The Clinical Significance of Iron Overload and Iron Metabolism in Myelodysplastic Syndrome and Acute Myeloid Leukemia
鐵過載和鐵代謝在骨髓增生異常綜合症和急性骨髓性白血病中的臨床意義

Abstract 摘要

Introduction 介绍

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) represent heterogeneous clonal hematopoietic stem cells disorders. MDS is characterized by dysplasia of hematopoietic cells, AML by uncontrolled proliferation of poorly differentiated hematopoietic cells (blasts). Both diseases lead to insufficient hematopoiesis. Chronic fatigue due to anemia, bleeding due to thrombocytopenia and infection due to neutropenia are typical consequences. MDS bone marrow is prone to leukemic transformation with approximately 30% of MDS patients developing secondary AML over time (1). AML, being the most common acute leukemia in adults, is a disease that in most cases needs immediate treatment to avoid death within months or even weeks. Although our knowledge about the molecular drivers of AML is rapidly increasing, and recently resulted in the development of novel drugs and of molecularly informed treatment stratification, the 5-year overall survival (OS) rate is still below 30% (2).
骨髓增生异常综合征（MDS）和急性髓系白血病（AML）代表异质性克隆性造血干细胞疾病。MDS 的特征是造血细胞的畸形，AML 则是由未分化造血细胞（幼稚细胞）的不受控增殖所表现。这两种疾病都会导致造血不足。慢性疲劳是由贫血引起的，出血是由血小板减少引起的，感染是由中性粒细胞减少引起的，这些都是典型后果。MDS 骨髓容易发生白血病转化，大约 30%的 MDS 患者随着时间发展会发展为继发性 AML。AML 是成人中最常见的急性白血病，大多数情况下需要立即治疗，以避免在几个月甚至几周内死亡。尽管我们对 AML 的分子驱动因素的了解正在迅速增加，并最近导致了新药物的开发和分子信息治疗分层，但 5 年总生存率仍然低于 30%。

MDS and AML patients may develop primary iron overload arising from insufficient erythropoiesis (3). Repeated transfusions, which aim at ameliorating the symptoms of anemia, often lead to secondary iron overload. Iron overload in MDS and AML patients may lead to multiple cellular and systemic changes and therefore plays a crucial role in these hematologic malignancies ( Figure 1 ). Besides the importance of iron and proteins involved in iron metabolism for multiple cellular functions, iron is tightly connected to the production of reactive oxygen species (ROS) and can lead to cell death when in excess (4). Iron overload in the bone marrow and other tissues can result in alterations of the microenvironment and contribute to increased morbidity (5). In this respect, iron has been demonstrated to participate in aggravating the symptoms of MDS and AML patients by contributing to bone marrow failure (6). Excess iron can also alter the components of the immune system and result in an increased susceptibility to various infections (7). Therefore, serum and cellular iron levels have a prognostic value at initial diagnosis, might influence the response to chemotherapy and predict the outcome after hematopoietic stem cell transplantation (HSCT) (8–10). The involvement of iron in diverse metabolic processes and its special necessity for malignant cells makes it an interesting therapeutic target (11).
MDS 和 AML 患者可能由于红细胞生成不足而发展出原发性铁过载（3）。旨在改善贫血症状的反复输血往往导致继发性铁过载。MDS 和 AML 患者的铁过载可能导致多种细胞和系统变化，因此在这些血液恶性肿瘤中起着至关重要的作用（图 1）。除了铁及参与铁代谢的蛋白对多种细胞功能的重要性外，铁与活性氧自由基（ROS）的产生密切相关，当过量时可导致细胞死亡（4）。骨髓和其他组织中的铁过载可能导致微环境的改变，并导致发病率增加（5）。在这方面，铁已被证明通过促进骨髓衰竭而加重 MDS 和 AML 患者的症状。过量的铁还可以改变免疫系统的组成，并导致对各种感染的易感性增加（7）。 因此，血清和细胞铁水平在初步诊断时具有预后价值，可能影响化疗的反应，并且可以预测造血干细胞移植（HSCT）后的结果。铁在多种代谢过程中的参与以及对恶性细胞的特殊必要性使其成为一个有趣的治疗靶点。

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Figure 1 图 1

Potential cellular and systemic consequences of iron overload in patients with MDS or AML. Many of these factors are interwoven and may all together contribute to patient outcome.
MDS 或 AML 患者铁过载的潜在细胞和系统后果。许多这些因素相互交织，可能共同影响患者的预后。

In this review, we will first give an overview of the molecular basis of iron metabolism and its role in hematopoiesis. We will then focus on the altered iron metabolism in MDS and AML patients including clinical consequences. Subsequently, we will elucidate the effect of iron overload on the pathophysiology of MDS and AML, clinical consequences of the altered iron metabolism and its use as a potential target for therapy.
在这篇评论中，我们将首先概述铁代谢的分子基础及其在造血过程中的作用。然后，我们将重点关注 MDS 和 AML 患者中铁代谢的改变，包括临床后果。随后，我们将阐明铁过载对 MDS 和 AML 病理生理学的影响，改变后的铁代谢的临床后果以及其作为潜在治疗靶点的用途。

Iron Homeostasis and its Role for Normal Hematopoiesis
铁稳态及其在正常造血过程中的作用

Iron is an essential micronutrient for fundamental metabolic processes in all cells and organisms and is therefore a crucial element for terrestrial life. A vital iron-binding protein of the human body is hemoglobin, which is crucial for the transport, storage and distribution of oxygen. Hemoglobin in circulating erythrocytes and erythroid precursors in the bone marrow contains about two thirds of the total body iron (12). Besides, iron is bound to myoglobin in the muscles. Iron is also part of prosthetic groups such as in cytochrome proteins and Fe-S clusters due to its ability to facilitate electron transfer. Thereby, it is essential for the function of the citric acid cycle (TCA), the respiratory chain, DNA synthesis and DNA repair.
铁是所有细胞和生物体中基本代谢过程的必需微量营养素，因此对陆地生命至关重要。人体中一种重要的铁结合蛋白是血红蛋白，对氧气的运输、储存和分配至关重要。循环红细胞和骨髓中的红细胞前体中的血红蛋白含有大约全身铁的三分之二。此外，铁结合在肌肉中的肌红蛋白中。铁还是辅基团的一部分，如细胞色素蛋白和 Fe-S 簇，由于其促进电子转移的能力。因此，它对柠檬酸循环（TCA）、呼吸链、DNA 合成和 DNA 修复的功能至关重要。

Systemic iron homeostasis is maintained by a balance of iron uptake, recycling and loss ( Figure 2A ). Nutritional iron is mainly available as ferric iron, which can be reduced by ferrireductases. Subsequently, ferrous iron can be internalized into enterocytes via need-oriented gastrointestinal active transport mechanisms by the divalent metal ion transporter (SLC11A2). Iron may also be internalized through siderophore-associated binding to lipocalin-2 (LCN2) and subsequent endocytosis (13). Moreover, nutritional heme and possibly also ferritin can be absorbed by enterocytes via mechanisms not fully determined yet (14). Efflux of iron across the basolateral membrane into the bloodstream via ferroportin (SLC40A1), the only known iron exporter, is usually followed by its oxidation to ferric iron by the membrane-bound ferroxidase hephaestin. Ferric iron can be loaded to transferrin (TF) and then be used for the needs of the body. Excess iron is stored via ferritin (FTH and FTL) mainly in the liver. The body loses iron via exfoliation of cells on the inner and outer surfaces of the body with stool, urine, sweat and blood loss in menstruating women, but there are no physiological active excretion mechanisms to release an excess of iron in mammals and humans and the iron excretion cannot physiologically be increased beyond these values. High iron levels lead to systemic secretion of hepcidin, the most relevant regulator of the systemic iron metabolism, by the liver. Hepcidin binds to ferroportin on enterocytes and iron-storing cells like macrophages, resulting in an internalization and degradation of the hepcidin-ferroportin complex and thus effectively shuts down nutritional iron absorption and iron release from internal iron storage. Hepcidin expression is controlled by regulatory feedback mechanisms that involve active erythropoiesis: erythroblast-derived erythroferrone (ERFE), growth differentiation factor 11 (GDF11), growth differentiation factor 15 (GDF15) and twisted gastrulation protein homolog 1 (TWSG1) have been shown to influence hepatic hepcidin secretion, thus linking erythropoietic iron demand to iron supply (15–18).
系统铁稳态是通过铁的摄取、回收和丢失的平衡来维持的（图 2A）。营养铁主要以三价铁的形式存在，可以被铁还原酶还原。随后，二价铁可以通过双价金属离子转运蛋白（SLC11A2）的需要性胃肠活跃转运机制内化到肠细胞中。铁也可以通过铁载体相关结合到脂联素-2（LCN2）并随后内吞而内化（13）。此外，营养血红素和可能也是铁蛋白可以通过尚未完全确定的机制被肠细胞吸收（14）。铁通过铁转运蛋白（SLC40A1）跨基底侧膜进入血液，这是已知的唯一铁输出蛋白，通常随后通过膜结合的铁氧化酶赫费斯汀氧化为三价铁。三价铁可以装载到转铁蛋白（TF）中，然后用于身体的需求。多余的铁主要储存在肝脏中的铁蛋白（FTH 和 FTL）中。 身体通过排便、尿液、汗液和女性月经期间的血液流失，通过身体内外表面细胞的脱落而丢失铁元素，但在哺乳动物和人类中没有生理活跃的排铁机制来释放过量的铁元素，铁元素的排泄在生理上不能超出这些值。高铁水平导致肝脏系统性分泌肝铁素调节蛋白（hepcidin），这是系统性铁代谢最相关的调节因子。肝铁素调节蛋白结合到肠细胞和铁储存细胞（如巨噬细胞）上的铁转运蛋白，导致肝铁素调节蛋白-铁转运蛋白复合物的内化和降解，从而有效地关闭营养铁的吸收和内部铁储存的释放。肝铁素调节蛋白的表达受到调节性反馈机制的控制，这些机制涉及活跃的红细胞生成：已经显示红细胞前体细胞衍生的红细胞铁素（ERFE）、生长分化因子 11（GDF11）、生长分化因子 15（GDF15）和扭曲胃肠蛋白同源物 1（TWSG1）影响肝脏肝铁素调节蛋白的分泌，从而将造血铁需求与铁供应联系起来。

