Novel therapies for bone marrow failure and Diamond-Blackfan Anemia

我们开发了一项转化和临床研究项目，研究通过药物艾曲泊帕（EPAG）体内刺激造血的疗效和安全性，EPAG是一种与造血干细胞和祖细胞上的c-mpl受体结合的小分子，证明了在难治性重度再生障碍性贫血、新发重度再生障碍性贫血、中度再生障碍性贫血和骨髓增生异常中的疗效。在本报告期内，我们专注于缓解和分子/细胞遗传学进展和结局的预测因素，并与Neal Young博士团队就这些主题进行了合作研究。 我们最初研究EPAG在骨髓衰竭中作用的主要焦点是Diamond-Blackfan贫血患者对药物的显著反应，Diamond-Blackfan贫血是一种遗传性严重低增殖性贫血，由一组核糖体蛋白基因之一的突变导致单倍不足。这种反应令人惊讶，并引导我们研究其机制，并寻求EPAG作为DBA的可能新疗法。 根据我们所有EPAG骨髓衰竭试验中产生的数据，我们观察到EPAG是一种有效的铁螯合剂。在我们的大型队列研究中，我们发现EPAG治疗导致快速且具有临床意义的铁卸载。 缓解率和复发率不受初始铁状态的影响，表明再生障碍性贫血中EPAG的活性与HSC刺激有关，而不是铁卸载。然而，一些患者在长期EPAG治疗期间需要口服补铁，以避免临床相关的铁缺乏症（Young et al，2022） 在中度AA/血细胞减少试验中，EPAG逆转了单个DBA患者中遗传性核糖体病DBA的贫血，沿着关于可能的DBA病理生理学的新知识，这一观察结果使我们假设EPAG在DBA中的可能有效性可能是由于EPAG的强效细胞内螯合活性。最近的实验室研究表明，DBA中红系细胞发育受到抑制，这是由于红系祖细胞中蛋白质合成减慢，导致全球链生产与血红素生物合成失衡，导致游离血红素/细胞内铁增加和活性氧的毒性积累。我们已经设计并完成了一项临床试验的招募和随访，以研究EPAG在DBA中的安全性和活性（20-H-0021）。仅1/15例患者有应答，但大多数患者因血小板增多需要减量或停药。现在两个总的病人的反应是令人鼓舞的关于血红素耗竭可能如何提高DBA的红细胞输出的基本假设。我们现在正致力于开发一种新药，这种新药可以减缓血红素的合成，而不会引起血小板增多。口服Gly 1 T甘氨酸转运抑制剂中的双蝶呤，它减缓红系前体中血红素的合成，红系前体在产生血红素的第一步中依赖于甘氨酸。我们已经表明，这种药物在体外和体内小鼠模型中有效地改善DBA红系祖细胞成熟。 这项试验现已开始招募患者。我们正在进行相关的实验室和影像学研究，以评估铁的状态和机制的EPAG和bitopertin的作用，对样本的患者参加了最初的DBA试验。 我们还注意到，在我们先前的试验中，对EPAG有反应的最初DBA患者是嵌合体，在早期HSC中具有体细胞回复，导致野生型HSC造血输出的一部分。尽管存在这种嵌合现象，但患者在EPAG前仍存在严重贫血和输血依赖性，这表明突变型红系细胞发育可抑制成红细胞岛内的野生型细胞。我们在分析嵌合体的谱系特性和EPAG的影响以及与华盛顿大学的Janis Abkowitz合作的鼠竞争性再增殖模型中探索了这一假设，记录了突变DBA细胞对实验鼠移植嵌合体中WT细胞的显著抑制作用，这项工作现已发表（Doty等人，Blood，2022）。我们正在对DBA患者（包括嵌合体患者）和嵌合体小鼠的红系细胞进行单细胞RNASeq和基因分型，以研究可能的机制和相关途径。这些研究对于DBA基因治疗的发展具有重要意义，如果残留的突变细胞可以抑制野生型红细胞生成，那么DBA的基因治疗将无效

We developed a translational and clinical research program investigating the efficacy and safety of in vivo stimulation of hematopoiesis via the drug eltrombopag (EPAG), a small molecular that binds to the c-mpl receptor on hematopoetic stem and progenitor cells, demonstrating efficacy in refractory severe aplastic anemia, de novo severe aplastic anemia, moderate aplastic anemia, and myelodysplasia. During the current reporting period, we focused on predictors for response and molecular/cytogenetic progression and outcomes, participating in collaborative research with the group of Dr Neal Young on these topics. A major focus that resulted from our initial program studying the role of EPAG in marrow failure was the striking response to the drug in a patient with Diamond-Blackfan anemia, an inherited severe hypoproliferative anemia shown to result from mutations resulting in haploinsufficiency of one of a a group of ribosomal protein genes. This response was surprising, and led us to investigate the mechanism and pursue EPAG as a possible new therapy for DBA. Based on data generated in all of our EPAG bone marrow failure trials, we observed that EPAG is a potent iron chelator. In our large cohort study, we found that EPAG treatment results in rapid and clinically-significant iron unloading. Response rates and relapse rates were not impacted by initial iron status, suggesting that in aplastic anemia the activity of EPAG is linked to HSC stimulation not iron unloading. However, several patients have required oral