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: 8763293
• 负责人: Peter Aplan
• 金额: $ 28.65万
• 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/8763293
• 关键词: Address; Allogenic; Angiogenesis Inhibitors; Animal Model; Apoptosis Inhibitor; Azacitidine; Binding; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cause of Death; Cell Death; Cell Line; Cell Therapy; Cells; Chimeric Proteins; Collaborations; Cytosine; Cytotoxic Chemotherapy; DNA; DNA Methyltransferase Inhibitor; Data; Decitabine; Disease; Disease remission; Disulfiram; Doctor of Medicine; Dose; Dysmyelopoietic Syndromes; Genes; Goals; 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; Mouse Cell Line; 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; Solutions; Sorting - Cell Movement; T-Lymphocyte; Techniques; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Transplantation; Xenograft Model; cytopenia; cytotoxic; deep sequencing; demethylation; effective therapy; human disease; in vivo; inhibitor/antagonist; killings; leukemia; malignant phenotype; 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 has shown clear differences in global cytosine methylation between the NHD13 and WT samples, and partial reversal of this hypermethylation with DAC treatment, using a deep sequencing technique. The major portion of this data is unpublished, a small amount has been published in 2010. A manuscript describing these findings is currently being prepared. 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. As stated in the goals, we have generated leukemic mice and cell lines in which the leukemia is driven by the binding of a leukemic fusion protein (NP23) to H3K4Me3 residues. We have treated these NP23 cell lines with compounds that have been shown to inhibit binding of PHD domains (including that present in the NP23 fusion) to H3K4Me3 in solution. NP23 cell lines are completely killed by a 12 hr treatment with one of these compounds (disulfiram), whereas control cell lines are not killed by this compound. Furthermore, cell death is associated with a marked (more than 5-fold) decrease in binding of the NP23 fusion protein to selected H3K4Me3 residues. A manuscript describing these findings is currently under review.