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Figure 2 图 2

Iron metabolism under physiological conditions (A) and in case of MDS/AML (B). Black arrows indicate direct iron metabolism, gray arrows represent regulatory mechanisms. LPI, labile plasma iron; MPS, mononuclear phagocyte system; NTBI, non-transferrin-bound iron; TFBI, transferrin-bound iron.
在生理条件（A）和 MDS/AML 情况下（B）的铁代谢。黑箭头表示直接铁代谢，灰箭头代表调节机制。LPI，易变性血浆铁；MPS，单核吞噬细胞系统；NTBI，非转铁蛋白结合铁；TFBI，转铁蛋白结合铁。

Overall, only 4% to 10% of the daily iron need is supplied by uptake of nutritional iron, whereas the majority of iron gets recycled by different cell types originating from the bone marrow. Cells within the mononuclear phagocyte system (MPS) remove senescent blood cells via phagocytosis and digestion. Afterwards, iron is released into the blood, from where it is transported by transferrin back to the bone marrow for recurrent use in hematopoiesis. About ten times the amount of serum transferrin iron is recycled through this bone marrow-MPS-bone marrow cycle per day (19).
总体而言，每天摄入的营养铁只能满足日常需求的 4%至 10%，而大部分铁都是由源自骨髓的不同细胞类型循环利用的。单核吞噬细胞系统（MPS）内的细胞通过吞噬和消化去除老化的血细胞。随后，铁释放到血液中，通过转铁蛋白被输送回骨髓，用于再生造血。每天通过这种骨髓-MPS-骨髓循环回收的血清转铁蛋白铁量是血清转铁蛋白铁量的十倍（19）。

Intracellular iron acquisition is provided by ferrous iron importers (SLC11A2, SLC39A8, SLC39A14) or by binding of diferric transferrin to the cell surface transferrin receptors (TFR: TFRC and TFR2α) resulting in an internalization of the complex by clathrin-mediated endocytosis. Acidification of the endosome results in the release of ferric iron from transferrin. Additionally, circulating FTH can bind transferrin-independently to TFRC and be internalized in this way (20). Endosomal ferric iron is reduced to ferrous iron via ferrireductases. Ferrous iron can then be transported to the cytosol, where it represents the labile cellular iron (LCI) pool. This non-bound, redox-active and chelatable iron pool can be utilized in cellular metabolic processes, or, when in excess, be stored in ferritin or excreted via ferroportin. NCOA4 can mediate ferritinophagy, while it is degraded via HERC2 ubiquitination-mediated induction of the proteasomal degradation machinery in the presence of iron (21). Intracellular iron proteins are post-transcriptionally regulated by the IRP/IREs regulatory network. Therefore, mRNAs of regulated proteins harbor specific hairpin stem loops, called iron-responsive elements (IRE), situated in the 3’ or 5’ untranslated regions. In iron-deplete cells, the iron-responsive element binding proteins ACO1 and IREB2 bind to the IREs of specific mRNAs resulting in mRNA stabilization or translational repression of these mRNAs. In this way, they modulate the expression of iron-regulating proteins, which subsequently leads to an increase of the labile iron pool. In iron-replete cells, ACO1 works instead as aconitase in the TCA cycle and IREB2 undergoes SCF^FBXL5 E3 ubiquitin ligase mediated ubiquitination and proteasomal degradation (22).
细胞内铁的获取由亚铁铁导入蛋白（SLC11A2、SLC39A8、SLC39A14）提供，或者通过双铁转铁蛋白与细胞表面转铁蛋白受体（TFR: TFRC 和 TFR2α）的结合，导致复合物通过衬氨酸介导的内吞作用而内化。内体的酸化导致铁蛋白中的三价铁释放。此外，循环的 FTH 可以独立于转铁蛋白地结合到 TFRC 并以这种方式内化（20）。内体中的三价铁通过铁还原酶还原为亚铁，亚铁随后可以被运送到细胞质，其中它代表了易变的细胞铁（LCI）库。这种非结合的、氧化还原活性和可螯合的铁库可以在细胞代谢过程中被利用，或者在过量时被储存在铁蛋白中或通过铁转运蛋白排泄。NCOA4 可以介导铁蛋白吞噬，同时在铁存在的情况下通过 HERC2 泛素化介导诱导蛋白酶体降解机制而被降解（21）。细胞内铁蛋白受 IRP/IREs 调控网络的后转录调控。 因此，受调控蛋白的 mRNA 携带特定的发夹状茎环，称为铁响应元件（IRE），位于 3'或 5'非翻译区。在缺铁细胞中，铁响应元件结合蛋白 ACO1 和 IREB2 结合到特定 mRNA 的 IRE，导致这些 mRNA 的稳定或翻译抑制。通过这种方式，它们调节铁调控蛋白的表达，从而导致可变铁库的增加。在富铁细胞中，ACO1 反而作为 TCA 循环中的顺式异柠檬酸脱水酶，IREB2 经历 SCF E3 泛素连接酶介导的泛素化和蛋白酶体降解。

Both, iron deficiency and iron overload lead to impaired hematopoietic functions. Iron deficiency resulting in microcytic anemia due to impaired hemoglobin production is a common nutritional deficiency disorder affecting especially women and children worldwide. As a consequence of iron overload, dysplastic changes and detrimental effects on erythroblast differentiation and maturation resulting in a reduction of the proliferative capacity of erythropoiesis and of erythroblast apoptosis in vitro have been described (3, 23). Additionally, iron overload has been shown to induce growth arrest and cell death due to oxidative stress via ROS-mediated activation of p38MAPK, JNK and p53 pathways in immature hematopoietic cells (24, 25). Thereby, the IRP/IRE regulatory network is essential in maintaining hematopoietic stem cells in their physiological self-renewal process. While Ireb2(-/-) mice develop microcytic anemia, deletion of Fbxl5 in murine hematopoietic stem cells leads to impaired hematopoiesis due to Ireb2 overexpression and subsequent iron overload (26, 27).
缺铁和铁过载都会导致造血功能受损。由于受损的血红蛋白产生而导致的微小细胞性贫血是一种常见的营养缺乏症，特别影响全球妇女和儿童。铁过载的结果是造血功能异常，对红细胞前体细胞的分化和成熟产生不良影响，导致红细胞生成能力和体外红细胞前体细胞凋亡的降低（3, 23）。此外，铁过载已被证明通过 ROS 介导的 p38MAPK、JNK 和 p53 途径诱导生长停滞和细胞死亡，由于氧化应激在未成熟造血细胞中的激活（24, 25）。因此，IRP/IRE 调控网络对于维持造血干细胞的生理自我更新过程至关重要。虽然 Ireb2(-/-)小鼠会发展微小细胞性贫血，但在小鼠造血干细胞中删除 Fbxl5 会导致由于 Ireb2 过度表达和随后的铁过载而导致造血功能受损（26, 27）。

Elevated Iron Levels in MDS and AML Patients
MDS 和 AML 患者的铁水平升高

Measurement of a patient’s iron status is difficult due to various pitfalls of the available methods. Most commonly, iron status is measured based on serum iron indicators such as serum ferritin, transferrin saturation and soluble transferrin receptor (sTFR). However, the results may be influenced by external factors including inflammation, growth factors and organ dysfunctions (28). In case of acute iron overload, exceedance of the transferrin binding capacity leads to detectable amounts of non-transferrin-bound iron (NTBI) in the serum. A subfraction of NTBI is chemically labile plasma iron (LPI), which is toxic due to its redox-activity and can cause oxidative damage to cellular membranes, proteins and DNA (29). NTBI including LPI are loosely bound to serum components as albumin and citrate (30). Thereby, the presence and dynamics of active iron forms as NTBI and LPI may be accountable for direct toxic effects, whereas steady iron markers as ferritin may reflect mainly systemic changes in iron metabolism. Iron overload can also be measured via organ biopsies or imaging methods as biomagnetic susceptometry or magnetic resonance imaging (MRI) although these methods are rarely applied due to their invasiveness, costs or insufficient validation (31).
患者铁状态的测量由于现有方法的各种缺陷而变得困难。最常见的是，铁状态是基于血清铁指标（如血清铁蛋白、转铁蛋白饱和度和可溶性转铁蛋白受体（sTFR））来衡量的。然而，结果可能会受到包括炎症、生长因子和器官功能障碍在内的外部因素的影响。在急性铁超载的情况下，超过转铁蛋白结合能力会导致血清中可检测到非转铁蛋白结合铁（NTBI）的量。NTBI 的一个亚分数是化学不稳定的血浆铁（LPI），由于其氧化还原活性，可能对细胞膜、蛋白质和 DNA 造成氧化损伤。NTBI 包括 LPI 松散地结合在血清成分如白蛋白和柠檬酸中。因此，活性铁形式如 NTBI 和 LPI 的存在和动态可能对直接毒性效应负责，而稳定的铁标记物如铁蛋白可能主要反映铁代谢中的系统变化。 铁过载也可以通过器官活检或成像方法来测量，如生物磁感应法或磁共振成像（MRI），尽管这些方法很少被应用，因为它们具有侵入性、成本高或验证不足（31）。