iron supplementation while on long-term EPAG to avoid clinically-relevant iron deficiency (Young et al, 2022) The observation that EPAG reversed anemia in the inherited ribosomopathy DBA in the single DBA patient enrolled in the moderate AA/cytopenia trial along with new knowledge regarding possible DBA pathophysiology led us to hypothesize that the possible effectiveness of EPAG in DBA may be due to the potent intracellular chelating activity of EPAG. Recent laboratory studies suggest that erythroid development is inhibited in DBA due to slowed protein synthesis in erythroid progenitors, with a resulting imbalance in global chain production versus heme biosynthesis, leading to free heme/increased intracellular iron and toxic accumulation of reactive oxygen species. We have designed and now completed accrual and follow-up for a clinical trial to investigate the safety and activity of EPAG in DBA (20-H-0021). Only 1/15 patients responded, however the majority of patients required dose reductions or drug discontinuation due to thrombocytosis. The now two total patient responses is encouraging regarding the underlying hypothesis regarding how heme depletion might improve erythroid output in DBA. We are now focusing on a new drug that can slow heme synthesis without inducing thrombocytosis. Bitopterin in an oral Gly1T glycine transport inhibitor, It slows heme synthesis in erythroid precursors that are dependent on glycine for the first step in producing heme. We have shown that this drug is active in improving DBA erythroid progenitor maturation in vitro and in vivo in murine models. This trial has now begun enrolling patients. We are carrying out correlative laboratory and imaging studies to assess iron status and mechanism of EPAG and bitopertin action on samples from patients enrolled in the initial DBA trial. We have also noted that the initial DBA patient responding to EPAG in our prior trial was a mosaic, with somatic reversion in an early HSC, resulting in a fraction of wild type HSC hematopoietic output. Despite this mosaicism, the patient remained severely anemic and transfusion-dependent prior to EPAG, suggesting that mutant developing erythroid cells could inhibit wild-type cells within erythroblastic islands. We have explored this hypothesis in analyzing the lineage properties of the mosaicism and impact of EPAG, as well as a murine competitive repopulation model collaboratively with Janis Abkowitz at the University of Washington, documenting a marked inhibitory effect of mutant DBA cells on WT cells in experimental murine transplant chimeras, and this work has now been published (Doty et al, Blood, 2022). We are carrying out single cell RNASeq and genotyping of developing erythroid cells from both DBA patients (including the mosaic patient) and mosaic mice to investigate the likely mechanism and pathways that are involved. These studies have great relevance for the development of gene therapies for DBA, which would not be effective if residual mutant cells can inhibit wild type erythropoiesis