难以开发针对骨髓增生异常综合征的有效治疗方法的原因之一是没有可用于建模或研究该疾病的骨髓增生异常综合征细胞系。尽管许多研究人员尝试开发骨髓增生异常综合征的异种移植模型，但这些尝试收效甚微。鉴于 NUP98-HOXD13 (NHD13) 小鼠会发展出一种高度渗透性骨髓增生异常综合征，与人类疾病非常相似，我们已开始研究以确定这些小鼠是否是骨髓增生异常综合征的有用临床前模型。我们最初的研究使用了 DNA 甲基转移酶抑制剂 5-氮杂胞苷。我们的初步研究包括 3 组小鼠 [注射 5-氮杂胞苷的 NHD13 小鼠 (n=6)、注射生理盐水的 NHD13 小鼠 (n=4) 和注射 5-氮杂胞苷的 WT 小鼠 (n=4)]。经过 16 周的治疗后，结果似乎很有希望，因为与盐水治疗的 NHD13 小鼠相比，治疗的 NHD13 小鼠的血红蛋白显着增加（2.21 +/- 1.47 g/dL vs 0.13 +/- 0.66 g/dL，p= 0.02）。不幸的是，三只接受 NHD13 治疗的小鼠在接下来的两个月内被发现死亡，我们无法确定这些小鼠的死因。剩下的三只小鼠的血红蛋白在接下来的 12 周内逐渐下降，并接近基线血红蛋白水平。尽管如此，28 周后这些小鼠的血红蛋白水平保持稳定的观察结果还是令人鼓舞的。为了确定我们是否达到足以引起胞嘧啶去甲基化的 5-氮杂胞苷水平，我们与 J.P. Issa 博士（M.D. Anderson）合作检查了整体和基因特异性甲基化状态；这些实验证明，与对照组相比，NHD13 小鼠存在高甲基化，并且 5-氮杂胞苷治疗部分逆转了高甲基化。因为上面讨论的实验使用转基因小鼠，所以用5-氮杂胞苷的有效治疗不能用完全正常（即野生型或WT）骨髓替代MDS骨髓，因为所有骨髓都是转基因的。因此，为了区分由于MDS克隆的分化和MDS克隆的消除而导致的外周血血细胞减少症的改善，我们使用具有WT和NHD13骨髓的嵌合小鼠重复了实验。这些重复实验是使用地西他滨 (DAC)（一种相关的 DNA 甲基转移酶抑制剂）进行的。与用盐水治疗的对照小鼠相比，用 DAC 治疗的小鼠表现出血液学改善和生存优势；这个实验现在已经重复了三次。我们将来自治疗小鼠的 BM 细胞分选为 WT 和 NHD13 群体，并将这些样本中的 DNA 发送给我们的合作者（J.P. Issa 博士），他显示了 NHD13 和 WT 样本之间整体胞嘧啶甲基化的明显差异，以及部分逆转使用深度测序技术，通过 DAC 处理实现这种高度甲基化。该数据的大部分尚未发表，一小部分已于 2010 年发表。目前正在准备描述这些发现的手稿。尽管FDA最近批准了三种治疗MDS的药物，但MDS患者唯一的治疗选择仍然是同种异体造血干细胞移植（HSCT）。同种异体 HSCT 有两个基本组成部分：高剂量细胞毒性化疗和免疫介导的移植物抗宿主 (GVH) 或移植物抗白血病 (GVL) 效应。然而，高剂量细胞毒治疗和 GVL 在 MDS 患者中的相对作用尚未明确。我们建议使用 NHD13 小鼠移植作为研究这个问题的方法。我们最初的实验使用 650 或 1000 CGy 的辐射，表明 650 CGy 无效，但 1000 CGy 可以诱导 26-38 周的缓解，定义为外周血计数正常化和循环宿主细胞少于 2%。然而，尽管与非移植小鼠相比，缓解期延长，存活时间延长，但所有小鼠最终都复发了，这表明这种清髓疗法没有疗效。为了解决 GVL 效应的问题，我们将 C57bl6 小鼠与 C3h.SW 小鼠杂交。 C3H.SW 小鼠在主要组织相容性基因座上与 C57Bl6 小鼠相同，但在次要组织相容性基因座上存在大量错配108。因此，将 C3H.SW 供体细胞移植到 C57Bl6 宿主小鼠中已用于研究 GVH 和 GVL。我们将 C57Bl6/C3HSW 小鼠的 BM 移植到 C57Bl6 NHD13 受体中。在这些条件下，小鼠几乎没有产生 GVH 或 GVL。随后使用更高剂量的 BM 和 5 x 10E06 外周 T 细胞进行的实验显示出严重的 GVH，导致 5 只小鼠中的 3 只死亡。第三项试验使用更高剂量的骨髓细胞 (10E07)，前景乐观，因为受者已出现轻度 GVH，并且在移植后可存活长达 36 周。这些研究尚未发表。除了上述实验外，我们还将 NHD13 小鼠转移给几个学术机构的同事，并将 NHD13 小鼠授权给至少两家独立的生物技术公司进行临床前研究。这些同事计划用多种药物治疗 NHD13 小鼠，包括组蛋白脱乙酰酶抑制剂、细胞凋亡抑制剂和血管生成抑制剂。正如目标中所述，我们已经生成了白血病小鼠和细胞系，其中白血病是由白血病融合蛋白 (NP23) 与 H3K4Me3 残基的结合驱动的。我们用化合物处理这些 NP23 细胞系，这些化合物已被证明可以抑制溶液中 PHD 结构域（包括存在于 NP23 融合体中的结构域）与 H3K4Me3 的结合。用其中一种化合物（双硫仑）处理 12 小时可完全杀死 NP23 细胞系，而对照细胞系不会被该化合物杀死。此外，细胞死亡与 NP23 融合蛋白与选定 H3K4Me3 残基的结合显着（超过 5 倍）减少有关。描述这些发现的手稿目前正在审查中。