Eltrombopag for the Treatment of Bone Marrow Failure Syndromes

艾曲波帕治疗骨髓衰竭综合征

• 批准号: 10012690
• 负责人: Andre LaRochelle
• 金额: $ 67.72万
• 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/10012690
• 关键词: Adverse event; Affect; Affinity; Age; Androgens; Aplastic Anemia; BFU-E; BRCA2 gene; Binding; Binding Sites; Bioavailable; Blood; Blood Platelets; Bone Marrow; Bypass; CD34 gene; CRISPR/Cas technology; Cell Culture Techniques; Cell Death; Cell Survival; Cells; Chromosome Breakage; Chromosome abnormality; Clinical; Clinical Trials; Colony-Forming Units Assay; Colony-forming units; Common Terminology Criteria for Adverse Events; DNA Double Strand Break; DNA Repair; DNA-dependent protein kinase; Data; Defect; Dependence; Diagnosis; Diamond-Blackfan anemia; Dose; Double Strand Break Repair; EPOR gene; Eligibility Determination; Enrollment; Erythroblasts; Erythrocyte Transfusion; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Exclusion Criteria; Exhibits; Exposure to; Extracellular Domain; Fanconi' s Anemia; Female; Fibroblasts; Genes; Genetic; Genome Stability; Genomic Instability; Globin; Goals; Harvest; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heme; Hemoglobin; Hour; Human; Hypersensitivity; In Vitro; Individual; Inflammation Mediators; Inflammatory; Inherited; Interferons; Investigation; Iron; Iron Chelating Agents; Karyotype determination procedure; Ligand Binding; Lymphocyte; Malignant Neoplasms; Mediating; Modality; Modeling; Molecular; Mus; Mutate; Mutation; Myelogenous; Nonhomologous DNA End Joining; Oral; PTPRC gene; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Phosphorylation; Plasmids; Platelet Transfusion; Point Mutation; Population; Positioning Attribute; Prevention; Production; Pronormoblasts; Property; Proteins; Publishing; RPS19 gene; Recurrence; Reporter; Reporting; Research Design; Reserve Cell; Residual state; Ribosomal Proteins; Ribosomes; Safety; Signal Pathway; Signal Transduction; Signal Transduction Inhibition; Skin; Stem cells; Syndrome; System; TFRC gene; TNF gene; Testing; Therapeutic; Thrombopoietin; Time; Toxic effect; Translations; Transplantation; Western Blotting; Work; base; bone marrow failure syndrome; cancer therapy; cell injury; clinical efficacy; cytokine; cytopenia; erythroid differentiation; genome integrity; hematopoietic differentiation; improved; inclusion criteria; induced pluripotent stem cell; inhibitor/antagonist; insight; mimetics; monolayer; novel; novel therapeutics; outcome forecast; phase 2 study; polysome profiling; preclinical study; primary endpoint; progenitor; receptor; response; self-renewal; small molecule

Ojective 1: Characterize the mechanisms by which Epag promotes trilineage hematopoiesis. 1.1 Epag evades IFN blockade of signal transduction from c-MPL in human HSPCs. By directly comparing the effect of IFN on human HSPCs in the presence of TPO or Epag in vitro, we made two observations. First, we found that IFNg perturbed TPO-induced signaling pathways in human HSPCs and that Epag could bypass this inhibition, resulting in enhanced progenitor activity and long-term HSPC repopulating potential in the presence of IFNg. Second, we showed that IFNg disrupted the low-affinity interaction between TPO and the extracellular domain of c-MPL, delineating a novel molecular mechanism by which IFNg inhibits TPO signaling in HSPCs. Epag may bypass the inflammatory inhibition of signal transduction by avoiding capture by IFNg in the bone marrow, thus providing an explanation for its clinical efficacy in SAA, despite already elevated levels of TPO in these patients. This work was published (Blood 133:2043-55 (2019)). 