Using this variety of methods, over the years several characteristics of an altered iron metabolism in MDS and AML patients have been found together indicating a state of iron overload in these diseases ( Figure 2B ).
利用这种各种方法，多年来已经发现了 MDS 和 AML 患者铁代谢异常的几个特征，表明这些疾病存在铁过载的状态（图 2B）。

The most common reason for iron overload in patients with hematologic diseases is the administration of multiple red blood cell transfusions representing a massive excess of iron uptake with about 200 mg of iron in one unit of packed red blood cells (32). Transfusion-associated iron is processed by hepatic and splenic macrophages, which recycle heme iron from erythrocytes and release it into the extracellular space, thereby increasing the serum iron pool.
患有血液学疾病的患者出现铁过载的最常见原因是多次输注红细胞制剂，这代表了大量的铁摄入，每单位浓缩红细胞中含有约 200 毫克铁。输血相关的铁由肝脏和脾脏的巨噬细胞处理，这些细胞从红细胞中回收血红素铁并释放到细胞外空间，从而增加血清铁库。

Independent of red blood cell transfusions, disease-immanent factors can contribute to the iron overload phenotype. Dysplastic ineffective erythropoiesis is one of the cardinal manifestations of MDS, leading to an insufficient production of mature erythrocytes and potentially to a higher turnover of erythroid progenitors. This insufficient erythropoiesis leads to the secretion of hepcidin-suppressing cytokines and thus might result in further iron overload. A vicious cycle is formed, in which primary bone marrow dysfunction causes iron overload, which in turn amplifies bone marrow dysfunction. The presence of this mechanism is supported by data from Cui et al., who found elevated hepcidin and ferritin levels, but a reduced hepcidin/ferritin ratio compared to healthy controls in a study including 107 MDS patients without prior transfusions (33). In the same study, elevated ferritin levels correlated with decreased proliferative potential of erythropoiesis ex vivo. However, the extent of these mechanisms seems to differ between MDS subtypes. MDS subtypes with a high presence of ring sideroblasts (RARS, corresponding to MDS-RS according to the present WHO classification) as a morphological correlate of iron-loaded mitochondria, have been shown to have the lowest hepcidin/ferritin ratio (34, 35). Therefore, inefficient erythropoiesis might be more prominent in these subtypes than in other MDS patients. Correspondingly, MDS-RS is typically associated with a mutation in the splicing factor gene SF3B1. An SF3B1 mutation was recently identified by Bondu et al. to lead to the expression of an alternative ERFE transcript, which suppresses hepcidin transcription and thereby provides an explanation for the increased iron load especially in these patients (36). The European MDS registry (EUMDS) investigated the occurrence of iron overload in MDS patients prospectively (37). Here, clinical data and iron metabolism-associated parameters including serum levels of ferritin, transferrin, hepcidin, GDF15, sTFR, NTBI and LPI were analyzed in newly diagnosed lower-risk MDS patients from 148 centers in 16 countries in Europe and Israel since 2008. The results indicate that the above-mentioned concept of primary, disease-immanent iron overload may not be of strong relevance for the majority of MDS patients: markers of iron overload were elevated over all MDS subtypes. However, occurrence specifically correlated with transfusion-dependent MDS and with the MDS-RS subtype.
独立于红细胞输血，疾病固有因素可能导致铁过载表型。畸形无效的造血是 MDS 的主要表现之一，导致成熟红细胞的生产不足，可能导致红细胞祖细胞的更高周转率。这种不足的造血导致肝素抑制细胞因子的分泌，从而可能导致进一步的铁过载。形成了一种恶性循环，即原发性骨髓功能障碍导致铁过载，进而增强骨髓功能障碍。此机制的存在得到了 Cui 等人的数据支持，他们在一项研究中发现，与未接受输血的 107 名 MDS 患者相比，肝素和铁蛋白水平升高，但肝素/铁蛋白比值降低（33）。在同一研究中，升高的铁蛋白水平与体外红细胞增殖潜力降低相关。然而，这些机制的程度似乎在 MDS 亚型之间有所不同。 MDS 亚型中具有高比例环状铁染色体（RARS，根据目前的 WHO 分类对应于 MDS-RS）作为铁负荷线粒体的形态学相关物，已被证明具有最低的肝铁蛋白/铁蛋白比值（34, 35）。因此，这些亚型中的效率低下的造血可能比其他 MDS 患者更为突出。相应地，MDS-RS 通常与剪接因子基因 SF3B1 的突变相关联。最近 Bondu 等人发现 SF3B1 突变导致表达一种替代 ERFE 转录本，抑制肝铁蛋白转录，从而解释了特别是这些患者中铁负荷增加的原因（36）。欧洲 MDS 登记处（EUMDS）前瞻性调查了 MDS 患者中铁过载的发生（37）。自 2008 年以来，148 个中心在欧洲和以色列的 16 个国家中对新诊断的低风险 MDS 患者进行了临床数据和与铁代谢相关的参数的分析，包括铁蛋白、转铁蛋白、肝铁蛋白、GDF15、sTFR、NTBI 和 LPI 的血清水平。 结果表明，上述关于原发性、疾病内在性铁过载概念可能对大多数 MDS 患者并不具有强相关性：铁过载标志物在所有 MDS 亚型中均升高。然而，发生特别与依赖输血的 MDS 和 MDS-RS 亚型相关。

During chemotherapy and foremost during hematopoietic stem cell transplantation (HSCT) the iron homeostasis might be further disturbed as a result of erythroid cell lysis and suppressed erythropoiesis. This theory matches data from the German prospective multicenter study ALLIVE including 22 MDS and 90 AML patients and some smaller studies, which show an increase in NTBI and LIP levels during allogeneic HSCT (38–40).
在化疗期间，尤其是在造血干细胞移植（HSCT）期间，由于红细胞溶解和红细胞生成受抑制，铁稳态可能会进一步受到干扰。这一理论与德国前瞻性多中心研究 ALLIVE 的数据相吻合，该研究包括 22 例 MDS 和 90 例 AML 患者以及一些较小的研究，这些研究显示在同种异体 HSCT 期间 NTBI 和 LIP 水平增加（38-40）。

During the course of AML, signs of iron overload have also been described. Frequently, serum ferritin is elevated at initial AML diagnosis. The extent correlates with the leukemic burden, normalizes in remission, and increasing levels may signify a relapse (41). Increased hepcidin serum levels at diagnosis and pre- as well as post-HSCT were described in two small cohorts including exclusively or mostly AML patients (42, 43). However, hepcidin and ferritin are acute-phase proteins and might not only indicate iron overload but may also reflect a state of inflammation. Correspondingly, ferritin and hepcidin serum levels in one of these studies correlated with serum levels of CRP and IL-6 (42). In another study, ferritin levels were also elevated in CRP-low patients and ferritin and hepcidin levels correlated with the number of blood transfusions (43). Overall, valid data including definitive measures of iron overload and investigations in the systemic iron state in AML are missing. Specifically, there are no data available from investigating the interplay of insufficient hematopoiesis and iron metabolism in AML. Presumably, ineffective erythropoiesis due to dysplastic changes applies only to an AML subgroup (especially AML with myelodysplasia-related changes), whereas in other AML subtypes, insufficient erythropoiesis may rather be driven by other pathomechanisms as the suppression of erythropoiesis by inflammatory cytokines (44).
在 AML 的过程中，也描述了铁过载的迹象。通常，在初步 AML 诊断时，血清铁蛋白水平升高。其程度与白血病负担相关，缓解时会恢复正常，而升高的水平可能表明复发（41）。两个小队列中描述了诊断时和 HSCT 前后铁调蛋白血清水平升高，这两个队列仅或主要包括 AML 患者（42, 43）。然而，铁调蛋白和铁蛋白是急性期蛋白，可能不仅指示铁过载，还可能反映炎症状态。相应地，其中一项研究中，铁蛋白和铁调蛋白血清水平与 CRP 和 IL-6 的血清水平相关（42）。在另一项研究中，CRP 低的患者铁蛋白水平也升高，铁蛋白和铁调蛋白水平与输血次数相关（43）。总的来说，缺乏包括铁过载的明确措施和 AML 系统铁状态调查在内的有效数据。具体来说，目前没有可用于调查 AML 中造血不足和铁代谢相互作用的数据。 据推测，由于发育异常变化导致的无效红细胞生成仅适用于 AML 亚组（特别是具有骨髓增生异常相关变化的 AML），而在其他 AML 亚型中，红细胞生成不足可能更多地受到其他病理机制的驱动，如炎症细胞因子对红细胞生成的抑制。

