; Inflammation; Interferon Type II; JAK2 gene; Janus kinase 2; Laboratories; Licensing; Link; Mononuclear; Monosomy 7; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Proliferating; Proteasome Inhibitor; Skin; T-Cell Activation; Telomere Shortening; Therapeutic immunosuppression; Tyrosine Kinase Inhibitor; graft vs host disease; improved; killings; leukemia; multicatalytic endopeptidase complex; rat Piga protein

We found that c-myc, encoded by a gene present on chromosome 8, was upregulated in trisomy 8 cells, likely a gene-dosage effect and a direct result of the trisomy. Upregulation of cyclin D1 mRNA and protein in these cells results from upregulated c-myc. Cyclin D1 increases cell proliferation and inhibits apoptosis via upregulated anti-apoptotic proteins. WT1 m-RNA is also high in trisomy 8 and may elicit an immune response directed against trisomy 8 cells. Knock-down studies of these proteins in trisomy 8 showed preferential killing of the trisomy 8 clone in vitro, suggesting that a potential avenue for therapeutic intervention might be inhibition of these transcription factors. A drug developed by Onconova Pharmaceuticals, ON1910, suppresses c-myc as well as cyclin D1. In vitro, this compound inhibited trisomy 8 cell proliferation and promoted apoptosis while increasing growth of normal diploid cells and promoting their maturation. Monosomy 7 cells express increased amounts of the granulocyte colony-stimulating factor receptor (GCSFR) isoform IV, which accounts for the cells inferior responses to physiologic levels of granulocyte colony stimulating factor. However, monosomy 7 cells proliferate at higher concentrations of this cytokine, as occur in bone marrow failure or with pharmacologic administration of GCSF. Recently, we have attempted to develop targeted therapy for each of these cytogenetic abnormalities using laboratory data linking regulation of important transcription factors favoring survival of trisomy 8 cells or monosomy 7 cells. Potential therapies for monosomy 7 include drugs that inhibit tyrosine kinase and Jak2, while inhibitors of cyclin D or c-myc might be effective in trisomy 8. We conducted a phase I study of an anti-cyclin D1 drug, ON1910, in MDS. ON 01910.Na inhibited cyclin D1 accumulation in MDS bone marrow-derived mononuclear cells, and was selectively toxic to trisomy 8 cells while promoting maturation of diploid cells. Three of four patients with refractory anemia with excess blasts and trisomy 8 and 2 of 2 patients with monosomy 7 treated on a pilot clinical study with ON 01910.Na had transient and two had sustained responses to the drug. Responders showed decreased bone marrow blasts and improved blood counts. Patients who exhibited hematologic responses to ON 01910.Na had decreased cyclin D1 levels in their CD34+ cells. Modulation of cell cycle control enzymes such as cyclin D1 therefore may represent a novel targeted approach for trisomy 8 MDS. We previously demonstrated abnormalities in the granulocyte colony stimulating factor receptor in monosomy 7 cells, which result in abnormal transduction of growth factor signals. This altered signaling results in resistance to apoptosis and increased proliferation in the presence of high concentrations of GCSF, ultimately resulting in a survival advantage compared to normal diploid cells. We assesed the effect