Novel therapies for bone marrow failure and Diamond-Blackfan Anemia

我们开发了一项转化和临床研究计划，调查通过药物eltrombopg(EPAG)在体内刺激造血的有效性和安全性，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，显示了对难治性重度再生障碍性贫血、新出现的重度再生障碍性贫血、中度再生障碍性贫血和骨髓发育不良的疗效。在本报告所述期间，我们专注于反应和分子/细胞遗传学进展和结果的预测因素，参与了与Neal Young博士小组就这些主题进行的合作研究。 我们最初研究EPAG在骨髓衰竭中的作用的一个主要焦点是钻石-布莱克凡贫血患者对药物的显著反应，这是一种遗传性的严重低增殖性贫血，被证明是由于一组核糖体蛋白基因中的一个突变导致的。这一反应令人惊讶，并促使我们研究EPAG的机制，并将EPAG作为治疗DBA的可能的新疗法。 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是一种有效的铁络合剂。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负荷。反应率和复发率不受初始铁状态的影响，这表明在再生障碍性贫血中，EPAG的活性与HSC的刺激有关，而不是铁卸载。然而，一些患者在长期服用EPAG时需要口服补铁，以避免临床相关的铁缺乏(Young等人，2022) 在参加中度AA/细胞减少试验的单个DBA患者中，观察到EPAG逆转了遗传性核糖体病DBA的贫血，以及关于DBA可能的病理生理学的新知识，使我们假设EPAG在DBA中的可能有效性可能是由于EPAG具有强大的细胞内螯合活性。最近的实验室研究表明，由于红系祖细胞蛋白质合成减慢，DBA的红系发育受到抑制，导致全球链生产与血红素生物合成的不平衡，导致游离血红素/细胞内铁增加，以及活性氧的毒性积累。我们已经设计并完成了一项临床试验，以调查DBA中EPAG的安全性和活性(20-H-0021)。只有1/15的患者有反应，但大多数患者因血小板增多而需要减少剂量或停药。关于血红素耗竭如何改善DBA中红系输出的基本假设，现在总共有两个患者的反应是令人鼓舞的。我们现在正专注于一种新药，它可以减缓血红素的合成，而不会导致血小板增多。Bitopterin在口服Gly1T甘氨酸转运抑制剂中，它减缓红系前体的血红素合成，而红系前体依赖甘氨酸作为产生血红素的第一步。我们已经证明，该药在体外和体内对小鼠DBA红系祖细胞的成熟具有促进作用。这项试验现在已经开始招募患者。我们正在进行相关的实验室和成像研究，以评估EPAG和bitpertin对参加DBA初始试验的患者样本的铁状态和作用机制。 我们还注意到，在我们之前的试验中，最初对EPAG有反应的DBA患者是马赛克，早期HSC出现体细胞逆转，导致一部分野生型HSC的造血量。尽管有这种嵌合体，患者在EPAG之前仍然存在严重的贫血和输血依赖，这表明突变的发育中的红系细胞可以抑制红细胞岛内的野生型细胞。我们在分析EPAG嵌合体的谱系特性和EPAG的影响时探索了这一假设，以及与华盛顿大学的Janis Abkowitz合作的小鼠竞争再种群模型，记录了突变的DBA细胞在实验小鼠移植嵌合体中对WT细胞的显著抑制作用，这项工作现在已经发表(Doty等人，血液，2022)。我们正在对DBA患者(包括马赛克患者)和马赛克小鼠的发育中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途径。这些研究对DBA的基因治疗的发展具有很大的相关性，如果残留的突变细胞可以抑制野生型红细胞生成，则DBA的基因治疗将不会有效

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