Signs of iron overload show a prognostic impact in both, MDS and AML patients in many studies. One of the open questions in the field is, whether iron overload is just a consequence of increased transfusion frequency, which is a well-known measure of disease severity, which would explain the worse prognosis, or, whether iron overload per se has a negative impact on the course of the disease. In both diseases, the degree of transfusion dependency was associated with a worse patient outcome (45–48). However, high levels of LPI were associated with inferior overall and progression-free survival in lower-risk MDS patients irrespective of the transfusion status in the study of the European MDS registry (37). The ALLIVE study revealed that in patients undergoing allogeneic HSCT, pretransplant NTBI was associated with an increased incidence of non-relapse mortality and a worse overall survival, which is hard to explain by the pretransplant disease severity alone (38). Besides, high serum ferritin levels at AML diagnosis were associated with a worse outcome (9, 49, 50). The same is true for ferritin levels before and after allogeneic HSCT in cohorts including mainly MDS and AML patients (51, 52). Data on the prognostic impact of the liver iron content measured by MRI for patients receiving allogeneic HSCT are ambiguous. While a meta-analysis of four studies with mixed patient cohorts including overall 50% AML and 16% MDS patients found that increased liver iron was not indicative for bad patient outcome (53), the ALLIVE study showed an association of high pretransplant liver iron with increased early non-relapse mortality (NRM) (38). Despite different compositions of the patient cohorts with older, more severely iron-overloaded patients in the ALLIVE study, the role for liver iron overload in NRM remains inconclusive.
铁过载的迹象在许多研究中显示出对 MDS 和 AML 患者的预后影响。该领域中一个未解之谜是，铁过载是否仅是增加输血频率的结果，这是疾病严重程度的一个众所周知的衡量标准，这可以解释更糟糕的预后，或者铁过载本身是否对疾病进程产生负面影响。在这两种疾病中，输血依赖程度与患者预后更差相关（45-48）。然而，在欧洲 MDS 登记研究中，无论输血状况如何，高水平的 LPI 与低风险 MDS 患者的整体生存率和无进展生存率不利相关（37）。ALLIVE 研究表明，在接受同基因异体 HSCT 的患者中，移植前 NTBI 与非复发死亡率的增加和整体生存率的恶化相关，这很难仅通过移植前疾病严重程度来解释（38）。此外，在 AML 诊断时高血清铁蛋白水平与更糟糕的预后相关（9, 49, 50）。 同样适用于主要包括 MDS 和 AML 患者的队列在异基因 HSCT 之前和之后的铁蛋白水平（51,52）。关于通过 MRI 测量的肝铁含量对接受异基因 HSCT 患者的预后影响的数据是模棱两可的。虽然一项涵盖总体 50% AML 和 16% MDS 患者的混合患者队列的四项研究的荟萃分析发现增加的肝铁并不预示患者预后不良（53），但 ALLIVE 研究显示了移植前高肝铁与早期非复发死亡率（NRM）增加的关联（38）。尽管 ALLIVE 研究中患者队列的组成不同，包括年龄较大、铁过载严重的患者，但肝铁过载在 NRM 中的作用仍然没有定论。

Taking the data on the prognostic impact of different iron overload markers together, the overall correlation with patient outcome is striking. However, it is difficult to exclude that this is merely the reflection of disease severity. Despite these doubts, clinical correlation data and studies on the consequences of iron overload from other diseases, led to the widespread recommendation to treat transfusion-induced iron overload in patients with hematological malignancies. Several therapeutic options are available that will be reviewed in section Therapies Aiming at Iron Metabolism as a Possible Target in MDS and AML.
将不同铁过载标志物的预后影响数据综合在一起，与患者预后的整体相关性显著。然而，很难排除这仅仅是疾病严重程度的反映。尽管存在这些疑虑，临床相关数据和关于其他疾病引起的铁过载后果的研究，导致广泛推荐在患有血液恶性肿瘤的患者中治疗输血诱导的铁过载。有几种治疗选择可供选择，将在《治疗以铁代谢为可能靶点的 MDS 和 AML 部分》中进行审查。

Potential Roles of Iron-Related Intracellular Proteins in AML and MDS
AML 和 MDS 中与铁相关的细胞内蛋白质的潜在作用

To further understand the iron metabolism in MDS and AML, investigating the role of iron-related intracellular proteins might help explaining the interplay between iron and essential intracellular networks in MDS and AML cells.
为了进一步了解 MDS 和 AML 中的铁代谢，调查与铁相关的细胞内蛋白质的作用可能有助于解释 MDS 和 AML 细胞中铁与必要细胞内网络之间的相互作用。

Expression of iron-importing proteins might be an indicator for the iron need of the cells. It has been appreciated for almost 40 years that AML cells strongly bind to transferrin (54). In humans, two transferrin-binding receptors have been identified: TFRC is a ubiquitously expressed high affinity receptor and TFR2α is restricted to certain cell types as hepatocytes and erythroblasts and has an approximately 25-fold lower affinity for transferrin than TFRC (55, 56). The alternative TFR2 isoform, TFR2β, lacks the transmembrane and cytoplasmic domain but might be involved in the regulation of iron efflux in the MPS (57). Overexpression of TFRC was demonstrated in AML cells (58–60) and supports the hypothesis of a higher iron consumption of these cells. Thereby, TFRC expression was higher in undifferentiated than in more differentiated AML subtypes and decreased with terminal differentiation (59, 61). Neither high TFRC mRNA nor TFRC protein levels in AML cells correlated with patient outcome although a correlation was found with increased anemia, thrombopenia and complex cytogenetics (62, 63). On the contrary, higher TFR2 mRNA levels in bone marrow samples were surprisingly associated with a favorable outcome in AML and MDS patients (64, 65). However, the increase of TFR2 mRNA levels in MDS and AML bone marrow samples were shown to roughly correlate with the proportion of erythroid cells in the marrow and might therefore only to a minor extent reflect the expression of MDS or AML cells themselves (13, 66). This association with the erythroid cell number might be the explanation for the favorable outcome. Deducing from these data, higher TFRC expression of AML cells might reflect an undifferentiated blast status whereas higher TFR2 mRNA expression in the bone marrow of MDS and AML patients might be a marker for the number of erythroid cells. However, there is also evidence for a need of higher iron amounts due to overall higher TFRC expression and the necessity of TFR for leukemic cell growth as shown in TFR antibody studies described in section Perspectives.
铁进口蛋白的表达可能是细胞对铁需求的指标。近 40 年来，人们已经认识到 AML 细胞强烈结合转铁蛋白（54）。在人类中，已经鉴定出两种转铁蛋白结合受体：TFRC 是广泛表达的高亲和力受体，TFR2α仅限于某些细胞类型，如肝细胞和红细胞前体细胞，并且对转铁蛋白的亲和力约为 TFRC 的 25 倍（55, 56）。另一种 TFR2 亚型 TFR2β缺乏跨膜和胞质结构域，但可能参与 MPS 中铁外流的调节（57）。在 AML 细胞中已证明 TFRC 的过表达（58-60），并支持这些细胞对铁的更高消耗的假设。因此，TFRC 的表达在未分化 AML 亚型中高于更为成熟的亚型，并随着终末分化而降低（59, 61）。AML 细胞中的 TFRC mRNA 或 TFRC 蛋白水平高与患者预后无关，尽管发现与贫血、血小板减少和复杂的细胞遗传学有关（62, 63）。 相反，骨髓样本中较高的 TFR2 mRNA 水平与 AML 和 MDS 患者的良好预后惊人地相关（64, 65）。然而，MDS 和 AML 骨髓样本中 TFR2 mRNA 水平的增加似乎与骨髓中红细胞的比例大致相关，因此可能只在很小程度上反映 MDS 或 AML 细胞本身的表达（13, 66）。这种与红细胞数量的关联可能是有利结果的解释。根据这些数据推断，AML 细胞的较高 TFRC 表达可能反映未分化的爆发状态，而 MDS 和 AML 患者骨髓中较高的 TFR2 mRNA 表达可能是红细胞数量的标志。然而，也有证据表明，由于整体 TFRC 表达较高以及 TFR 对白血病细胞生长的必要性，需要更多铁量，如在“展望”部分描述的 TFR 抗体研究中所示。

