Pre-clinical Studies of Therapy for Myelodysplastic Syndrome

骨髓增生异常综合征治疗的临床前研究

• 批准号: 8349259
• 负责人: Peter Aplan
• 金额: $ 37.04万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8349259
• 关键词: Address; Allogenic; Angiogenesis Inhibitors; Apoptosis Inhibitor; Azacitidine; Biological Assay; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cause of Death; Cell Line; Cell Therapy; Cells; Collaborations; Cytosine; Cytotoxic Chemotherapy; DNA; DNA Methyltransferase Inhibitor; Data; Decitabine; Disease; Disease remission; Doctor of Medicine; Dose; Dysmyelopoietic Syndromes; Genes; Graft-Versus-Tumor Induction; Hematopoietic Stem Cell Transplantation; Hemoglobin; Hemoglobin concentration result; Histocompatibility; Histone Deacetylase Inhibitor; Hypermethylation; Immune; Institution; Licensing; Manuscripts; Mediating; Methylation; Minor; Modeling; Mus; NUP98 gene; Patients; Peripheral; Pharmaceutical Preparations; Pilot Projects; Population; Pre-Clinical Model; Publishing; Radiation; Radiation therapy; Relapse; Relative (related person); Research Personnel; Role; Saline; Sampling; Sorting - Cell Movement; T-Lymphocyte; Techniques; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Transplantation; Xenograft Model; abstracting; cytopenia; cytotoxic; demethylation; effective therapy; human disease; inhibitor/antagonist; leukemia; meetings; mouse model; peripheral blood; preclinical study; research study; response; small molecule; success

One reason for the difficulty in developing effective treatments for myelodysplastic syndrome is that there are no myelodysplastic syndrome cell lines which can be used to model or study the disease. Although numerous investigators have attempted to develop xenograft models for myelodysplastic syndrome, these attempts have met with little success. Given that the NUP98-HOXD13 (NHD13) mice develop a highly penetrant myelodysplastic syndrome that closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model for myelodysplastic syndrome. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine. Our pilot study included 3 groups of mice [NHD13 mice injected with 5-azacytidine (n=6), NHD13 mice injected with saline (n=4), and WT mice injected with 5-azacytidine (n=4)]. After 16 weeks of therapy, the results appeared promising, as the treated NHD13 mice showed a significant increase in hemoglobin compared to the saline treated NHD13 mice (2.21 +/- 1.47 g/dL vs 0.13 +/- 0.66 g/dL, p=0.02). Unfortunately, three of the NHD13 treated mice were found dead in the following two months, and we were not able to determine the cause of death for these mice. The three remaining mice had gradual decreases in hemoglobin over the next 12 weeks, and were close to baseline hemoglobin levels. Nonetheless the observation that these mice had stable hemoglobin levels after 28 weeks was encouraging. To determine whether we were achieving levels of 5-azacytidine adequate to cause cytosine demethylation, we examined global and gene-specific methylation status, in collaboration with Dr. J.P. Issa (M.D. Anderson); these experiments demonstrated hypermethylation in the NHD13 mice compared to controls, and partial reversal of the hypermethylation with 5-azacytidine treatment. Because the experiments discussed above used transgenic mice, effective treatment with 5-azacytidine could not replace the MDS bone marrow with completely normal (ie, wildtype or WT) bone marrow, since all of the bone marrow was transgenic. Therefore, in order to distinguish improvement in peripheral blood cytopenia due to differentiation of the MDS clone from elimination of the MDS clone, we have repeated the experiments using chimeric mice, that have both WT and NHD13 bone marrow. These repeat experiments have been performed using Decitabine (DAC), a related DNA methyltransferase inhibitor. Mice treated with DAC showed hematologic improvement and a survival advantage compared with saline-treated control mice; this experiment has now been repeated three times. We have sorted BM cells from treated mice into WT and NHD13 populations, and sent DNA from these samples to our collaborator (Dr. J.P. Issa), who will be assaying these samples for cytosine methylation differences using a deep sequencing technique. The major portion of this data is unpublished, a small amount has been published in 2010. We plan to submit a manuscript describing these findings in early 2012. Despite the recent FDA approval of three drugs for MDS, the only curative option for patients with MDS remains allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT has two essential components, high-dose cytotoxic chemo-radiotherapy, and an immune-mediated graft versus host (GVH), or graft versus leukemia (GVL) effect. However, the relative contributions of high dose cytotoxic therapy and GVL are not well established in MDS patients. We propose to use transplantation of the NHD13 mice as a means to investigate this question. Our initial experiments, using either 650 or 1000 CGy of radiation, indicated that 650 CGy was ineffective, but that 1000 CGy could induce a remission of 26-38 weeks, defined by normalization of peripheral blood counts and less than 2% circulating host cells. However, despite this period of prolonged remission, and prolonged survival compared to non-transplanted mice, all of the mice have ultimately relapsed, indicating that this myeloablative therapy was not curative. To address the question of a GVL effect, we crossed C57bl6 mice with C3h.SW mice. C3H.SW mice are identical to C57Bl6 mice at the major histocompatibility loci, but have numerous mismatches at minor histocompatibility loci108. For this reason, transplantation of C3H.SW donor cells into C57Bl6 host mice has been used to study GVH and GVL. We transplanted BM from C57Bl6/C3HSW mice into C57Bl6 NHD13 recipients. The mice developed little GVH or GVL under these conditions. Subsequent experiments using higher doses of BM and 5 x 10E06 peripheral T cells showed severe GVH, that was lethal to 3 of 5 mice. A third trial, using a higher dose of bone marrow cells (10E07) is promising, as the recipients have developed mild GVH, and have survived up to 36 weeks post-transplant. These studies have not yet been published. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at several academic institutions, and have licensed NHD13 mice to at least two separate biotech companies for pre-clinical studies. These colleagues have plans to treat NHD13 mice with a variety of agents, including histone deacetylase inhibitors, apoptosis inhibitors, and angiogenesis inhibitors. One of these companies has an abstract that will be presented in Dec. 2010.

