Investigation of the mechanisms of action of eltrombopag

艾曲波帕的作用机制研究

• 批准号: 9354134
• 负责人: Andre LaRochelle
• 金额: $ 39.15万
• 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/9354134
• 关键词: Address; Affect; Age; Agonist; American Society of Hematology; Androgens; Aplastic Anemia; Apoptosis; Apoptotic; BBC3 gene; Binding; Biological Assay; Bypass; CD34 gene; CISH gene; CRISPR/Cas technology; CSF3 gene; Cell Culture Techniques; Cell Cycle Arrest; Cell Line; Cell Survival; Cell Transplants; Cells; Chronic; Clinical; Clinical Data; Clinical Trials; Critical Pathways; Cultured Cells; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; Data; Defect; Disease; Dose; Double Strand Break Repair; Dyes; Engraftment; Equilibrium; Exposure to; FLT3 gene; Failure; Fanconi Anemia Complementation Group A Protein; Fanconi' s Anemia; Flow Cytometry; GADD45G; Genes; Growth Factor Gene; Hematopoiesis; Hematopoietic; Hematopoietic Cell Growth Factors; Hematopoietic stem cells; Hour; Human; Inflammatory; Inherited; Injection of therapeutic agent; Interferons; Investigation; Kinetics; Life; Ligands; MPL gene; Maintenance; Malignant Neoplasms; Manuscripts; Measures; Mus; Nonhomologous DNA End Joining; Outcome; PPM1D gene; PTPRC gene; Pancytopenia; Pathway interactions; Patients; Phosphorylation; Plasmids; Predisposition; Preparation; Proteins; Refractory; Regulation; Reporter; Role; STAT1 gene; Signal Pathway; Signal Transduction; Stat5 protein; Stem cells; Testing; Thrombopoietin; Transplantation; Work; annexin A5; bone marrow failure syndrome; homologous recombination; improved; insight; interest; intravenous injection; irradiation; knock-down; meetings; mimetics; pre-clinical; receptor; recombinational repair; repaired; response; self-renewal; signal processing; stem

Ojective 1: Demonstrate that Eltrombopag can bypass the TPO signaling defect induced by IFN To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 g/ml (Epag), with or without IFN 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 9.7% and 87.6 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFN (46.7 16.1% and 24.6 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFN, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFN similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFN. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 3.8% human CD45+ cells) than with TPO5 (21 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFN, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFN to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFN on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFN impaired TPO signaling in human HSPCs. In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFN. When Epag and TPO were combined, inhibition of TPO signaling by IFN was partially restored. By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFN on TPO signaling was abolished. Activation of IFN receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFN and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFN-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFN-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epags evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. This work will be presented at the Annual Meeting of the American Society of Hematology (ASH) in December 2016 and a manuscript is in preparation. Ojective 2: Demonstrate that Eltrombopag has DNA repair activity in human HSPCs