of inhibition of Jak2 on the survival of these cells, using two types of drugs: Jak-2 inhibitors currently available for treatment of myeloproliferative disorder and tyrosine kinase inhibitors which might have non-specific activity against phosphylated Jak2, which has tyrosine kinase activity. Dasatinib, licensed for treatment of chronic myelogenous leukemia, has non-specific activity against tyrosine kinases other than bcr-abl. These drugs have much less toxicity compared to chemotherapeutic drugs, particularly important in elderly and debilitated patients. We examined the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in marrow cells, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of marrow with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus controls, while diploid cell numbers were stable in flow cytometry experiments, incubation with TG101348 decreased the number of CD34+CD13- stem cells and increased more differentiated CD34-CD13+ myeloid cells. We have applied for a patent for the use of this drug in patients with monosomy 7 and excess blasts. In vitro studies of dasatinib demonstrated decreased numbers of monosomy 7 cells while dramatically increasing proliferation of diploid hematopoietic colony formation. We secured from Bristol Myers a donation of dasatinib and are currently preparing a phase II study of this drug in aplastic anemia patients who have developed monosomy 7. Similar to aplastic anemia, some individuals with MDS (usually younger patients) show features of immune activation and respond to immunosuppressive therapy. Wilms tumor antigen1 (WT1) is up-regulated in MDS but not in healthy CD34+ stem cells and expression levels increase as the disease progress8. We have recently identified cytotoxic T lymphocytes (CTL) directed against WT1 protein. These CTL characterize patients who recover hematopoietic function after immunosuppressive therapy, and they specifically recognize MDS cells with the trisomy 8 abnormality that express WT1. We plan to clone and sequence the WT1-specific TCR (T cell receptor) for use in gene transfer experiments. Much of the Branchs work has been directed toward evaluating the factors that favor survival of aneuploid cells in an inflammatory environment such as AA. Inflammatory conditions such as ulcerative colitis or Barretts esophagus predispose to chromosomal instability. We documented the presence of aneuploidy and tetraploidy in skin GVHD and were able to reproduce these genomic changes by adding mis-matched allogeneic lymphocytes or interferon gamma. Aplastic anemia also shows some features of chronic inflammation. We postulated that chromosomal abnormalities might be a consequence of genomic damage caused by effector T-cells as well as of telomere shortening from accelerated cell turnover. We were able to demonstrate aneuploidy, which appeared to be induced by the inflammatory environment, and which was ameliorated by addition of free radical scavengers, alpha-tocopherol, and desferrioxamine, and by an inhibitor of T cell activation, cyclosporine. In paroxysmal nocturnal hemoglobinuria, there is expansion of a clone of hematopoietic stem cells mutant in X chromosome gene, Pig-A, resulting in failure to present glycosylphilphasphinositol-anchored proteins on the cell surface. The most prominent feature of PNH is intravascular hemolysis