1.2 Epag promotes DNA DSB repair in human HSPCs. To assess whether Epag promotes DNA DSB repair in human HSPCs and to identify the pathways involved, CD34+ cells obtained from healthy individuals were first cultured for 24 hours in the presence or absence of Epag and the DNA-PK inhibitor NU7441, exposed to low dose (2Gy) IR, and either transfected with NHEJ or HR DNA DSB repair reporter plasmids or assessed for changes in H2AX phosphorylation (H2AX), an indicator of IR-induced DSBs, at various times after IR. We demonstrated that Epag specifically activates the canonical NHEJ (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Importantly, Epag-mediated DNA repair resulted in enhanced genome stability, survival and function of primary HSPCs, as demonstrated in karyotyping analyses, CFU assays and after transplantation in immunodeficient NSG mice. Eltrombopag may thus offer a new therapeutic modality for the prevention of HSPC injury induced by IR in cancer therapy, and could have implications for the treatment of genome instability syndromes such as FA. This work was published (Exp Hematol 73:1-6 (2019)). Objective 2: Evaluate the safety and efficacy of Epag in subjects with Fanconi anemia. We have initiated a clinical trial to investigate whether Epag may offer a novel therapeutic modality for subjects with FA. Our pre-clinical studies described above indicate that Epag evades blockade of signal transduction from c-MPL induced by inflammatory cytokines. Additionally, we found that Epag enhances DNA repair activity in human HSPCs. Thus, Epag may positively influence two of the main known mechanisms leading to BMF in FA. Study Design. This is a non-randomized, phase II study of Epag given to subjects with FA (NCT03204188). Subjects receive Epag for 6 months. Subjects who cannot tolerate the medication or fail to respond by 6 months are taken off study drug. Subjects who respond at 6 months are invited in the extension phase for an additional 3 years. Eligibility Assessment. Inclusion criteria: (1) Confirmed diagnosis of FA by a biallelic mutation in a known FANC gene and/or by positive chromosome breakage analysis in lymphocytes and/or skin fibroblasts; (2) One or more of the following cytopenias: platelets 30K/L or platelet transfusion dependence in the 8 weeks prior to study entry, ANC 500/L, Hgb 9.0 g/dL or red blood cell (RBC) transfusion dependence in the 8 weeks prior to study entry; (3) Failed or declined treatment with androgens; 4) Age > 4 years. Exclusion criteria: (1) Evidence of MDS or AML; (2) Cytogenetic abnormalities associated with poor prognosis in FA; (3) Known biallelic mutations in BRCA2; (4) Active malignancy or likelihood of recurrence of malignancies within 12 months; (5) Treatment with androgens 4 weeks prior to initiating EPAG. Primary Endpoints. The primary efficacy endpoint is the proportion of drug responders at 6 months. Response to Epag is defined by one or more of the following criteria: (1) Platelets increase by 20K/L above baseline, or platelet transfusion independence; (2) Hgb increase by > 1.5g/dL or a reduction in the units of RBC transfusions by at least 50%; (3) At least a 100% increase in ANC for subjects with a pretreatment ANC of < 0.5 x 109/L, or an ANC increase > 0.5 x 109/L. The primary safety endpoint is the toxicity profile assessed at 6 months using the CTCAE criteria. Enrollment. Two subjects have been enrolled to date. No drug-related adverse events have been observed. Subject #1 (7YO female) did not respond to 6 months of Epag, likely due to limited HSPC reserve in the context of profound cytopenias (ANC = 100/L, Hgb = 6g/dL, Plt = 0K/L). In contrast, subject #2 (49YO female) showed response to Epag at 3 months and will continue on the extension phase of the study. This study will continue in FY20 and is expected to provide important clinical information on safety and efficacy of Epag in subjects with FA. Objective 3: Evaluate the ability of Epag to improve erythropoiesis in Diamond Blackfan anemia. In this study, we investigated whether Epag could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced ROS and increased proerythroblast survival and maturation. To test this possibility, we first established an iPSC model of DBA by reprogramming MNCs from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line. RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTM monolayer-based approach. Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+ primitive erythroid progenitors (P1), CD71+CD45loEPOR- proerythroblasts (P2), and more mature CD71+CD45-EPOR- erythroblasts (P3). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage. Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitors inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 g/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations. Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA.

OXTIVE 1：表征EPAG促进三利甲基造血的机制。 1.1 EPAG在人HSPC中逃避了从C-MPL的IFN阻断信号转导。 通过直接比较在体外TPO或EPAG存在下IFN对人HSPC的影响，我们进行了两个观察结果。首先，我们发现IFNG扰动了人类HSPC中TPO诱导的信号通路，并且EPAG可以绕过这种抑制作用，从而在IFNG存在下增强了祖细胞活性和长期HSPC重新流动潜力。其次，我们表明IFNG破坏了TPO与C-MPL的细胞外结构域之间的低亲和力相互作用，从而描绘了一种新型分子机制，IFNG抑制了HSPC中的TPO信号传导。 EPAG可以通过避免在骨髓中捕获IFNG的捕获来绕过对信号转导的炎症抑制，从而为SAA的临床疗效提供了解释，尽管这些患者的TPO水平升高。这项工作发表了（血液133：2043-55（2019））。 1.2 EPAG促进人类HSPC中的DNA DSB修复。 为了评估EPAG是否促进了人类HSPC中的DNA DSB修复并确定所涉及的途径，在存在或不存在EPAG的情况下，首先将从健康个体获得的CD34+细胞培养24小时，而DNA-PK抑制剂NU7441则暴露于低剂量（2GY）IR，并与NHEJ或HR Reprast confrasts转移有关，并将其转移到DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DRAST中。 IR之后的不同时间，磷酸化（H2AX）是IR诱导的DSB的指标。我们证明了EPAG专门激活了规范NHEJ（C-NHEJ）DNA修复机制，这是一种已知支持基因组完整性的途径。重要的是，EPAG介导的DNA修复导致基因组稳定性，原代HSPC的生存和功能增强，如核分析分析，CFU分析和免疫缺陷NSG小鼠移植后所证明的那样。因此，Eltrombopag可以为预防癌症治疗中IR引起的HSPC损伤提供一种新的治疗方法，并可能对治疗基因组不稳定性综合症（例如FA）有影响。这项工作发表（Exp Hematol 73：1-6（2019））。 目标2：评估EPAG在Fanconi贫血受试者中的安全性和功效。 我们已经开始了一项临床试验，以研究EPAG是否可以为FA受试者提供新的治疗方式。我们上面描述的临床前研究表明，EPAG逃避了炎症细胞因子诱导的C-MPL的信号转导的阻断。此外，我们发现EPAG增强了人类HSPC中的DNA修复活性。因此，EPAG可能会积极影响导致FA中BMF的两种主要已知机制。 研究设计。这是对具有FA受试者（NCT03204188）的EPAG的非随机II期研究。受试者接受EPAG 6个月。无法忍受药物或无法通过6个月反应的受试者被取消研究药物。在延长阶段邀请6个月反应的受试者再邀请3年。 资格评估。纳入标准：（1）通过在淋巴细胞和/或皮肤成纤维细胞中通过阳性染色体破裂分析中的双重突变和/或通过阳性染色体破裂分析确认了FA的诊断； （2）在研究入学前的8周内，血小板30K/L或血小板输血依赖性ANC 500/L，HGB 9.0 g/dl或红细胞（RBC）输血依赖性在研究入学前8周； （3）雄激素的治疗​​失败或下降； 4）年龄> 4岁。排除标准：（1）MDS或AML的证据； （2）与FA的预后不良有关的细胞遗传异常； （3）BRCA2中已知的双重突变； （4）在12个月内复发恶性肿瘤的主动恶性肿瘤或可能性； （5）在启动EPAG前4周对雄激素进行处理。 主要终点。主要疗效终点是6个月时药物反应者的比例。对EPAG的响应由以下一个或多个标准定义：（1）血小板在基线以上增加20k/L或血小板输血独立性； （2）HGB增加> 1.5g/dl或将RBC输血单位减少至少50％； （3）预处理ANC <0.5 x 109/L或ANC增加> 0.5 x 109/L的ANC至少增加100％。 主要的安全终点是使用CTCAE标准在6个月评估的毒性特征。 注册。迄今为止，已经注册了两个主题。尚未观察到与药物有关的不良事件。受试者＃1（7yo女性）对6个月的EPAG没有响应，这可能是由于在深度细胞质的背景下HSPC储备有限（ANC = 100/L，HGB = 6G/DL，PLT = 0K/L）。相反，受试者2（49yo女性）在3个月时显示对EPAG的反应，并将继续进行研究的扩展阶段。这项研究将在20财年继续进行，预计将提供有关EPAG在FA受试者中的安全性和功效的重要临床信息。 目标3：评估EPAG改善钻石黑芬贫血中红细胞生成的能力。 在这项研究中，我们调查了EPAG是否可以在DBA营救红细胞生成。我们假设EPAG可能通过限制铁的稳健细胞内铁螯合特性来抑制血红素的合成，从而导致铁诱导的ROS的降低并增加培养基细胞存活和成熟。为了测试这种可能性，我们首先通过对RPS19中失活突变的患者进行重新编程，这是DBA的IPSC模型，这是DBA中最常见的突变基因。我们还通过CRISPR/CAS9介导的RPS19点突变校正在已建立的DBA IPSC系列中生成了ISENIC IPSC线。 RPS19通过Western印迹证实了RPS19的单倍不足，并且通过DBA IPSC的多核体分析检测到40S/60S核糖体亚基比的预期降低。该表型在同基因IPSC中归一化。然后，使用基于STEMDIFFTM单层的方法将DBA和等源性IPSC系和来自健康供体衍生的IPSC进行21天进行造血分化。当该系统中观察到最大的红细胞产生时，在培养的第19天到第21天之间收获造血细胞。正常和异源IPSC有效地在各个成熟阶段产生了红细胞细胞，包括CD71+CD45+EPOR+EPOR+原始的红细胞祖细胞（P1），CD71+CD45LOEPOR-培养基 - 原生蛋白酶 - 培养基 - P2），以及更多成熟的CD71+CD71+CD45-EPOR-ERYYCOR-ERYYCRAST（P3）。相反，在DBA IPSC分化后检测到的大多数红细胞细胞在P1中组成，成熟到P2和P3的成熟度有限，这与红骨早期祖细胞阶段的分化块一致。此外，在菌落成型单元（CFU）测定中，DBA IPSC产生的髓样菌落数量（CFU-G，CFU-M和CFU-GM）与正常和异基因IPSC相当，但红骨菌落（BFU-E和CFU-E）（BFU-E和CFU-E）不可能与DBA相当不可用。接下来，从培养的第10天到21号EPAG 3 g/ml的存在，DBA IPSC被区分了。 EPAG的添加改善了晚期成熟，如早期祖细胞百分比（P1）的百分比降低，并且更成熟的P2和P3成熟的细胞细胞群体的增加表明。正在进行研究以确认EPAG介导的铁限制并减少血红素合成，因为这是本研究中观察到的改善的红细胞成熟的主要分子机制。总体而言，我们的数据表明，DBA IPSC的定向分化会在体外概括了早期的红斑成熟缺陷，并且可以通过在培养过程中添加EPAG来部分挽救红细胞生成。这些结果表明，EPAG可以改善DBA患者的红细胞产生。