Only recently, the roles of LCN2 and BDH2 have attracted attention in MDS and AML patients. LCN2 can bind to siderophores and thereby lead to iron internalization via endocytosis or to the secretion of iron via endosome recycling thereby potentially enabling iron overload or iron deficiency (67). BDH2 catalyzes the rate-limiting step for the formation of the mammalian siderophore 2,5-dihydroxybenzoic acid (68). This might facilitate LCN2-mediated iron uptake but also prevent iron overload in the cytoplasm and iron depletion in mitochondria. In cytogenetically normal AML patients, LCN2 mRNA was reduced (69). Thereby, high LCN2 mRNA expression in the bone marrow was associated with a favorable outcome especially in combination with wild-type FLT3 showing an enhanced apoptosis under hydrogen peroxide and cytarabine treatment whereas showing a protective effect under DFO treatment. On the contrary, BDH2 overexpression has been associated with poor overall survival in cytogenetically normal AML (70) and with elevated ferritin levels as well as an increased risk for progression to leukemia in MDS (71). As further mechanistical analyses are missing, it can only be speculated that in this case LCN2 overexpressing cells might have an increased LPI pool predisposing them to oxidative stress, whereas BDH2 overexpression might prevent cytoplasmatic iron overload. Further studies validating these results and unraveling the underling mechanisms are highly needed.
最近，LCN2 和 BDH2 在 MDS 和 AML 患者中的作用引起了关注。LCN2 可以结合铁载体，从而通过内吞作用导致铁的内化，或通过内体回收导致铁的分泌，从而可能导致铁过载或铁缺乏（67）。BDH2 催化了哺乳动物铁载体 2,5-二羟基苯甲酸形成的速率限制步骤（68）。这可能有助于 LCN2 介导的铁摄取，但也可以防止细胞质中的铁过载和线粒体中的铁耗竭。在细胞遗传学正常的 AML 患者中，LCN2 mRNA 减少（69）。因此，骨髓中高 LCN2 mRNA 表达与有利的预后相关，特别是与野生型 FLT3 结合，显示在过氧化氢和环磷酰胺处理下增强的细胞凋亡，而在 DFO 处理下显示保护作用。相反，BDH2 过表达与细胞遗传学正常的 AML 患者总生存率较低（70），以及铁蛋白水平升高以及 MDS 进展为白血病风险增加相关（71）。 由于缺乏进一步的机械分析，只能推测在这种情况下，LCN2 过表达的细胞可能具有增加的 LPI 池，使其易受氧化应激的影响，而 BDH2 过表达可能防止细胞质铁超载。迫切需要进一步研究来验证这些结果并揭示潜在机制。

The intracellular conversion of insoluble ferric to soluble ferrous iron is mediated by ferric reductases including STEAP protein members. Although STEAP1 has no iron reducing function, it co-localizes with transferrin and TFRC suggesting also a role in iron homeostasis. In AML, STEAP1 was shown to be overexpressed and associated with an adverse OS (72).
胞内不溶性三价铁转化为可溶性二价铁是由包括 STEAP 蛋白成员在内的铁还原酶介导的。虽然 STEAP1 没有铁还原功能，但它与转铁蛋白和 TFRC 共定位，也暗示在铁稳态中起作用。在 AML 中，已经显示 STEAP1 被过度表达，并与不良 OS 相关。

Systemically elevated levels of the iron storage protein ferritin suggest a role for intracellular ferritin levels in MDS and AML as well. FTH1 was reported to be expressed particularly in erythroid blasts measured by immunohistochemistry (73). In another study, FTH1 and FTL mRNA overexpression and FTH1 protein overexpression measured by immunoblot were shown in AML primary cells compared to peripheral mononuclear cells (9). The presence of ferritin may reduce the LPI pool and therefore prevent ROS formation. In line with this, a decreased in vitro cytotoxic activity of cytarabine was detected in FTH1 overexpressing AML. Additionally, analyses of the erythroleukemia cell line K562 indicate that FTH1 expression might prevent ROS-induced protein misfolding (74) and ROS-induced activation of the HIF1A/CXCR4 pathway leading to an epithelial-to-mesenchymal transition (EMT)-like phenotype (75). Besides, FTH1 might regulate RAF1 downregulation and activate pERK1/2 through downregulation of the expression of distinct microRNAs (76). Therefore, intracellular ferritin expression might play a role in MDS and AML especially in erythroid blasts on many levels.
系统性升高的铁贮存蛋白铁蛋白水平表明细胞内铁蛋白水平在 MDS 和 AML 中也起作用。据报道，FTH1 在免疫组织化学检测中特别在红细胞前体细胞中表达（73）。在另一项研究中，与外周单核细胞相比，AML 原代细胞中显示了 FTH1 和 FTL mRNA 过表达以及通过免疫印迹检测到的 FTH1 蛋白过表达（9）。铁蛋白的存在可能减少 LPI 池，从而防止 ROS 形成。与此一致，检测到 FTH1 过表达的 AML 中细胞杂交素的体外细胞毒活性降低。此外，对红白血病细胞系 K562 的分析表明，FTH1 表达可能防止 ROS 诱导的蛋白质错误折叠（74），并防止 ROS 诱导的 HIF1A/CXCR4 通路激活，导致上皮-间质转化（EMT）样表型（75）。此外，FTH1 可能通过下调 RAF1 并通过下调不同 microRNA 的表达来激活 pERK1/2。因此，细胞内铁蛋白表达可能在 MDS 和 AML 中发挥作用，特别是在多个水平上的红细胞前体细胞中。

Expression of the iron exporter FPT is also suggested to reduce the LPI pool and thereby the formation of ROS. Low FPT levels in AML cells correlated with good risk cytogenetics, increased sensitivity to cytarabine treatment and favorable outcomes (10) but a causal relationship could not be deduced from this data.
铁出口蛋白 FPT 的表达也建议减少 LPI 池，从而减少 ROS 的形成。AML 细胞中低 FPT 水平与良好风险细胞遗传学、对阿糖胞苷治疗的增加敏感性和有利结果相关（10），但从这些数据中无法推断因果关系。

Overall, several changes in proteins associated with iron metabolism have been detected in MDS and AML cells. Mutually, the iron status and these proteins as well as several intracellular signaling pathways influence each other. Thereby, especially proteins directly regulating the intracellular iron pool seem to have an impact on cell viability and patient outcome.
总的来说，在 MDS 和 AML 细胞中检测到了与铁代谢相关的蛋白质的几处变化。铁状态和这些蛋白质以及几种细胞内信号传导途径相互影响。因此，特别是直接调节细胞内铁库的蛋白质似乎对细胞的存活能力和患者预后产生影响。

Iron and ROS Homeostasis in Leukemogenesis
白血病发生中的铁和 ROS 稳态

Iron and ROS homeostasis are closely entangled. Iron contributes to ROS formation by the production of hydroxyl radicals via the Haber-Weiss and Fenton reaction. Moreover, iron is involved in indirect ROS production. Multiple iron-containing enzymes and those which require iron as an indispensable cofactor contribute to ROS production under normal conditions (77). So, as an important component of the respiratory chain iron is involved in the formation of mitochondrial ROS during oxidative phosphorylation (78). Vice versa, ROS can interact with iron sulfur clusters ([4Fe-4S]), turning them into their inactive form ([3Fe-4S]+). This leads to a switch in the function of the iron-sulfur cluster protein ACO1 from its role as aconitase in the TCA cycle to its function as an IRE-binding protein regulating the expression of various proteins involved in iron metabolism and other pathways (79).
铁和 ROS 稳态密切相关。铁通过 Haber-Weiss 和 Fenton 反应产生的羟基自由基促进 ROS 的形成。此外，铁参与间接 ROS 的产生。多种含铁酶和需要铁作为不可或缺辅因子的酶在正常条件下促进 ROS 的产生。因此，作为呼吸链的重要组成部分，铁参与了氧化磷酸化过程中线粒体 ROS 的形成。反之，ROS 可以与铁硫簇（[4Fe-4S]）相互作用，将其转化为其无活性形式（[3Fe-4S]+）。这导致铁硫簇蛋白 ACO1 的功能发生转变，从其在 TCA 循环中的丙酮酸脱水酶角色转变为其作为 IRE 结合蛋白的功能，调节涉及铁代谢和其他途径的各种蛋白的表达。

Elevated ROS levels have been detected in MDS and AML patients compared to controls (80, 81). Moreover, iron overload is accompanied by increased ROS levels in this patient cohort (82–84). Therefore, iron may contribute to leukemogenesis via its effect on the ROS homeostasis.
相对于对照组，MDS 和 AML 患者中检测到了升高的 ROS 水平（80, 81）。此外，铁过载伴随着该患者群体中 ROS 水平的增加（82-84）。因此，铁可能通过对 ROS 稳态的影响促进白血病发生。