due to absence of complement inactivating proteins on the red cell surface, but PNH clonal expansion occurs in the setting of bone marrow failure and many patients concurrently suffer aplastic anemia. The mechanisms by which PNH and aplastic anemia associate and the pathophysiology of GPI-anchored protein negative cell clonal expansion in aplastic anemia are not known. Recent experiments have tested a hypothesis that failure to produce the GPI moiety would affect protein degradation, as large quantities of proteins would be anchorless and require degradation via the proteasome. Experiments using paired hematopoietic cell lines and isolated human bone marrow cells have disclosed that proteasome inhibitors selectively affect the PNH clone, inducing an unfolded protein response and apoptosis. Treatment with proteasome inhibitors might eliminate PNH clones in patients.

我们发现由 8 号染色体上存在的基因编码的 c-myc 在 8 三体细胞中表达上调，这可能是基因剂量效应和三体性的直接结果。这些细胞中细胞周期蛋白 D1 mRNA 和蛋白质的上调是 c-myc 上调的结果。 Cyclin D1 通过上调抗凋亡蛋白来增加细胞增殖并抑制细胞凋亡。 WT1 mRNA 在 8 三体性中也很高，可能引发针对 8 三体细胞的免疫反应。对 8 三体性中这些蛋白质的敲低研究表明，在体外优先杀死 8 三体性克隆，这表明治疗干预的潜在途径可能是抑制这些转录因子。 Onconova Pharmaceuticals 开发的一种药物 ON1910 可抑制 c-myc 和细胞周期蛋白 D1。在体外，该化合物抑制 8 三体细胞增殖并促进细胞凋亡，同时增加正常二倍体细胞的生长并促进其成熟。单体 7 细胞表达的粒细胞集落刺激因子受体 (GCSFR) 亚型 IV 量增加，这说明细胞对生理水平的粒细胞集落刺激因子的反应较差。然而，单体 7 细胞在该细胞因子浓度较高时会增殖，如骨髓衰竭或 GCSF 药物给药时发生的情况。 最近，我们尝试利用实验室数据将有利于 8 三体细胞或 7 单体细胞存活的重要转录因子的调节联系起来，为每种细胞遗传学异常开发靶向治疗。 7 号单体的潜在疗法包括抑制酪氨酸激酶和 Jak2 的药物，而细胞周期蛋白 D 或 c-myc 的抑制剂可能对 8 三体有效。我们对抗细胞周期蛋白 D1 药物 ON1910 治疗 MDS 进行了 I 期研究。 ON 01910.Na 抑制 MDS 骨髓来源的单核细胞中细胞周期蛋白 D1 的积累，并选择性地对 8 三体细胞产生毒性，同时促进二倍体细胞的成熟。在一项用 ON 01910.Na 进行的试点临床研究中，4 名患有原始细胞过多和 8 三体性难治性贫血的患者中有 3 名患者以及 2 名 7 号单体患者中的 2 名患者对该药物有短暂反应，另外两名患者对该药物有持续反应。应答者表现出骨髓原始细胞减少和血细胞计数改善。对 ON 01910.Na 表现出血液学反应的患者 CD34+ 细胞中的细胞周期蛋白 D1 水平降低。 因此，细胞周期控制酶（例如细胞周期蛋白 D1）的调节可能代表一种针对 8 三体 MDS 的新型靶向方法。我们之前证明了7号单体细胞中粒细胞集落刺激因子受体的异常，这导致生长因子信号的异常转导。这种改变的信号传导导致细胞凋亡抵抗，并在高浓度 GCSF 存在的情况下增加增殖，最终导致与正常二倍体细胞相比的生存优势。我们使用两种类型的药物评估了抑制 Jak2 对这些细胞存活的影响：目前可用于治疗骨髓增殖性疾病的 Jak-2 抑制剂和酪氨酸激酶抑制剂，后者可能对具有酪氨酸激酶活性的磷酸化 Jak2 具有非特异性活性。达沙替尼被批准用于治疗慢性粒细胞性白血病，对除 bcr-abl 之外的酪氨酸激酶具有非特异性活性。与化疗药物相比，这些药物的毒性要小得多，这对于老年和虚弱的患者尤其重要。我们检查了高选择性 JAK2 抑制剂 TG101348 对骨髓细胞中单体 7 非整倍性的影响，以及该化合物对培养的 CD34+ 干细胞和 CD13+ 骨髓细胞以及 JAK-2 信号传导装置的活性。与对照相比，将骨髓与 TG101348 一起孵育 5 天，以浓度依赖性方式显着降低了单体 7 非整倍体细胞的绝对数量，而流式细胞术实验中二倍体细胞数量稳定，与 TG101348 一起孵育减少了 CD34+CD13- 干细胞的数量，并增加了更多分化的 CD34-CD13+ 骨髓细胞。我们已经申请了将该药物用于 7 号单体和原始细胞过多患者的专利。达沙替尼的体外研究表明，单体 7 细胞数量减少，同时显着增加二倍体造血集落形成的增殖。我们从百时美施贵宝公司获得了达沙替尼的捐赠，目前正在准备对患有 7 号单体的再生障碍性贫血患者进行该药物的 II 期研究。 与再生障碍性贫血类似，一些MDS患者（通常是年轻患者）表现出免疫激活的特征并对免疫抑制治疗有反应。 Wilms 肿瘤抗原 1 (WT1) 在 MDS 中上调，但在健康 CD34+ 干细胞中则不然，并且表达水平随着疾病进展而增加8。我们最近发现了针对 WT1 蛋白的细胞毒性 T 淋巴细胞 (CTL)。这些CTL是免疫抑制治疗后恢复造血功能的患者的特征，它们特异性识别表达WT1的具有8三体异常的MDS细胞。我们计划对 WT1 特异性 TCR（T 细胞受体）进行克隆和测序，用于基因转移实验。 该分支的大部分工作旨在评估有利于非整倍体细胞在炎症环境（如 AA）中存活的因素。溃疡性结肠炎或巴雷特食管等炎症容易导致染色体不稳定。我们记录了皮肤 GVHD 中非整倍体和四倍体的存在，并能够通过添加不匹配的同种异体淋巴细胞或干扰素 γ 来重现这些基因组变化。再生障碍性贫血还表现出慢性炎症的一些特征。我们推测染色体异常可能是效应 T 细胞引起的基因组损伤以及加速细胞周转导致的端粒缩短的结果。我们能够证明非整倍性，这种非整倍性似乎是由炎症环境诱导的，并且通过添加自由基清除剂、α-生育酚和去铁胺以及T细胞活化抑制剂环孢菌素可以改善这种非整倍性。 在阵发性睡眠性血红蛋白尿症中，X染色体基因Pig-A中的造血干细胞突变体克隆扩增，导致细胞表面无法呈现糖基磷脂酰肌醇锚定蛋白。 PNH 最突出的特征是由于红细胞表面缺乏补体失活蛋白而导致的血管内溶血，但 PNH 克隆扩张发生在骨髓衰竭的情况下，许多患者同时患有再生障碍性贫血。 PNH 与再生障碍性贫血相关的机制以及再生障碍性贫血中 GPI 锚定蛋白阴性细胞克隆扩增的病理生理学尚不清楚。最近的实验验证了一个假设，即无法产生 GPI 部分会影响蛋白质降解，因为大量蛋白质将是无锚定的，需要通过蛋白酶体降解。使用配对造血细胞系和分离的人骨髓细胞进行的实验表明，蛋白酶体抑制剂选择性地影响 PNH 克隆，诱导未折叠蛋白反应和细胞凋亡。蛋白酶体抑制剂治疗可能会消除患者体内的 PNH 克隆。