难以开发有效治疗的骨髓增生综合征的原因之一是，没有可用于建模或研究该疾病的骨髓增生综合征细胞系。尽管许多研究人员试图为骨髓增生综合征开发异种移植模型，但这些尝试却几乎没有成功。 鉴于NUP98-HOXD13（NHD13）小鼠会形成高度渗透性的骨髓增生综合征，与人类疾病非常相似，因此我们已经开始研究以确定这些小鼠是否是骨髓增生性综合征的有用前模型。 我们的最初研究使用了DNA-甲基转移酶抑制剂5-氮杂丁胺。我们的试点研究包括3组小鼠[NHD13小鼠注射了5-氮杂丁胺（n = 6），注射盐水（n = 4）的NHD13小鼠，并注入了5-氮杂丁胺（n = 4）的WT小鼠]。经过16周的治疗后，与盐水治疗的NHD13小鼠相比，治疗的NHD13小鼠的血红蛋白显着增加，结果似乎很有希望（2.21 +/- 1.47 g/dl vs 0.13 +/- 0.13 +/- 0.66 g/dl，p = 0.02）。不幸的是，在接下来的两个月中发现了NHD13治疗的小鼠中的三只死亡，我们无法确定这些小鼠的死亡原因。剩下的三只小鼠在接下来的12周内血红蛋白逐渐降低，并且接近基线血红蛋白水平。尽管如此，这些小鼠在28周后具有稳定的血红蛋白水平令人鼓舞。为了确定我们是否达到5-氮杂丁丁的水平足以引起胞嘧啶脱甲基化，我们与J.P. Issa博士（M.D. Anderson）合作检查了全球和基因特异性甲基化状态；这些实验表明，与对照组相比，NHD13小鼠的高甲基化以及通过5-氮杂替丁治疗的高甲基化的部分逆转。 由于上面讨论的实验使用了转基因小鼠，因此用5-氮杂丁丁的有效治疗无法用完全正常的（即野生型或WT）骨髓替代MDS骨髓，因为所有骨髓都是转基因。因此，为了区分由于MDS克隆与消除MDS克隆的分化，为了区分外周血细胞质的改善，我们使用嵌合小鼠重复了实验，这些嵌合小鼠具有WT和NHD13骨髓。这些重复实验已使用Decitabine（DAC），一种相关的DNA甲基转移酶抑制剂。与盐水处理的对照小鼠相比，用DAC处理的小鼠表现出血液学改善和生存优势。该实验现已重复三次。我们已经将经过处理的小鼠的BM细胞分类为WT和NHD13群体，并将这些样品的DNA发送给我们的合作者（J.P. ISSA博士），后者将使用深层的测序技术分析这些样品，以实现胞质甲基化差异。该数据的主要部分未发表，在2010年发表了少量。我们计划在2012年初提交描述这些发现的手稿。尽管最近FDA批准了三种MDS的FDA批准，这是MDS患者的唯一治疗选择，这是同种异体同种异体血管血细胞细胞转移（HSCT）。同种异体HSCT具有两个必不可少的成分，高剂量的细胞毒性化学疗法，以及免疫介导的移植物与宿主（GVH），或移植物与白血病（GVL）效应。但是，高剂量细胞毒性疗法和GVL的相对贡献在MDS患者中尚未很好地确定。我们建议将NHD13小鼠的移植作为研究这个问题的一种手段。 我们使用650或1000 CGY辐射的最初实验表明650 CGY无效，但是1000 CGY可以诱导26-38周的缓解，这是通过外周血计数的归一化定义的，且循环宿主细胞少于2％。然而，尽管与非移植小鼠相比，尽管长期缓解且生存率延长，但所有小鼠最终都已经复发，这表明这种骨髓性疗法尚未治愈。 为了解决GVL效应的问题，我们用C3H.SW小鼠越过C57BL6小鼠。 C3H.SW小鼠与主要组织相容性基因座的C57BL6小鼠相同，但在较小的组织相容性基因座上有许多不匹配。因此，将C3H.SW供体细胞移植到C57BL6宿主小鼠中已被用于研究GVH和GVL。我们将BM从C57BL6/C3HSW小鼠移植到C57BL6 NHD13接收者中。在这些条件下，小鼠几乎没有GVH或GVL。随后使用较高剂量的BM和5 X 10E06外周T细胞的实验显示出严重的GVH，这对5只小鼠中的3只致命。第三次使用较高剂量的骨髓细胞（10E07）的试验很有希望，因为受体发展了轻度的GVH，并且移植后长达36周存活。这些研究尚未发表。 除了上面概述的实验外，我们还将NHD13小鼠转移到了几个学术机构的同事，并已将NHD13小鼠许可到至少两家独立的生物技术公司进行临床前研究。这些同事计划用各种药物治疗NHD13小鼠，包括组蛋白脱乙酰基酶抑制剂，凋亡抑制剂和血管生成抑制剂。这些公司之一的摘要将于2010年12月提出。