Due to this connection, iron overload has been discussed to be involved in mutagenesis and leukemic transformation. Highly reactive hydroxyl radicals can directly interact with DNA leading to DNA damage (85). Moreover, ROS can stimulate the generation of lipid peroxyl radicals especially of polyunsaturated fatty acids (PUFAs) leading to reactive aldehydes that are mutagenic and genotoxic (86). In a mouse model for myelodysplastic syndrome using NUP98-HOXD13 (NHD13) transgenic mice, increased levels of ROS were detected in bone marrow nucleated cells accompanied by increased DNA double strand breaks supporting a connection between ROS and malignant transformation (87). In this line, a 5-year prospective registry study including 599 MDS patients revealed a deceased rate of progression to AML in patients treated with iron chelators (60). On the contrary, an earlier meta-analysis of Zeidan et al. did not confirm differences in the progression of MDS to AML with or without administration of iron chelators (88). Thereby, analyses might differ due to different MDS subgroups, observation periods and a potential selection bias for patients with longer predicted survival receiving iron chelation. Deducing from these results, leukemic transformation as a result of iron overload is a valid hypothesis but data are still ambiguous and more prospective trials are required. Possibly, disease related risk factors in MDS may overcome the influence of iron overload on progression to AML. The fact that mutations in the hereditary hemochromatosis protein (HFE) have not been found to increase the risk of AML (89, 90) may also indicate that de novo AML development is not induced by systemic iron overload.
由于这种联系，铁过载被讨论涉及到突变和白血病转化。高度活性的羟基自由基可以直接与 DNA 相互作用，导致 DNA 损伤（85）。此外，ROS 可以刺激脂质过氧基自由基的生成，特别是多不饱和脂肪酸（PUFAs），导致具有突变原性和遗传毒性的反应性醛类（86）。在使用 NUP98-HOXD13（NHD13）转基因小鼠模型研究骨髓增生异常综合征时，发现骨髓核细胞中 ROS 水平升高，伴随着 DNA 双链断裂的增加，支持 ROS 与恶性转化之间的联系（87）。在这方面，一项包括 599 名 MDS 患者的 5 年前瞻性登记研究显示，接受铁螯合剂治疗的患者 AML 进展的死亡率降低（60）。相反，Zeidan 等人早期的荟萃分析并未证实 MDS 进展为 AML 时是否使用铁螯合剂有差异（88）。 因此，由于不同的 MDS 亚组、观察期和长期预测存活接受铁螯合治疗的患者可能存在潜在的选择偏倚，分析结果可能会有所不同。根据这些结果推断，铁过载导致白血病转化是一个有效的假设，但数据仍然模棱两可，需要更多前瞻性试验。可能，MDS 中与疾病相关的危险因素可能会克服铁过载对 AML 进展的影响。遗传性血色病蛋白（HFE）突变未被发现增加 AML 风险的事实（89, 90）也可能表明，系统性铁过载不会诱导新发 AML 的发展。

ROS is known to highly influence hematopoiesis including hematopoietic stem cell state and function (91, 92). ROS is also involved in the regulation of various intracellular processes and signaling pathways (e.g. NF-κB, MAPK, PI3K-Akt, ubiquitination) as it is able to interact directly with proteins, ions and other molecules (93). Therefore, ROS might also influence stemness and proliferation of MDS and AML cells. Many molecular lesions related to MDS and AML development including mutations in FLT3, NRAS/KRAS and IDH1/2 affect intracellular ROS production, thus potentially promoting ROS-mediated oncogenic signaling (94). Therefore, iron might impact intracellular signaling and cell fate decisions also by its influence on intracellular ROS signaling. Indeed, iron and associated proteins are involved in some of these signaling pathways as described in section Potential Roles of Iron-Related Intracellular Proteins in AML and MDS. However, studies further investigating this theory are needed.
ROS 被认为对造血过程产生高度影响，包括造血干细胞的状态和功能（91, 92）。ROS 还参与调节各种细胞内过程和信号通路（例如 NF-κB、MAPK、PI3K-Akt、泛素化），因为它能够直接与蛋白质、离子和其他分子相互作用（93）。因此，ROS 可能也会影响 MDS 和 AML 细胞的干性和增殖。许多与 MDS 和 AML 发展相关的分子病变，包括 FLT3、NRAS/KRAS 和 IDH1/2 的突变，影响细胞内 ROS 产生，从而潜在地促进 ROS 介导的致癌信号（94）。因此，铁可能通过影响细胞内 ROS 信号传导来影响细胞内信号传导和细胞命运决定。事实上，铁及其相关蛋白参与了一些这些信号通路，如《AML 和 MDS 中铁相关细胞内蛋白的潜在作用》部分所述。然而，需要进一步研究这一理论。

In the extreme, iron overload with subsequent overwhelming accumulation of ROS can lead to ferroptosis, a non-apoptotic form of programmed cell death dependent on iron that differs from other regulated cell death mechanisms as apoptosis. First labeled by Dixon in 2012, ferroptosis is the consequence of a reduced antioxidant defense leading to uncontrolled lipid peroxidation and subsequent oxidative cell death (95). Depending on the activation of ROS-connected signaling pathways, cells are at a different risk for ferroptosis. Treatment of NRAS-Q61L mutated AML cells with the ferroptosis-inducing molecule erastin resulted in enhanced ROS levels and cytosolic translocation of HMGB1 leading to cell death, whereas this effect was not seen in unmutated cell lines (96). Importantly, the effect was iron-dependent and HMGB1 knock-down lead to lower expression of TFRC.
在极端情况下，铁过载导致随后的 ROS 过度积累可能导致铁死亡，这是一种依赖于铁的非凋亡形式的程序化细胞死亡，与其他调节的细胞死亡机制如凋亡不同。由 Dixon 在 2012 年首次命名，铁死亡是抗氧化防御减少导致未受控制的脂质过氧化和随后的氧化细胞死亡的结果。根据 ROS 连接的信号通路的激活程度，细胞对铁死亡的风险不同。用诱导铁死亡分子 erastin 处理 NRAS-Q61L 突变的 AML 细胞导致 ROS 水平增加和 HMGB1 的胞浆转位导致细胞死亡，而在未突变的细胞系中未见到这种效应。重要的是，这种效应是依赖于铁的，HMGB1 敲除导致 TFRC 的表达降低。

Leukemic cells seem especially exposed to iron overload with the risk of undergoing ferroptosis. This indicates that they may have gained some ferroptosis evasion strategies. Indeed, Hole et al. could show that higher levels of NOX-derived ROS (ROS) in AML blasts were tolerated by evading oxidative stress response through suppression of p38MAPK signaling (97). Additionally, glutathione peroxidases, which can protect cells from oxidative damage by reducing lipid hydroperoxides and free hydrogen peroxide are overexpressed in AML patient samples and associated with an adverse OS (98). Moreover, Yusuf et al. show a dependency of murine and human AML cells on ALDH3A2, which can detoxify fatty aldehydes and thereby prevent oxidative damage due to lipid peroxidation (99). In mouse models, reduction of Aldh3a2 induced ferroptosis in leukemic cells and was synergistically lethal combined with the inhibitor of glutathione peroxidase 4 (GPX4) RSL3, whereas it was dispensable for normal hematopoiesis. In this line, the transcription factor NFE2L2 also seems to strengthen the oxidative stress defense in leukemic cells by regulating the expression of many antioxidative proteins especially in case of additional chemotherapeutic treatment (100, 101). Parallelly, NFE2L2 also regulates the expression of iron-related proteins as FTH1, FTL and HMXO1 again supporting a close connection between ROS and iron homeostasis. All these findings support the hypothesis that AML cells might benefit from the toleration of higher iron and ROS levels. To which extent iron is involved in this pathomechanism and if this is also the case for MDS cells has to be further elucidated.
白血病细胞似乎特别容易受铁过载的影响，存在发生铁死亡的风险。这表明它们可能已经获得了一些铁死亡逃避策略。事实上，Hole 等人发现 AML 爆发细胞中 NOX 源 ROS（ROS）水平较高，通过抑制 p38MAPK 信号传导来规避氧化应激反应（97）。此外，谷胱甘肽过氧化物酶可以通过还原脂质过氧化物和游离过氧化氢来保护细胞免受氧化损伤，在 AML 患者样本中过度表达，并与不良 OS 相关（98）。此外，Yusuf 等人展示了小鼠和人类 AML 细胞对 ALDH3A2 的依赖性，ALDH3A2 可以解毒脂肪醛，从而防止由脂质过氧化引起的氧化损伤（99）。在小鼠模型中，减少 Aldh3a2 会导致白血病细胞发生铁死亡，并与谷胱甘肽过氧化物酶 4（GPX4）抑制剂 RSL3 联合使用具有协同致命作用，而对正常造血过程则是可有可无的。 在这一行中，转录因子 NFE2L2 似乎也通过调节许多抗氧化蛋白的表达，特别是在额外的化疗治疗情况下，加强了白血病细胞的氧化应激防御（100, 101）。与此同时，NFE2L2 还调节与铁相关的蛋白的表达，如 FTH1、FTL 和 HMXO1，再次支持 ROS 和铁稳态之间密切联系的假设。所有这些发现都支持 AML 细胞可能受益于对更高铁和 ROS 水平的耐受性的假设。铁在这一病理机制中的作用程度以及这是否也适用于 MDS 细胞，还需要进一步阐明。

Iron and the Microenvironment
铁和微环境

Hematopoietic and leukemic blasts reside and proliferate in bone marrow niches interacting with their microenvironment. The microenvironment including mesenchymal cells, endothelial cells, sympathetic neurons, other hematopoietic and immune cells and the extracellular matrix is considered to be a key regulator of MDS and AML pathogenesis and recurrence (25, 102). Leukemic cells seem to adopt the bone marrow microenvironment according to their needs and suppress normal hematopoiesis via secretion of cytokines, microRNAs and exosomes.
造血和白血病原始细胞寄居并在骨髓微环境中增殖。微环境包括间质细胞、内皮细胞、交感神经元、其他造血和免疫细胞以及细胞外基质，被认为是 MDS 和 AML 发病和复发的关键调节因子。白血病细胞似乎根据自身需求采用骨髓微环境，并通过细胞因子、微 RNA 和外泌体的分泌抑制正常造血。

