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: 9556418
• 负责人: Peter Aplan
• 金额: $ 52.95万
• 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/9556418
• 关键词: Address; Allogenic; Angiogenesis Inhibitors; Apoptosis Inhibitor; Azacitidine; Binding; Biotechnology; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Death; Cell Line; Cell Therapy; Cells; Chimeric Proteins; Collaborations; Cytosine; Cytotoxic Chemotherapy; DNA; DNA Methyltransferase Inhibitor; Data; Decitabine; Disease; Disease remission; Disulfiram; Donor Lymphocyte Infusion; Dose; Dysmyelopoietic Syndromes; Genome; Goals; Graft-Versus-Tumor Induction; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Histocompatibility; Histone Deacetylase Inhibitor; Hypermethylation; Immune; Institution; Malignant Neoplasms; Manuscripts; Mediating; Methylation; Minor; Modeling; Mouse Cell Line; Mus; NUP98 gene; Patients; Peripheral; Phase I Clinical Trials; Phase II Clinical Trials; Pre-Clinical Model; Preparation; Publishing; Relapse; Research Personnel; Role; Saline; Sampling; T-Lymphocyte; T-Lymphocyte Subsets; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Transplantation; Xenograft Model; cancer diagnosis; cancer therapy; chemoradiation; cytopenia; cytotoxic; effective therapy; experimental study; human disease; in vivo; inhibitor/antagonist; killings; leukemia; malignant phenotype; mouse model; novel; peripheral blood; preclinical study; predicting response; small molecule; small molecule inhibitor

One reason for the difficulty in developing effective treatments for myelodysplastic syndrome (MDS) is that there are no MDS cell lines which can be used to model or study the disease. Although numerous investigators have attempted to develop xenograft models for MDS, these attempts have been unable to engraft cells that will produce MDS in mice. Given that the NUP98-HOXD13 (NHD13) mice develop a highly penetrant MDS which closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model to evaluate MDS therapies. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine; some of these results have recently been published (Genome Res. 4:580-91, 2014).However, since our initial studies 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. Sorted BM cells from treated mice show clear differences in global cytosine methylation between the NHD13 and WT samples, and partial reversal of this hypermethylation with DAC treatment (collaboration with Dr. J.P. Issa). A manuscript describing these findings is in preparation. Drs. Difillipantonio, Doroshow, and colleagues from the Division of Cancer Treatment and Diagnosis (DCTD) have developed two novel DNMT1 inhibitors, one of which is now in phase I clinical trials. In collaboration with Dr. Difillipantonio and colleagues, we are now treating chimeric NHD13/WT mice with these compounds to assess efficacy in treatment of MDS. Preliminary results show a survival benefit as well as improvement in hematopoiesis in mice receiving one of these DNMT1 inhibitors. 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. 1000 CGy induced 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 mice 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 or 5 x 10E06 peripheral T cells showed severe GVH. A third trial, using a higher dose of bone marrow cells (10E07)has led to survival 52 weeks post-transplant. These results demonstrate a survival benefit for bone marrow transplant; ongoing experiments are aimed at determining whether donor lymphocyte infusion or co-tranplantation of specific T cell subsets will lead to yet greater survival. A portion of this data has been presented in abstract form and a manuscript describing these results is in preparation. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at many academic institutions, and have licensed NHD13 mice to several 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 compounds (ACE-536) is now in phase II clinical trials for patients with MDS. As stated in the goals, we have generated leukemic mice and cell lines in which 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 was recently published (Cancer Discov. 5:564-77, 2014), and an in vivo trial of disulfiram for NP23 leukemias has begun.

骨髓增生异常综合征 (MDS) 的有效治疗方法难以开发的原因之一是没有可用于建模或研究该疾病的 MDS 细胞系。尽管许多研究人员尝试开发MDS异种移植模型，但这些尝试无法在小鼠体内移植产生MDS的细胞。鉴于 NUP98-HOXD13 (NHD13) 小鼠会产生一种高度渗透性的 MDS，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评估 MDS 疗法的有用的临床前模型。我们最初的研究使用了 DNA 甲基转移酶抑制剂 5-氮杂胞苷；其中一些结果最近已发表（Genome Res. 4:580-91, 2014）。然而，由于我们最初的研究使用转基因小鼠，5-氮杂胞苷的有效治疗不能取代完全正常的 MDS 骨髓（即，野生型或WT）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。来自治疗小鼠的分选 BM 细胞显示出 NHD13 和 WT 样本之间整体胞嘧啶甲基化的明显差异，并且通过 DAC 治疗（与 J.P. Issa 博士合作）部分逆转了这种高甲基化。描述这些发现的手稿正在准备中。博士。 Difillipantonio、Doroshow 和癌症治疗和诊断部门 (DCTD) 的同事开发了两种新型 DNMT1 抑制剂，其中一种目前正在进行 I 期临床试验。我们与 Difillipantonio 博士及其同事合作，目前正在用这些化合物治疗嵌合 NHD13/WT 小鼠，以评估 MDS 的治疗效果。初步结果显示，接受其中一种 DNMT1 抑制剂的小鼠可提高生存率并改善造血功能。 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。 1000 CGy 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，这段缓解期延长了，存活时间也延长了，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 或 5 x 10E06 外周 T 细胞进行的实验显示出严重的 GVH。第三项试验使用更高剂量的骨髓细胞 (10E07)，使患者在移植后存活 52 周。这些结果证明了骨髓移植对生存的益处；正在进行的实验旨在确定供体淋巴细胞输注或特定 T 细胞亚群的共同移植是否会导致更高的存活率。该数据的一部分已以摘要形式呈现，描述这些结果的手稿正在准备中。除了上述实验外，我们还将 NHD13 小鼠转移给许多学术机构的同事，并将 NHD13 小鼠授权给多家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。其中一种化合物 (ACE-536) 目前正处于针对 MDS 患者的 II 期临床试验中。正如目标中所述，我们已经培育出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。最近发表了一篇描述这些发现的手稿（Cancer Discov. 5:564-77, 2014），并且双硫仑治疗 NP23 白血病的体内试验已经开始。