G-CSF mobilized human CD34+ cells from 5 independent healthy donors were cultured in the presence of SCF and Flt3-L (SF), SF and TPO (SFT), or SF and Eltrombopag (SFE) for 24 hours before exposure to 2Gy -irradiation, and then cultured for an additional 5 to 24 hours. DNA damage was quantified by flow cytometric determination of H2AX expression, a marker of irradiation-induced DNA double-strand breaks (DSB), and CD34+ cell survival was measured by flow cytometry using Annexin V and a viability dye. There were significantly fewer H2AX+ cells 5 hours post-irradiation when the culture included TPO or Eltrombopag than with SF alone (n=5). Five hours post-irradiation, cultures containing TPO or Eltrombopag had significantly increased percentages of live cells (n=5), as well as decreased percentages of cells undergoing apoptosis compared to cultures with SF alone (SFT 12.6 0.5% p=0.003; SFE 12.4 2.1% p=0.012; SF 21.5 3.7%, n=5). RT-qPCR arrays performed at 5 hours after irradiation on CD34+ cells cultured as above with SFT or SFE showed a significant decrease (p<0.05) of at least two-fold in several pro-apoptotic or cell cycle arrest genes (BBC3, CCNO, GADD45G, PPM1D) compared to CD34+ cells cultured with SF alone. Twenty-four hours post-irradiation, cells cultured with TPO or Eltrombopag had significantly increased percentages of live cells (n=3), and decreased percentages of dead cells compared to cells cultured with SF alone (SFT 9.75 1.0% p=0.013; SFE 16.3 0.6% p=0.032; SF 36.5 6.2%, n=3). Progenitor cell survival was assessed using the CFU assay. The number of colony-forming cells was 5.9 ( 0.4) and 3.6 ( 0.2) fold higher when cultured with TPO or Eltrombopag, respectively, before -irradiation than when cultured with SF alone (p=0.005 and 0.006, respectively, n=2). Survival of long-term repopulating HSCs was assessed by quantifying human CD45+ cell engraftment at least 2 months after intravenous injection of NSG mice with irradiated human CD34+CD38- cells pre-cultured for 24 hours with SF, SFT or SFE. Engraftment of cells cultured with TPO or Eltrombopag was significantly higher than engraftment obtained after injection of cells cultured with SF alone before -irradiation. To gain insights into the mechanisms of DNA repair, the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways were examined by transfecting a linearized reporter plasmid which, upon repair by NHEJ or HR, expresses GFP. A tdTomato plasmid was used for normalization and efficiency was calculated by the ratio of GFP+tdTomato+ to total tdTomato+ cells. TPO and eltrombopag significantly increased NHEJ efficiency (mean 0.26 0.01 and 0.25 0.02, respectively) compared with cells cultured with SF alone (mean 0.18 0.01, p=0.002 and p=0.02, respectively, n=3). Because CD34+ cells do not use HR, the c-Mpl expressing HEL 92.1.7 cell line was used to investigate HR repair pathways. No improvement in HR was observed with TPO or Eltrombopag. We conclude that, analogous to TPO, Eltrombopag favors NHEJ DNA DSB repair and, consequently, survival of both hematopoietic stem and progenitor cells after -irradiation. These pre-clinical data suggest that Eltrombopag may be of benefit in the treatment of patients with Fanconi Anemia (FA). In FY17, we will also confirm the utility of Eltrombopag in FA by directly testing DNA repair activity in FA HSPCs. The latter, inaccessible in FA patients due to bone marrow failure, will be obtained via CRISPR/Cas9-knockdown of FANCA gene in normal CD34+ cells. A clinical trial testing Eltrombopag in patients with FA is also in preparation and will also be initiated in FY17.