Excess iron in AML and MDS patients is deposited in various organs including the bone marrow thereby altering the composition of the hematopoietic niche and potentially leading to hematopoietic niche defects. A murine iron overload model revealed elevated ROS levels and increased bone resorption leading to changes in the bone microarchitecture with trabecular and cortical thinning of the bone (103). This loss of bone substance seems related to changes in the bone marrow mesenchymal stem cells (BM-MSCs). Several alterations in the bone marrow stroma cell number and composition have been reported which concur in the fact that iron overload reduces the differentiation into osteoblasts relative to other cell subtypes and reduces matrix calcification (103–105). Cheng et al. could also demonstrate a ROS-mediated cell death of mesenchymal cells due to iron overload mediated by the AMPK/MFF/DNM1L pathway triggering mitochondrial fragmentation and reducing ATP production (106). The alterations of the mesenchymal cell compartment were also shown to influence their supporting function for hematopoiesis. Thereby, the expression of several adhesion molecules and cytokine secretion was altered in bone marrow stroma cells under overload conditions impairing their capacity to support hematopoietic cells growth (24, 105, 107). This might also be important for transplant engraftment during HSCT, as transplantation from normal donor mice to mice with iron overload resulted in a delayed hematopoietic reconstitution (107). Therefore, the effects of iron overload on bone marrow structure and mesenchymal cells might attribute to the defective hematopoiesis found in MDS and AML patients.
AML 和 MDS 患者体内的过量铁被沉积在包括骨髓在内的各种器官中，从而改变造血微环境的组成，可能导致造血微环境缺陷。小鼠铁过载模型显示提高的 ROS 水平和增加的骨吸收，导致骨微结构发生变化，骨骼的梁状和皮质变薄（103）。这种骨质物质的流失似乎与骨髓间充质干细胞（BM-MSCs）的变化有关。已报道骨髓基质细胞数量和组成的多个改变，这些改变都表明铁过载减少了成骨细胞相对于其他细胞亚型的分化，并减少了基质钙化（103-105）。程等人还证明了一种 ROS 介导的细胞死亡，这是由于 AMPK/MFF/DNM1L 途径介导的铁过载引发了线粒体碎裂并减少了 ATP 产生（106）。已经显示，间充质细胞区的改变也影响了它们对造血的支持功能。 因此，在过载条件下，骨髓基质细胞中几种粘附分子的表达和细胞因子分泌发生改变，影响了它们支持造血细胞生长的能力（24, 105, 107）。这对于 HSCT 期间移植的移植结果可能也很重要，因为从正常供体小鼠到铁过载小鼠的移植导致造血重建延迟（107）。因此，铁过载对骨髓结构和间充质细胞的影响可能导致 MDS 和 AML 患者中发现的造血功能缺陷。

Macrophages in the bone marrow of MDS patients were shown to have higher FTH expression (108). Additionally, expression of HMOX1 in macrophages was associated with an adverse patient outcome. In the microenvironment of solid tumors, tumor-associated macrophages (TAMs) are thought to contribute to tumor progression via delivery of iron to the tumor cells by an iron-release macrophage phenotype (109). However, it has not been investigated if there might be a similar role of leukemia-associated macrophages.

Normal cells of the hematopoietic and especially erythropoietic system are also highly affected by changes in the iron homeostasis as already described in section Iron Homeostasis and its Role for Normal Hematopoiesis. Morbidity and mortality in iron overloaded MDS and AML patients might therefore largely be explained by the toxicity of iron to these cells. In a murine iron overload model using RUNX1S291fs-induced MDS mice, the survival of iron overloaded MDS mice decreased as a result of an impaired frequency and colony-forming capacity of normal hematopoietic stem cells (110).
造血系统，尤其是红细胞生成系统的正常细胞也受铁稳态变化的影响，正如在铁稳态及其对正常造血作用部分所描述的那样。因此，铁过载的骨髓增生异常综合征（MDS）和急性髓系白血病（AML）患者的发病率和死亡率可能主要是由于铁对这些细胞的毒性所致。在使用 RUNX1S291fs 诱导的 MDS 小鼠铁过载模型中，铁过载的 MDS 小鼠的存活率降低，这是由于正常造血干细胞的频率和克隆形成能力受损所致。

The iron household in the bone marrow might also affect endothelial cells and the vascular architecture. Cellular iron deficiency increases VEGF-induced angiogenesis (111, 112). Moreover, it has been shown that ferritin promotes the assembly of endothelial cells by antagonizing the antiangiogenic effects of cleaved high molecular weight kininogen (113).
骨髓中的铁家庭可能也会影响内皮细胞和血管结构。细胞铁缺乏增加了 VEGF 诱导的血管生成（111, 112）。此外，已经表明铁蛋白通过拮抗剪切高分子量激肽原的抗血管生成作用来促进内皮细胞的组装（113）。

Beside the bone marrow, an altered iron metabolism can also impact other organs. Iron overload due to multiple transfusions has been demonstrated to be toxic to various organs as liver, heart, pancreas, thyroid and pituitary gland leading to an increased morbidity and mortality (114). The influence of organ iron overload on patient outcome in MDS and AML patients is not fully determined yet. As described in section Elevated Iron Levels in MDS and AML Patients, data on a potential correlation of liver iron overload with NRM are ambiguous.
除了骨髓外，改变的铁代谢也可能影响其他器官。由于多次输血导致的铁过载已被证明对肝脏、心脏、胰腺、甲状腺和垂体等各种器官具有毒性，导致患病率和死亡率增加。器官铁过载对 MDS 和 AML 患者预后的影响尚未完全确定。正如在 MDS 和 AML 患者中铁水平升高部分所述，关于肝脏铁过载与 NRM 之间潜在相关性的数据是模棱两可的。

Iron, Inflammation, and Infection
铁、炎症和感染

Iron and proteins related with iron overload are closely connected to local or systemic inflammation and might also influence the occurrence of infections by effects on the immune system and various pathogens.
铁和与铁过载有关的蛋白质与局部或全身性炎症密切相关，也可能通过对免疫系统和各种病原体的影响影响感染的发生。

Patients with AML and MDS are often immunocompromised due to a suppression of normal hematopoiesis by the disease and bone marrow toxicity of applied chemotherapies. Additionally, the patients frequently undergo multiple medical interventions including placements of catheters, which further increase the risk of inflammation and infection. Patients receiving an allogeneic HSCT have also a risk of inflammation due to a Graft-versus-Host Disease (GvHD) and need immunosuppressive therapy.
AML 和 MDS 患者常因疾病对正常造血功能的抑制和应用化疗药物的骨髓毒性而免疫功能受损。此外，患者经常接受多种医疗干预措施，包括置管术，这进一步增加了炎症和感染的风险。接受异基因造血干细胞移植的患者也存在由于移植物抗宿主病（GvHD）引起的炎症风险，并需要免疫抑制疗法。

Inflammatory stimuli lead to an upregulation of hepcidin and other acute-phase proteins as ferritin and caeruloplasmin as well as a down-regulation of negative acute-phase-proteins as transferrin. The resulting downregulation of available plasma iron may withhold iron from pathogenic microorganisms and protect healthy tissues from ROS damage at the site of infection. Many microorganisms require iron for electron transport, glycolysis, genome synthesis and defense, making it an essential nutrient. Excess iron has shown to stimulate the growth of many gram-positive and gram-negative bacteria, fungi and single-celled eukaryotes as well as the replication of viruses (115, 116). Correspondingly, patients with hemochromatosis or hemoglobinopathies are at increased risk for infectious diseases due to iron overload (117, 118).
炎症刺激导致肝铁蛋白和其他急性期蛋白（如铁蛋白和铜蓝蛋白）的上调，以及负急性期蛋白（如转铁蛋白）的下调。可用血浆铁的下调可能会阻止病原微生物获取铁，并保护健康组织免受感染部位的 ROS 损伤。许多微生物需要铁来进行电子传递、糖酵解、基因组合成和防御，使其成为一种必需营养素。过量的铁已被证明会刺激许多革兰氏阳性和革兰氏阴性细菌、真菌和单细胞真核生物的生长，以及病毒的复制。相应地，血色病或血红蛋白病患者由于铁过载而增加感染疾病的风险。

In patients undergoing allogeneic HSCT, high pre-transplant ferritin levels have been associated with an increased risk for invasive fungal pneumonia (119, 120) and hepatosplenic candidiasis (121). Patients suffering from mucormycosis in allogeneic HSCT recipients were found to have a severe iron overload compared with a matched control population (122). Moreover, early bacterial infections in allogeneic HSCT recipients were increased in patients with elevated pre-transplant hepcidin levels (123). A large metanalysis demonstrated a higher incidence of blood stream infections, a lower incidence of chronic GvHD and no effect concerning acute GvHD to be associated with high-pretransplant ferritin levels (52). Additionally, Pullarkat et al. reported in a prospective study, that iron overload measured by pre-transplant ferritin was a risk factor for mortality and blood stream infections but also for acute GvHD (124). Thereby, all these studies point towards a prognostic impact of iron overload markers as ferritin and hepcidin for fungal and bacterial infections as well as for the occurrence of GvHD. Although an elevation of these markers was measured before the onset of the diseases, a bias for patients that were already initially prone to inflammation and infection cannot be excluded.
在接受同种异体造血干细胞移植的患者中，高于移植前铁蛋白水平与侵袭性真菌性肺炎（119, 120）和肝脾念珠菌病（121）的风险增加相关。与配对对照人群相比，同种异体造血干细胞移植受体中患有粘膜毛霉病的患者发现存在严重的铁超载（122）。此外，同种异体造血干细胞移植受体中早期细菌感染在具有升高的移植前肝铁蛋白水平的患者中增加（123）。一项大型荟萃分析显示，高于移植前铁蛋白水平与血流感染的发生率增加、慢性移植物抗宿主病发生率降低以及急性移植物抗宿主病无影响相关（52）。此外，Pullarkat 等人在一项前瞻性研究中报告，通过移植前铁蛋白测量的铁超载是死亡和血流感染的危险因素，也是急性移植物抗宿主病的危险因素（124）。因此，所有这些研究都指向铁超载标志物如铁蛋白和肝铁蛋白对真菌和细菌感染以及移植物抗宿主病的发生具有预后影响。 尽管在疾病发作之前测量到这些标记物的升高，但不能排除对那些最初就容易发炎和感染的患者存在偏见。

