Myelodysplasia

我们发现，由8号染色体上的一个基因编码的c-myc在8号三体细胞中被上调，可能是基因剂量效应和三体的直接结果。细胞周期蛋白D1 mRNA和蛋白的上调是由c-myc上调引起的。Cyclin D1通过上调抗凋亡蛋白，促进细胞增殖，抑制细胞凋亡。WT1 m-RNA在8三体中含量也很高，可能引发针对8三体细胞的免疫反应。这些蛋白在8号三体中的敲除研究显示出体外对8号三体克隆的优先杀伤，这表明抑制这些转录因子可能是治疗干预的潜在途径。Onconova制药公司开发的一种药物，ON1910，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，促进细胞凋亡，同时促进正常二倍体细胞的生长和成熟。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，这说明细胞对粒细胞集落刺激因子生理水平的反应较差。然而，单体7细胞在较高浓度的这种细胞因子下增殖，如骨髓衰竭或GCSF药物管理时发生的情况。最近，我们试图利用实验室数据开发针对这些细胞遗传学异常的靶向治疗，这些数据连接了有利于8三体细胞或7单体细胞存活的重要转录因子的调节。7号单体的潜在治疗方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号三体有效。我们在MDS患者中进行了一项抗cyclin D1药物ON1910的I期研究。在01910年。Na抑制了MDS骨髓源性单核细胞中cyclin D1的积累，对8三体细胞具有选择性毒性，同时促进二倍体细胞的成熟。4例难治性贫血伴过多原细胞和8三体患者中的3例和2例7单体患者中的2例接受了on 01910的试点临床研究。1名患者对该药有短暂反应，2名患者有持续反应。应答者显示骨髓原细胞减少，血细胞计数提高。对on01910有血液学反应的患者。Na降低了CD34+细胞的cyclin D1水平。因此，细胞周期控制酶如cyclin D1的调节可能代表了一种新的靶向治疗8三体MDS的方法。我们之前证明了单体7细胞中粒细胞集落刺激因子受体的异常，导致生长因子信号的异常转导。这种改变的信号导致在高浓度GCSF存在下对凋亡的抵抗和增殖增加，最终导致与正常二倍体细胞相比的生存优势。我们评估了Jak2抑制对这些细胞存活的影响，使用两种类型的药物：目前可用于治疗骨髓增生性疾病的Jak2抑制剂和酪氨酸激酶抑制剂，后者可能对磷酸化Jak2具有非特异性活性，而Jak2具有酪氨酸激酶活性。达沙替尼，获准用于治疗慢性骨髓性白血病，对酪氨酸激酶（bcr-abl）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，这对老年人和虚弱的患者尤其重要。我们研究了高选择性JAK2抑制剂TG101348对骨髓细胞单体7非整倍体的影响，以及该化合物对培养的CD34+干细胞和CD13+骨髓细胞以及JAK2信号传导装置的活性。与对照组相比，TG101348在骨髓中孵育5天显著降低了单体7非整倍体细胞的绝对数量，且呈浓度依赖性，而在流式细胞术实验中，二倍体细胞数量保持稳定，TG101348在骨髓中孵育5天减少了CD34+CD13-干细胞的数量，增加了分化程度更高的CD34-CD13+骨髓细胞。我们已经申请了一项专利，将这种药物用于单体7和过多原细胞的患者。体外研究表明，达沙替尼减少了单体7细胞的数量，同时显著增加了二倍体造血集落形成的增殖。我们从Bristol Myers获得了dasatinib的捐赠，目前正在准备该药物在患有7号单体的再生障碍性贫血患者中的II期研究。

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.