目标1：证明艾曲波帕可以绕过IFN诱导的TPO信号传导缺陷 为了解决骨髓衰竭中内源性 TPO 水平较高时 Epag 功效的悖论，在 SCF、FLT3 和 TPO 5 ng/ml (TPO5) 或 Epag 3 存在的情况下培养来自 6 名健康供体的 G-CSF 动员的人 CD34+ 细胞g/ml (Epag)，有或没有 100 ng/ml IFN。培养 7 天后，通过流式细胞术、CFU 测定和免疫缺陷 (NSG) 小鼠移植来表征细胞。单独含有 TPO5 或 Epag 的培养物中 CD34+ 细胞的百分比相似（分别为 83.3±9.7% 和 87.6±7.1%），但在 IFN 存在下，Epag 比 TPO5 保存得更好（分别为 46.7±16.1% 和 24.6±15.0%，p <0.05）。因此，当比较有和没有 IFN 的 7 天培养物时，使用 TPO5 时 CD34+ 细胞的绝对数量显着减少（平均 7.6 倍，p<0.005），但使用 Epag 时仅略有减少（平均 1.6 倍，p = n.s.） 。与不含 IFN 的培养物相比，在存在 IFN 的情况下 7 天后，TPO5 的调整后的 CFU 数量同样减少了 2.7 倍，但 Epag 的调整后的 CFU 数量保持不变。当将相同起始数量的 CD34+ 细胞的 7 天扩增后代移植到 NSG 小鼠中时，Epag（34 个 3.8% 人 CD45+ 细胞）的人细胞植入优于 TPO5（21 个 1.8% 人 CD45+ 细胞，p<0.05） ) 存在 IFN 的培养物，表明 Epag 对最原始的长期重新增殖的 HSPC 产生影响。为了研究 Epag 在炎症条件下积极影响 HSPC 维持的潜在机制，我们检查了 TPO、Epag 和 IFN 与人 CD34+ 细胞中各自受体结合时诱导的细胞信号传导途径。在浓度为 5ng/mL 时，TPO 诱导 STAT5 磷酸化快速（峰值 < 1 小时）高效上升，随后信号快速（< 2 小时）衰减。相比之下，Epag 诱导 STAT5 磷酸化缓慢（4 小时达到峰值）低效上升，并且信号持续至少 10 小时。 TPO 和 Epag 之间的细胞信号传导效力和动力学差异可能与它们与 c-MPL 不同区域的结合有关，从而导致受体构象的交替变化。接下来我们研究了 IFN 对 TPO 和 Epag 诱导的 STAT5 在信号峰值（分别小于 1 小时和 4 小时）磷酸化的影响。正如之前在小鼠 HSPC 中所显示的，IFN 损害了人类 HSPC 中的 TPO 信号传导。相反，Epag 诱导的 STAT5 磷酸化在 IFN 存在下得以保留或增加。当 Epag 和 TPO 联合使用时，IFN 对 TPO 信号传导的抑制部分恢复。通过将 TPO 剂量从 5 ng/mL 减少至 1 ng/mL，从而将信号传导效力降低至与 Epag 相似的水平，IFN 对 TPO 信号传导的抑制作用被消除。 IFN 受体通过其配体激活，诱导 STAT1 磷酸化，并随后表达细胞因子信号传导抑制因子 1 (SOCS-1)，SOCS-1 分别通过抑制 STAT1 和 STAT5 磷酸化，作为 IFN 和 c-MPL 受体的负调节因子。我们发现，在 TPO 5ng/mL 存在的情况下，IFN 诱导的 STAT1 磷酸化增加（增加 1.5 倍，p<0.05），但不受 Epag（增加 1.1 倍，p = n.s.）或 TPO 1ng/mL（1.1 - 增加倍，p = n.s.）。我们的数据表明，Epag 可以对抗 IFN 诱导的人类 HSPC 中 TPO 信号传导的干扰。 Epag 产生无对抗的低效、缓慢动力学正信号，并将 c-Mpl 激活至对 HSPC 自我更新至关重要的阈值水平以上。 Epags 逃避 IFN 对生长因子细胞信号转导关键通路的阻断，这可能解释了它在改善 SAA 造血功能方面的功效。 这项工作将于 2016 年 12 月在美国血液学会 (ASH) 年会上发表，手稿正在准备中。 目标 2：证明艾曲波帕对人类 HSPC 具有 DNA 修复活性 将来自 5 名独立健康供体的 G-CSF 动员的人 CD34+ 细胞在 SCF 和 Flt3-L (SF)、SF 和 TPO (SFT) 或 SF 和艾曲波帕 (SFE) 存在下培养 24 小时，然后暴露于 2Gy 辐射，然后再培养5至24小时。通过流式细胞术测定 H2AX 表达（辐射诱导的 DNA 双链断裂 (DSB) 的标记）来量化 DNA 损伤，并使用膜联蛋白 V 和活性染料通过流式细胞术测量 CD34+ 细胞存活率。当培养物包含 TPO 或艾曲波帕时，照射后 5 小时的 H2AX+ 细胞明显少于仅使用 SF (n=5)。照射后五小时，与单独使用 SF 的培养物相比，含有 TPO 或艾曲波帕的培养物的活细胞百分比显着增加 (n=5)，并且凋亡细胞的百分比下降（SFT 12.6 0.5% p=0.003；SFE 12.4 2.1% p=0.012；SF 21.5 3.7%，n=5)。辐射后 5 小时对用 SFT 或 SFE 培养的 CD34+ 细胞进行 RT-qPCR 阵列显示，几种促凋亡或细胞周期停滞基因（BBC3、CCNO、 GADD45G、PPM1D）与仅用 SF 培养的 CD34+ 细胞相比。照射后 24 小时，与单独使用 SF 培养的细胞相比，使用 TPO 或艾曲波帕培养的细胞的活细胞百分比显着增加 (n=3)，死细胞百分比降低 (SFT 9.75 1.0% p=0.013；SFE 16.3 0.6% p=0.032；SF 36.5 6.2%，n=3)。使用 CFU 测定评估祖细胞存活率。照射前与 TPO 或艾曲波帕一起培养时，集落形成细胞数分别比单独与 SF 培养时高 5.9 ( 0.4) 和 3.6 ( 0.2) 倍（分别为 p=0.005 和 0.006，n=2） 。向 NSG 小鼠静脉注射用 SF、SFT 或 SFE 预培养 24 小时的经照射的人 CD34+CD38- 细胞后至少 2 个月，通过量化人 CD45+ 细胞植入来评估长期重新增殖的 HSC 的存活率。用TPO或艾曲波帕培养的细胞的植入显着高于在辐射前注射单独用SF培养的细胞后获得的植入。为了深入了解 DNA 修复机制，通过转染线性化报告质粒来检查非同源末端连接 (NHEJ) 和同源重组 (HR) DNA 修复途径，该质粒经 NHEJ 或 HR 修复后表达 GFP。使用 tdTomato 质粒进行标准化，并通过 GFP+tdTomato+ 与总 tdTomato+ 细胞的比率计算效率。与单独使用 SF 培养的细胞相比（平均值分别为 0.18 ± 0.01，p=0.002 和 p=0.02，n=3），TPO 和艾曲波帕显着提高了 NHEJ 效率（平均值分别为 0.26 ± 0.01 和 0.25 ± 0.02）。由于 CD34+ 细胞不使用 HR，因此使用表达 c-Mpl 的 HEL 92.1.7 细胞系来研究 HR 修复途径。 TPO 或艾曲波帕未观察到 HR 改善。我们的结论是，与 TPO 类似，艾曲波帕有利于 NHEJ DNA DSB 修复，从而有利于造血干细胞和祖细胞在辐射后的存活。这些临床前数据表明，艾曲波帕可能对范可尼贫血 (FA) 患者的治疗有益。 2017 财年，我们还将通过直接测试 FA HSPC 中的 DNA 修复活性来确认艾曲波帕在 FA 中的效用。后者由于骨髓衰竭而在 FA 患者中无法获得，将通过 CRISPR/Cas9 敲低正常 CD34+ 细胞中的 FANCA 基因来获得。在 FA 患者中测试艾曲波帕的临床试验也在准备中，也将于 2017 财年启动。