The function of cells belonging to the immune system may also be influenced by iron homeostasis. In MDS patients with iron overload measured by elevated ferritin and transferrin saturation, Chen et al. found a lower percentage of CD3+ T-cells and disrupted T-cell subsets accompanied by higher ROS-levels in these cells (125). Using a murine iron overload model, Chen et al. showed that iron overload could reduce peripheral T-cells, decrease Th1/Th2 as well as Tc1/Tc2 ratio and increase CD4/CD8 ratio as well as the fraction of regulatory T-cells by inducing ROS-mediated oxidative stress and apoptosis of T-lymphocytes. The impact of these alterations on the anti-leukemic defense, inflammation and infection as well as patient outcome is yet unclear.
免疫系统细胞的功能也可能受铁稳态的影响。在通过升高的铁蛋白和转铁蛋白饱和度测量出的铁过载的 MDS 患者中，Chen 等人发现 CD3+ T 细胞的百分比较低，并伴随着这些细胞中较高的 ROS 水平。Chen 等人利用小鼠铁过载模型显示，铁过载可以减少外周 T 细胞，降低 Th1/Th2 以及 Tc1/Tc2 比率，并增加 CD4/CD8 比率以及调节性 T 细胞的比例，通过诱导 ROS 介导的氧化应激和 T 淋巴细胞的凋亡。这些改变对抗白血病防御、炎症和感染以及患者预后的影响尚不清楚。

Therapies Aiming at Iron Metabolism as a Possible Target in MDS and AML
针对 MDS 和 AML 中可能的靶点铁代谢的治疗方法

Features of iron overload, a differential iron metabolism and changes in proteins associated with iron have been found in MDS and AML patients. Markers of iron overload correlate with a worse prognosis in both patient cohorts. There is a rational for potential pathomechanisms explaining detrimental effects on the patient outcome by consequences of the altered iron metabolism. However, markers of iron overload are in many ways subject to the chicken-and-egg problem making it impossible to discriminate between cause and consequence. Therefore, interventional studies might cast light on the causative impact of the altered iron metabolism.
铁过载的特征，在 MDS 和 AML 患者中发现了不同的铁代谢和与铁相关的蛋白质的变化。铁过载的标志物与两组患者的预后不良相关。有一个合理的潜在病理机制来解释改变的铁代谢对患者结果的不利影响。然而，铁过载的标志物在许多方面都受到鸡生蛋问题的影响，这使得无法区分原因和后果。因此，干预性研究可能会揭示改变的铁代谢的原因性影响。

Iron-targeting strategies are based on the differential iron metabolism in case of MDS and AML compared to normal circumstances constituting a potential vulnerability in these diseases. Therapeutic strategies aiming at iron metabolism as a possible target in MDS and AML can be roughly distributed in four approaches: reduction of iron required for cellular functions via iron chelation, modulation of proteins involved in iron metabolism, induction of ferroptosis und exploitation of iron proteins for the delivery of antileukemic drugs ( Figure 3 ). Thereby, most studies have been conducted using iron chelators, whereas the other approaches are in the early stages of development.
铁靶向策略基于骨髓增生异常综合征（MDS）和急性髓系白血病（AML）相对于正常情况下的差异铁代谢，构成了这些疾病的潜在易感性。针对 MDS 和 AML 中铁代谢作为潜在靶点的治疗策略大致可分为四种方法：通过铁螯合减少细胞功能所需的铁、调节参与铁代谢的蛋白质、诱导铁死亡和利用铁蛋白传递抗白血病药物（图 3）。因此，大多数研究都是使用铁螯合剂进行的，而其他方法还处于早期开发阶段。

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Figure 3 图 3

Targets of different drugs interfering with iron metabolism. Simplified outline with colored arrows indicating the respective way of action. *loss of important protein functions can induce apoptosis and cell cycle arrest. ^†excessive ROS production leading to lipid peroxidation can lead to ferroptosis. LCI, labile cellular iron; TFR, transferrin receptor.
不同药物干预铁代谢的靶标。带有彩色箭头的简化轮廓，指示各自的作用方式。*重要蛋白功能的丧失可能诱导凋亡和细胞周期停滞。 ^† 过量的 ROS 产生导致脂质过氧化，可能导致铁死亡。LCI，不稳定的细胞铁；TFR，转铁蛋白受体。

Perspectives 观点

In this review, we demonstrated the clinical significance of iron homeostasis in MDS and AML patients. Iron metabolism has been shown to impact multiple intracellular functions, the production of ROS, the microenvironment as well as the susceptibility to infections. Markers of iron overload were demonstrated to have prognostic relevance although the impact of an altered iron metabolism on patient outcome in MDS and AML is still under debate as markers of iron overload are highly influenced by inflammatory signals and complicate the detection of causative associations. Supporting a partially causative connection between iron metabolism and patient outcome, therapeutics addressing the iron balance as ICT were found to improve the outcome especially in low/intermediate-1 risk MDS patients. As recurrent red blood cell transfusions constitute the major source of secondary iron overload in MDS and AML patients, a more restrictive application should be considered. Moreover, various agents targeting proteins involved in iron homeostasis or inducing ferroptosis are investigated preclinically or are in early clinical development. With a more detailed understanding of the pathophysiology of MDS and AML in the context of iron, future development of new iron-targeting strategies may lead to better patient outcomes. Therefore, basic research further investigating the processes involved in iron homeostasis linked with redox balance and leukemia is inevitable. Moreover, clinical studies analyzing reliable markers for pathophysiological active iron overload and prospective studies exploring function of iron-homeostasis targeting drugs are essential. Especially the combination of iron-homeostasis targeting drugs with other antileukemic agents constitutes a promising approach due to potential synergistic effects and should therefore be further elucidated.
在这篇综述中，我们展示了铁稳态在 MDS 和 AML 患者中的临床意义。已经显示铁代谢影响多个细胞内功能、ROS 的产生、微环境以及感染易感性。铁超载标志物已被证明具有预后相关性，尽管铁代谢异常对 MDS 和 AML 患者预后的影响仍在争论之中，因为铁超载标志物受炎症信号的影响很大，使得原因相关性的检测变得复杂。支持铁代谢与患者预后之间存在部分因果关系的连接，治疗以调节铁平衡的 ICT 被发现可以改善特别是低/中等-1 风险的 MDS 患者的预后。由于反复输注红细胞构成了 MDS 和 AML 患者继发性铁超载的主要来源，应考虑更为严格的应用。此外，正在临床前研究或早期临床开发中的各种靶向参与铁稳态的蛋白或诱导铁死亡的药物正在研究中。 通过对 MDS 和 AML 在铁元素背景下病理生理学的更详细了解，未来发展新的针对铁元素的策略可能会带来更好的患者预后。因此，进一步开展基础研究，探讨与氧化还原平衡和白血病相关的铁元素稳态过程是不可避免的。此外，分析可靠的标志物以评估病理活性铁元素过载的临床研究和探索铁元素稳态靶向药物功能的前瞻性研究是必不可少的。特别是将铁元素稳态靶向药物与其他抗白血病药物结合使用构成了一种有前景的方法，由于可能产生协同效应，因此应进一步阐明。

Author Contributions 作者贡献

Conceptualization was done by SW, AP, and HS. Investigation and writing of the draft was done by SW and AP. Review and editing of the draft was done by NK, FS, and HS. Supervision was done by HS. All authors contributed to the article and approved the submitted version.
概念化由 SW、AP 和 HS 完成。调查和起草工作由 SW 和 AP 完成。审查和编辑起草工作由 NK、FS 和 HS 完成。监督由 HS 完成。所有作者都为文章做出了贡献并批准了提交的版本。

Funding 资金

This work was supported by the German Cancer Consortium (DKTK) (SW, HS), the Alfred and Angeliga Gutermuth-Stiftung (SW), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—SFB1177 TP E07 (HS) and SFB815 TP A10 (HS, NK) and by the LOEWE Center Frankfurt Cancer Institute (FCI) funded by the Hessen State Ministry for Higher Education, Research and the Arts [III L 5-519/03/03.001-(0015)].
这项工作得到德国癌症联盟（DKTK）（SW，HS）、阿尔弗雷德和安吉利加·古特穆特基金会（SW）、德国研究基金会（DFG，德国研究基金会）—SFB1177 TP E07（HS）和 SFB815 TP A10（HS，NK）的支持，以及由黑森州高等教育、研究和艺术部（III L 5-519/03/03.001-(0015)）资助的 LOEWE 中心法兰克福癌症研究所（FCI）。

Conflict of Interest 利益冲突

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
作者声明，该研究在没有任何可能被解释为潜在利益冲突的商业或财务关系的情况下进行。

References